P = 0.05). This high circulating TNF-α bioactivity was strongly inhibited with the first infliximab infusion. The difference between IL-6 levels induced with plasma samples obtained before and 4 hours after the first infusion was greater in patients with a good clinical response . Similar findings were obtained for OPG production . Levels of circulating TNF-α bioactivity were predictive of clinical response to TNF-α inhibition, confirming a key role for TNF-α in these RA patients.Our objective was to clarify the heterogeneity in response to infliximab treatment in rheumatoid arthritis (RA); to this end, a bioassay was designed to explore the contribution of circulating tumour necrosis factor (TNF)-α bioactivity and its possible link to response. The bioassay is based on the induction of IL-6 and osteoprotegerin (OPG) production by synoviocytes in response to TNF-α. RA synoviocytes were cultured with TNF-α (5 ng/ml) and 42 RA plasma samples collected just before starting therapy. Levels of IL-6 and OPG were measured in supernatants. In 20 of the patients, plasma samples collected before and 4 hours after the first and the ninth infusions were tested in the same way. Plasma concentrations of TNF-α and p55 and p75 soluble receptors were measured using ELISA. TNF-α induced IL-6 and OPG production by synoviocytes, which was further increased with patient plasma dilutions and inhibited by infliximab. With plasma samples obtained before the first infusion, the IL-6-induced production was greater in patients with a good clinical response than in the poor responders (44.4 ± 23.3 ng/ml versus 27.4 ± 20.9 ng/ml;  Rheumatoid arthritis (RA) is a chronic disease characterized by synovial inflammation that leads to progressive joint damage. Knowledge concerning the role played by cytokines in mediating cell–cell interactions in rheumatoid synovium has led to the rational development of treatment with anticytokine agents. Among these proinflammatory cytokines, tumour necrosis factor (TNF)-α has emerged as a major therapeutic target, based on clinical studies with biological inhibitors such as monoclonal antibodies and soluble receptors. In large proportions of patients, TNF-α inhibitors strongly reduced symptoms of synovitis, biological markers of inflammation and bone destruction -4. HowevIn an attempt to explain these differences between patients, we explored whether heterogeneity exists in the contribution of circulating TNF-α bioactivity, with the hypothesis that patients with higher levels of bioactive TNF-α would be more sensitive to the systemic administration of a specific inhibitor. Such circulating TNF-α activity would reflect local joint production. The goal of the present study was to evaluate circulating TNF-α bioactivity in RA patients before infliximab treatment and to assess its acute modulation by infliximab. Indeed, the remaining TNF-α activity would represent the difference between total TNF-α and its fraction bound to specific and nonspecific inhibitors. Therefore, a bioassay was developed using the properties of synoviocytes to produce IL-6 and osteoprotegerin (OPG) in response to TNF-α ,6. Finaln = 24); and poor responders, with an ACR response equal to or less than 20 (n = 18). EDTA-treated venous blood was collected before infliximab therapy in all patients (n = 42). In 20 patients, blood samples were collected during infliximab treatment before and 4 hours after the first and ninth infusions. Plasma samples obtained by centrifugation were stored at -20°C and thawed before use. The main characteristics of the patients are summarized in Table Forty-two patients with RA , diagnosed according to the revised criteria of the American College of Rheumatology (ACR) [–31 yearsA functional assay for TNF-α activity was designed using the ability of RA synoviocytes to produce IL-6 and OPG in response to TNF-α . To isol4 cells/well) were cultured in 96-well plates  in a final volume of 200 μl in minimum essential medium (Life Technologies) supplemented with 10% heat-inactivated foetal calf serum (Life Technologies), 25 000 UI penicillin, 25 000 μg streptomycin and 250 μg fungizone.Synoviocytes were used at passages four to eight. RA synoviocytes (10TNF-α (0–10 ng/ml) was added to RA synoviocytes with or without infliximab (10 μg/ml). Then, TNF-α (5 ng/ml) was combined with 20 μl plasma per well in order to increase the sensitivity of the synoviocyte response. Plasma samples were collected just before the first infusion. In addition, RA synoviocytes were stimulated with plasma samples collected before and 4 hours after the first and the ninth infusions from 20 RA patients. TNF-α (5 ng/ml), with or without infliximab (10 μg/ml), was preincubated with these four plasma samples for 1 hour before being added to the culture.IL-6 and OPG production were measured by ELISA in 48-hour supernatants, as previously described ,6. At ba2 test was performed to detect differences between different subsets.Statistical analysis was performed using the Statview software . Means were compared using a nonparametric test. Spearman's correlation was used to determine a relationship between the changes in IL-6 and OPG levels and those of TNF-α or TNF soluble receptors detected by ELISA. χThe principle of the bioassay, as shown in Fig. n = 4), IL-6 production by synoviocytes increased in a dose-dependent manner .Addition of plasma further increased the effect of exogenous TNF-α. With RA plasma concentrations ranging from 0% to 20% used alone .Samples were obtained from 42 patients before the first infliximab infusion. The levels of IL-6 produced by stimulation with 10% plasma and TNF-α (5 ng/ml) are shown in Fig. For 20 of the patients, samples were collected before and after the first and the ninth infusions. There was no significant clinical difference between the 20 patients and the rest of the 42 patient cohort Table .in vivo administration of the TNF-α inhibitor. This pattern of reduction was associated with a very good clinical response at 54 weeks for all patients  except one (ACR 20). Conversely, no modulation in circulating bioactivity was observed in patients with a low level of TNF-α bioactivity .The difference between the IL-6 levels induced with TNF-α at 5 ng/ml and plasma obtained before and 4 hours after the first infusion correlated with good clinical response . In the second group, circulating bioactivity was still present before the ninth infusion but remained sensitive to further infliximab administration .Patients with high circulating bioactivity at the first infusion could be separated into two subsets. For the first group, before the ninth infusion, no circulating bioactivity was detected Fig. . This paion Fig. . With thP = 0.6).Various clinical and biological parameters were analyzed according to these patterns. Before treatment, no difference was observed between joint counts, erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor positivity, or Disease Activity Score 28 . Similar results were observed with p55  and p75  soluble receptors in good responders versus poor responders. No correlation was observed between circulating TNF-α bioactivity and TNF-α or soluble receptors, or the difference between TNF-α and p55 and p75 levels. Similarly, no negative correlation was observed between levels of circulating TNF-α bioactivity and of p55 or p75 soluble receptors.Levels of TNF-α and of soluble receptors (p55 and p75) were measured in plasma samples obtained at baseline from 20 RA patients. The mean TNF-α level was 144.87 ± 130.36 pg/ml in good responders and 153.75 ± 132.93 pg/ml in poor responders  measured by ELISA. This discrepancy further indicates the usefulness of a bioassay when function is the key. Such complexity has been observed when trying to explain loss of infliximab response by the induction of anti-mouse antibodies . Once agIn conclusion, this bioassay was able to predict correctly the clinical response in 69% of cases (29/42). Taking into account the effect of the first infusion increased the value to 90%. However, a simplified assay would need to be designed for routine application.ACR = American College of Rheumatology; ELISA = enzyme-linked immunosorbent assay; IL = interleukin; OPG = osteoprotegerin; RA = rheumatoid arthritis; TNF = tumour necrosis factor.The author(s) declare that they have no competing interests.HM conducted the experiments and wrote the paper. WM took OPG measurements. PM directed the research.


Large differences in tumour necrosis factor (TNF) productive ability 05337257erved among various strains of mice. DDY, CD-1, ICR and DBA/2 mice could produce a high titre of TNF activity, whereas Balb/c, C3H/HeJms and A/J mice produced a low titre of TNF activity. Administration of 200 micrograms/mouse of LPS to some strains of mice, i.e. DDD and C57B1/6J resulted in good production of TNF. ICR nu/nu mice produced the highest TNF activity among the nude mice. Balb/c nu/nu and DDD nu/nu mice exhibited very low titres of TNF activity. Nude mice required a rather higher dose of the priming agent, Propionibacterium acnes, than heterozygote littermates. Although it is commonly accepted that dual stimulation is necessary for TNF production, TNF activity was detected without the priming agent in SD rats and Golden hamsters by single injection of LPS. In these animals, much higher TNF production was observed after Propionibacterium acnes treatment than after a single injection of LPS. Large differences in TNF productive ability also existed among strains of rats. Although all animals receiving priming agents revealed hyperplasia of reticuloendothelial system, the sensitivity of the animals to LPS is considered to be the most important factor in their TNF productive ability.


The failure of chondrocytes to replace the lost extracellular matrix contributes to the progression of degenerative disorders of cartilage. Inflammatory mediators present in the joint regulate the breakdown of the established matrix and the synthesis of new extracellular matrix molecules. In the present study, we investigated the effects of tumor necrosis factor alpha (TNF-α) and epidermal growth factor (EGF) on chondrocyte morphology and matrix gene expression. Chondrocytes were isolated from distal femoral condyles of neonatal rats. Cells in primary culture displayed a cobblestone appearance. EGF, but not TNF-α, increased the number of cells exhibiting an elongated morphology. TNF-α potentiated the effect of EGF on chondrocyte morphology. Individually, TNF-α and EGF diminished levels of aggrecan and type II collagen mRNA. In combination, the effects of TNF-α and EGF were additive, indicating the involvement of discrete signaling pathways. Cell viability was not compromised by TNF-α or by EGF, alone or in combination. EGF alone did not activate NF-κB or alter NF-κB activation by TNF-α. Pharmacologic studies indicated that the effects of TNF-α and EGF alone or in combination were independent of protein kinase C signaling, but were dependent on MEK1/2 activity. Finally, we analyzed the involvement of Sox-9 using a reporter construct of the 48 base pair minimal enhancer of type II collagen. TNF-α attenuated enhancer activity as expected; in contrast, EGF did not alter either the effect of TNF-α or basal activity. TNF-α and EGF, acting through distinct signaling pathways, thus have additive adverse effects on chondrocyte function. These findings provide critical insights into the control of chondrocytes through the integration of multiple extracellular signals. The role of epidermal growth factor (EGF) in the development of articular cartilage and the pathogenesis of arthritis is poorly understood. During development, EGF produced by the apical ectodermal ridge promotes the outgrowth of the limb bud mesoderm; however, migration away from the apical ectodermal ridge and downregulation of EGF expression in the mesodermal cells is necessary for differentiation of this cell population into chondrocytes . We prev2 from synovial cells -3-(1H-indol-3-yl)-maleimide , or 2,3-bis(1H-indol-3-yl)-N-methylmaleimide , 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene , and 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)-butadiene . BIS I was used at a concentration that was greater than 500 times the inhibitory concentration 50% for conventional PKCs and twice the inhibitory concentration 50% for PKCζ. U0126 was used at a concentration previously found to be effective for inhibiting the phosphorylation of ERK1/2  butadiene.BIS = bisindolylmaleimide; EGF = epidermal growth factor; ERK = extracellular signal-regulated kinase; IL = interleukin; MAPK = mitogen-activated protein kinase; MEK1/2 = mitogen-activated protein kinase kinase 1 and 2; NF = nuclear factor; PARP = poly(ADP ribose) polymerase; PKC = protein kinase C; TNF-α = tumor necrosis factor alpha; TUNEL = terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling; U0124 = 1,4-diamino-2,3-dicyano-1,4-The author(s) declare that they have no competing interests.ARK, an MSc candidate, participated in the design of the study, performed all the experiments and analysis, prepared the figures, and contributed to the writing of the manuscript. SMB conceived of the study, participated in its design and analysis, and prepared and revised the manuscript. Both authors read and approved the final manuscript.


The effects on experimental melanoma of a combination of recombinant human tumour necrosis factor alpha  and carmustine (BCNU) were studied in vitro and in vivo. In vitro, BCNU alone was cytotoxic to murine B16 melanoma cells, and at all concentrations of BCNU this toxicity was increased by the addition of TNF. In vivo, BCNU and TNF, when given separately, caused tumour growth delay of B16 melanoma and of human melanoma xenografts in immune-deprived mice. The combination of TNF at low dose 2.5 x 10(5) U kg-1 = 122 ng kg-1) with BCNU (35 mg kg-1) resulted in significant growth delay  in B16 melanoma (P = 0.005). There was no significant increase in toxicity as assessed by weight loss and peripheral blood counts. Experiments with human melanoma xenografts yielded similar results (P = 0.001) but only at higher doses of TNF (1 x 10(6) U kg-1 = 489 ng kg-1). The enhancement of BCNU cytotoxicity by TNF may be important if it can be translated into patients with melanoma. A randomised study is now underway to investigate the clinical potential of this observation.


Arabidopsis thaliana FWA gene contains two tandem repeats and is an efficient target for RNA-directed de novo DNA methylation when it is transformed into plants. We showed that the FWA tandem repeats are necessary and sufficient for de novo DNA methylation and that repeated character rather than intrinsic sequence is likely important. Endogenous FWA can adopt either of two stable epigenetic states: methylated and silenced or unmethylated and active. Surprisingly, we found small interfering RNAs (siRNAs) associated with FWA in both states. Despite this, only the methylated form of endogenous FWA could recruit further RNA-directed DNA methylation or cause efficient de novo methylation of transgenic FWA. This suggests that RNA-directed DNA methylation occurs in two steps: first, the initial recruitment of the siRNA-producing machinery, and second, siRNA-directed DNA methylation either in cis or in trans. The efficiency of this second step varies depending on the nature of the siRNA-producing locus, and at some loci, it may require pre-existing chromatin modifications such as DNA methylation itself. Enhancement of RNA-directed DNA methylation by pre-existing DNA methylation could create a self-reinforcing system to enhance the stability of silencing. Tandem repeats throughout the Arabidopsis genome produce siRNAs, suggesting that repeat acquisition may be a general mechanism for the evolution of gene silencing.Tandem repeat sequences are frequently associated with gene silencing phenomena. The  Arabidopsis, methylation and silencing mediated by theFWA transgene are found to require the tandem repeat sequences upstream ofFWA and to be independent of siRNA association.In DRM2 isidopsis) . Howeveridopsis) .Arabidopsis by transforming plants with an additional copy of FWA [fwa endogenous allele, a transgenic copy of FWA introduced into wild-type plants by Agrobacterium-mediated transformation is always de novo methylated and silenced. Because both endogenous and transgenic FWA are silenced, transformed plants do not display a late-flowering phenotype. However, when drm2 mutant plants are transformed with FWA, the introduced transgenes remain unmethylated, and FWA overexpression causes a late-flowering phenotype. In drm2 transformants, endogenous FWA retains its methylation and silencing, because DRM2 only controls de novo methylation but not maintenance methylation of CG sites [FWA transgenes from drm2 transformants are outcrossed to reintroduce wild-type DRM2, the transgenes remain stably unmethylated [fwa endogenous alleles and suggests that FWA de novo DNA methylation only occurs during the process of plant transformation.De novo DNA methylation can be studied in y of FWA . DespiteCG sites . Intriguthylated . This isFWA transformation assay to demonstrate that de novo DNA methylation by DRM2 is guided by an RNAi pathway including the NUCLEAR RNA POLYMERASE IV A (NRPD1a) subunit of DNA-dependent RNA polymerase IV (RNA Pol IV), RNA-DEPENDENT RNA POLYMERASE2 (RDR2), DICER-LIKE3 (DCL3), and ARGONAUTE4 (AGO4) [RDR2 is required for paramutation demonstrates that RNA-directed DNA methylation at tandem repeats is conserved in both monocots and eudicots [Arabidopsis RNAi mutants reduce or eliminate CNG and asymmetric DNA methylation at endogenous tandem repeats and other sequences in a manner analogous to that of drm2 mutants. However, at most loci, the maintenance of CG methylation in drm2 or the RNAi mutants is generally unaffected. These results show that RNAi-related processes are required to propagate non-CG DNA methylation; meaning that, even after establishment, siRNA production likely persists at particular genomic loci.RNA interference (RNAi) is a versatile silencing pathway in which double-stranded RNA is processed by Dicer nucleases to generate 21–24-nucleotide small interfering RNAs (siRNAs) . siRNAs 4 (AGO4) . The receudicots . In addiFWA is targeted for DNA methylation, unlike the majority of single-copy Arabidopsis genes transformed into plants. Here we show that the tandem repeats of FWA are necessary and sufficient for triggering de novo DNA methylation, and that repeated character (rather than the FWA sequence itself) is likely critical. Transformed FWA can silence the unmethylated endogenous fwa gene in trans, and this property also requires tandem repeats. Surprisingly, the methylation state of endogenous FWA can affect silencing of an incoming transgene, because FWA transformed into fwa-1 is inefficiently methylated compared to wild type. The fact that DNA methylation enhances the ability of endogenous FWA to communicate with transformed FWA is particularly striking, because both methylated and unmethylated forms of endogenous FWA produce equivalent levels of siRNAs. Furthermore, we show that FWA siRNA synthesis is dependent on wild-type NRPD1a, RDR2, and DCL3, but not on the putatively downstream components NRPD1b, AGO4, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION1 (DRD1), and DRM2. Our results suggest a two-step model for DNA methylation, in which tandem repeats are sufficient to recruit NRPD1a, RDR2, and DCL3 to produce siRNAs, but a second triggering event such as plant transformation is required to allow siRNAs to target downstream DNA methylation. Finally, a genomic analysis suggests that unique tandem repeats, such as those at FWA, are common targets for the siRNA machinery, indicating the potential generality of this gene silencing mechanism on an evolutionary time scale.Although it seems clear that de novo DNA methylation derives its sequence specificity from siRNAs, it is not known why transformed fwa endogenous allele never becomes spontaneously methylated, de novo DNA methylation of transformed FWA occurs with 100% efficiency [FWA transgenes in drm2 transformants are not de novo methylated when crossed to plants containing functional DRM2. This suggests that de novo methylation of FWA occurs either during the transformation process, or shortly thereafter, and we sought to determine more precisely when this event occurs. During the floral dip procedure, Agrobacterium tumefaciens transforms the female gametophytic lineage of Arabidopsis (probably the egg cell itself), integrating its transferred DNA (T-DNA) only into the maternal genome [FWA into heterozygous rdr2–1/RDR2, ago4–1/AGO4, and drm1–1/DRM1 drm2–1/DRM2 plants , indicating that the genotype of the fertilized zygote or endosperm determines whether an FWA transgene is silenced. Thus, although FWA de novo DNA methylation requires plant transformation, the particular conditions that facilitate de novo DNA methylation must persist until sometime after fertilization.We found that all three experiments showed an early:late flowering ratio close to 3:1 amongst formants . This eaFWA is restricted to the two pairs of tandem repeats at the farthest 5' end of its transcribed region, suggesting that they play a key role in controlling gene silencing [FWA tandem repeats are 2 × 38 base pairs (bp) and 2 × 198 bp in length. To test the role of tandem repeats in FWA de novo DNA methylation, we cloned several deletion variants of FWA. Because some of these variants did not contain the FWA open reading frame, they would not be expected to cause late flowering when transformed into wild-type Arabidopsis, meaning that we could not use transgene-induced late flowering as an assay. Therefore, we took advantage of the observation that transforming the late-flowering fwa-1 mutant with a cosmid containing FWA causes the occasional appearance of early-flowering plants among first generation T1 transformants [fwa-1 plants never regain DNA methylation during normal plant growth, both transgenic and endogenous FWA genes contain DNA methylation in early-flowering fwa-1 + FWA plants [FWA can target de novo DNA methylation to the unmethylated endogenous fwa gene, albeit inefficiently.DNA methylation of ilencing . The FWAformants . DespiteA plants . This shfwa-1 plants with a 6.1 kilobase FWA transgene, and found that 5%–25% of transformants had an early-flowering phenotype  was unable to silence endogenous fwa. The “repeats deleted” and “single-copy” transgenes did not cause late flowering when transformed into the rdr2–1 mutant . To our surprise, however, we found that transgenic FWA was completely unmethylated in late-flowering fwa-1 + FWA T1 transformants, when assayed either by bisulfite genomic sequencing  is 18 in libraries made from floral tissue, in contrast to microRNA 167, which has a TPQ of 59951 in the same libraries [FWA siRNAs using conventional RNA probes [FWA siRNA signal in these previous experiments was very close to background levels, making it difficult to accurately assess the abundance of FWA siRNAs in different genetic backgrounds. To circumvent these problems, we synthesized a locked nucleic acid (LNA) oligonucleotide probe to FWA [FWA 24-nucleotide siRNAs . If this is the case, however, these alternative ARGONAUTEs are not capable of replacing the function of AGO4 in RNA-directed DNA methylation. The observation that NRPD1a is required for FWA siRNA production, yet NRPD1b is not, supports the conclusion of Kanno et al. and Pontier et al. that the two forms of RNA Pol IV play distinct roles in the process of RNA-directed DNA methylation [FWA siRNAs contrasts with that of siRNAs from the small euchromatic SINE transposon AtSN1, the levels of which are greatly reduced in ago4, nrpd1b, and drm1 drm2 mutants [AtSN1 may be a locus where feedback from downstream DNA methylation is required to maintain siRNA production.We observed that d dcl3–1 C. Howevehylation C. These pathways . It is phylation ,23. The  mutants ,28,29; AFWA siRNAs are produced from unmethylated fwa alleles and yet do not cause DNA methylation. This prompted us to ask if these siRNAs instead might be causing post-transcriptional mRNA destruction. To test this, we used reverse transcription coupled to real-time quantitative PCR (RT-qPCR) to compare the levels of mRNA produced in either fwa-1 plants that produce FWA siRNAs or in fwa-1 nrpd1a-1 plants, where FWA siRNAs are undetectable , tandem repeats at FWA and MEDEA INTERGENIC SUBTELOMERIC REPEAT (MEA-ISR) lose their non-CG DNA methylation but fully retain CG methylation [FWA remains silent in these plants (which have an early-flowering phenotype), because CG methylation is sufficient to suppress FWA expression. In nrpd1a-1 × rdr2–1 F1 plants, non-CG DNA methylation was immediately restored at both FWA and MEA-ISR /SUVH4, and the kyp-2 mutation strongly reduces H3K9me2 at the FWA tandem repeats [kyp-2 nrpd1a-1 and kyp-2 rdr2–1, then crossed these plants together to reintroduce wild-type NRPD1a and RDR2 in a background that is still homozygous for kyp-2. In the F1 of the kyp-2 nrpd1a-1 × kyp-2 rdr2–1 cross, asymmetric DNA methylation at FWA returned to the same levels as kyp-2 (FWA that have CG DNA methylation but lack KYP-dependent H3K9me2 are still capable of recruiting RNA-directed non-CG DNA methylation.Histone H3 lysine 9 dimethylation (H3K9me2) is a post-translational modification associated with gene silencing and has been shown to control non-CG DNA methylation in bidopsis ,32. Furt1 mutant . We ther repeats . We consas kyp-2 C. Thus, FWA as a model provide evidence that tandem repeats recruit RNA silencing proteins to produce siRNAs. To assess the generality of these findings, we correlated tandem repeat sequences throughout the Arabidopsis genome with a large database of siRNAs cloned by massively parallel signature sequencing [Our results using quencing . We usedquencing  to definquencing  to excluArabidopsis genome  from the euchromatic left arm of chromosome 1 using the same filtering procedure and threshold to remove repeated sequences or transposons. From a library of 5945 random windows, we found 14 that overlapped with dense or moderate siRNA clusters (0.24%). Thus, tandem repeats are nearly an order of magnitude more likely to produce siRNAs than random, nonrepeated sequences , and nucleotide sequence content. We found that tandem repeats with siRNAs were not significantly different in any of these characteristics . Thus, iThe euchromatic unique tandem repeats we analyzed represent only a small fraction of all tandem repeats that produce siRNAs. On a genome-wide scale, there is a strong correlation between tandem repeat character, siRNA production, and DNA methylation ,18,37; tFWA tandem repeats were proposed to arise from a SINE transposon insertion [FWA 5′ untranslated region [FWA tandem repeats and other Arabidopsis sequences (or any sequences in GenBank) suggests that other possibilities for their origin may be plausible. In particular, we propose a hypothetical yet parsimonious model in which the FWA tandem repeats could have arisen from an ancestral unrepeated sequence by a single internal duplication event , bypassing the requirement for preexisting CG DNA methylation in recruiting RNA-directed DNA methylation. Agrobacterium introduces its T-DNA into plant cells as a single-stranded DNA molecule, which is copied into double-stranded DNA before its integration into the plant genome [The process of plant transformation can also facilitate RNA-directed DNA methylation, because an t genome . Multiplt genome . AlternaAgrobacterium transformation to recruit molecular components that use siRNAs to cause de novo DNA methylation. In this way, the presence of siRNAs at unmethylated tandem repeats may potentiate DNA methylation during such expansions, or during periods of genome stress when chromatin is perturbed. Most tandem repeats are found at more than one genomic location and may be associated with transposons. In these cases, siRNA production may act as a kind of “immune memory” of dispersed repeats, allowing de novo DNA methylation to respond when selfish DNAs increase their copy number. The example of maize paramutation shows that tandem repeats in their natural context can indeed silence other genes in this manner [Tandem repeats within a chromosome can increase their copy number by unequal crossing over or by gene conversion. Because these processes involve DNA breakage and rejoining reactions, they may act in the same way as s manner .Our findings also highlight the molecular mystery of how tandem repeats are initially recognized by RNAi proteins. Double-stranded RNAs that provide a Dicer substrate can be produced from inverted repeats by monodirectional transcription and fold-back, or from high-copy sequences by transcription of dispersed repeats in opposite directions. However, the mechanism that distinguishes unique tandem repeats from single copies of the same sequence remains unclear. One possibility is that NRPD1a-containing Pol IV and/or RDR2 may recognize unusual DNA or RNA structures that can only be made by tandem repeats. Regardless of the exact recognition mechanisms involved, the cellular RNAi machinery may be poised to produce siRNAs from newly created tandem repeats. In addition to providing a defense against potentially harmful sequences, this molecular mechanism may facilitate the evolution of genes whose normal expression is controlled by siRNAs and DNA methylation.Plants were grown under continuous light. All mutants have been previously described ,29–31,33FWA transformation was performed as described [escribed . For hetFWA deletions were within the context of the XbaI-HindIII fragment from −2124 to 3876 (where the start codon is at +1) and were cloned into pCAMBIA1300. An FWA transgene where a BglII site is changed to EcoRI has been described [FWA promoter lacking the open reading frame contains nucleotides −2124 to 0, placed before a GFP-GUS open reading frame and cloned into pCAMBIA1300 from XbaI to HindIII (GFP-GUS was derived from pBGWFS7) [GFP-GUS and cloned into pCAMBIA1300.Unless noted, escribed . The “repBGWFS7) . The “reFWA, the region analyzed had the same boundaries as full-length FWA (nucleotides −1055 to −608). Bisulfite sequencing data are summarized in Bisulfite sequencing was performed as described ,28. For FWA LNA probes used in this study are listed in Northern blots were performed as described . The seqFWA and ACT7 PCR primer sequences are listed in RNA was extracted from rosette leaves with TRIzol reagent , DNAse treated with the DNA-free kit , and converted to cDNA with SuperScript II reverse transcriptase (Invitrogen) using a poly-T primer. Figure S1(126 KB PDF)Click here for additional data file.Figure S2(558 KB PDF)Click here for additional data file.Figure S3(233 KB PDF)Click here for additional data file.Figure S4(86 KB PDF)Click here for additional data file.Table S1(79 KB PDF)Click here for additional data file.Table S2(26 KB PDF)Click here for additional data file.


Drawing a pedigree is a prerequisite in genetic counseling. In medical records, a pedigree is useful to document the family history of the patient. Drawing a pedigree is also necessary in collecting genetic resources for medical research such as positional cloning. Currently, most pedigrees are drawn by hand or by drawing software. Due to the special requirements in a standardized pedigree, generating a pedigree by these methods is usually time-consuming and requires professionals. This limits the usage of a pedigree as demanded in remote diagnosis or online counseling from the counselees to send an electronic pedigree.We developed an online pedigree drawing tool, PediDraw, which enables users to generate pedigrees after inputting the family information step-by-step on web. It outputs a pedigree or table to present a family history to the counselors.PediDraw is a user-friendly web-based drawing tool. It is accessible via Internet. A pedigree is a genealogical graph or table which records the relationship of families by degrees. It is essential to genetic counseling. Drawing a pedigree is regarded as a trained skill of genetic counselors to obey the general rules of pedigree nomenclature . GeneticTo meet the above needs, several tools for drawing a pedigree or record the family history have been developed. Most of them are not web-based and can not be freely online accessible by users  as listed in the category of "Pedigree Drawing Programs/Software Reviews" compiled by a genetic counselor, Debra Collins, at University of Kansas Medical Center, although it is not comprehensive . A well-HyperText Markup Language (HTML), JavaScript (JS), Cascading Style Sheets (CSS), and Personal Home Page (PHP). All the data pages for storing the input information in a pedigree were converted to Structures Query Language (SQL) files based on the MySQL database system.EditPlus text editor, Macromedia Dreamweaver MX, Photoshop CS.PediDraw™ (version 1.0) is copyrighted by The National Copyright Administration of People's Republic of China. It is hosted in HP ProLiant ML150 dual Xeon 2.80-GHz server . It is running in Microsoft Windows XP environment. Apache provides a secure, efficient and extensible server for HTTP services.The following five modules are the operations the users have to complete sequentially.Before starting a pedigree drawing, a user has to register. When the registration is finished, the user can login by using his/her username and password. When the user re-logins, the profile that was input the last time is displayed.The first sheet inputs the registered user's information including age, sex, affection status, marital status. If the user is married, (s)he may specify the affection and survival status of her/his spouse and children. The user may edit the existing information when (s)he re-logins. When the user is not sure of the information, (s)he could check any box first, and modify or delete it later on. When finished, click the "Next" button for the next step.This sheet records the affection status and survival status of any user's ancestors, including parents and grandparents. When finished, click the "Next" Button for proceeding.In this sheet, PediDraw allows the user to input all the information  of her/his family members, including the user's brothers/sisters, and the brothers/sisters of the user's father and mother. To avoid the confusion, the name of a family member could be real or nickname, but any two members in the family should have different names. To extend the family members, underneath each selected family member, it allows the user to input the affection status and survival status of each family member's spouse and children. The information can be modified or deleted when editing. Choose "Modify" or "Delete" when the information of a member is filled. When verified, click the "Add" button, the added member will be showing below the main table in orange color. When all the family members are specified, click "CONFIRMATION" button above the main table to finish this step.Here displays a confirmation table of all the input information of the user's pedigree. In the table each member of the pedigree has been assigned an ID number automatically Table . UndernePediDraw is featured with "Help" information in each step. The major operations of a user are to make selections from the icons. A user may return to the previous page by using the backward button in the tool bar. All these features enable an untrained counselee to learn how to use PediDraw easily.PediDraw provides an online drawing tool for a pedigree that is recognized by any genetic counselors as it obeys the standardized nomenclature of a pedigree . AlthougAs described in the "Results" section, PediDraw allows the user to choose either a pedigree table or a pedigree tree for output, which can be saved in a computer or printed directly. This feature makes it feasible to send an electronic file to a genetic counselor. Currently, the Chinese version of PediDraw is implemented in the online genetic counseling in China Genetic Counseling Network (CGCN) . In CGCNPediDraw is providing a user-friendly web-based pedigree drawing tool. It has two optional output formats, a table and a graph. It is readable by any genetic counselors as PediDraw applies the standardized rules of drawing a pedigree in genetic counseling.PediDraw provides an open access  to any IThe author(s) declare that they have no competing interests.MH and WL designed the study. MH and WL wrote and tested the software. WL wrote the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Tumour necrosis factor (TNF) is crucial for the control of mycobacterial infection as TNF deficient (KO) die rapidly of uncontrolled infection with necrotic pneumonia. Here we investigated the role of membrane TNF for host resistance in knock-in mice with a non-cleavable and regulated allele (mem-TNF).M. tuberculosis H37Rv  and the survival, bacterial load, lung pathology and immunological parameters were investigated. Bone marrow and lymphocytes transfers were used to test the role of membrane TNF to confer resistance to TNF KO mice.C57BL/6, TNF KO and mem-TNF mice were infected with Mtb infection. However, during the chronic phase of infection mem-TNF mice succumbed to disseminated infection with necrotic pneumonia at about 150 days. Reconstitution of irradiated TNF-KO mice with mem-TNF derived bone marrow cells, but not with lymphocytes, conferred host resistance to Mtb infection in TNF-KO mice.While TNF-KO mice succumbed to infection within 4–5 weeks, mem-TNF mice recruited normally T cells and macrophages, developed mature granuloma in the lung and controlled acute Mtb infection. Bone marrow cells, but not lymphocytes from mem-TNF mice confer resistance to infection in TNF-KO mice. Long-term infection control with chronic inflammation likely disrupting TNF mediated cell-cell signalling, additionally requires soluble TNF.Membrane expressed TNF is sufficient to allow cell-cell signalling and control of acute  M. tuberculosis (Mtb) infection is regulated by T cells, macrophages and cytokines including IFNγ, IL-12 and TNF [M. tuberculosis and succumb to the infection [Protective immunity to nfection -6. A crinfection -10. TNF nfection -14.in vivo role in the control of Listeria and mycobacterial infection [TNF produced by macrophages, and a variety of other cells, is a major regulator of inflammation and leukocyte trafficking ,16. Althnfection ,21. Howenfection ,21. The nfection .Mtb infection. We compared the host resistance to acute mycobacterial infection in mem-TNF mice, eg. Δ1–9,K11E TNF knock-in mice [Here we asked whether membrane TNF is sufficient for containing -in mice , and TNF-in mice . We showMem-TNF  and TNF-M. tuberculosis H37Rv (Pasteur) was performed by delivering 100 bacteria into both nasal cavities (20 μl each) under xylazine-ketamine anaesthesia as described [Pulmonary infection with escribed . The bacM. tuberculosis H37Rv as described [Bacterial loads in organs of infected mice were evaluated at different time points after infection with escribed . For hisescribed .FACS analysis of inflammatory cells from infected lung was performed as described ,24. Rat IL-12p40 and IFNγ were quantified using commercial ELISA . Bioactive TNF was assessed using the WEHI 164 cells based bioassay .6 fresh unseparated bone marrow cells by intravenous injection in the lateral tail vein (n = 6 per group). Mice were left to fully reconstitute for at least three month prior to infection, reconstitution was verified by the analysis of the haematogram. In addition, freshly isolated splenic T lymphocytes (107 i.v.) from WT and mem-TNF mice were transferred into TNF KO mice (exposed to 4 Grey γ-irradiation) immediately before Mtb infection and survival was followed (n = 6 per group). The experiments were repeated once.Haemopoietic reconstitution of bone marrow was performed as described . BrieflyMurine bone marrow cells were isolated from femurs and differentiated into macrophages using 20% horse serum and 30% L929 cell-conditioned medium as a source of M-CSF . Alterna5 cells/well) and stimulated with LPS  or M. tuberculosis H37Rv , or infected with M. bovis BCG  as described before [Bone marrow derived macrophages (BMDM) and dendritic cells (BMDC) were plated  were compared using a log rank test for the comparison of the groups. A p value <0.05 was considered statistically significant.M. bovis BCG and Mtb infection or to LPS, TNF is undetectable in culture supernatants of BMDM from TNF-KO and strongly defective in mem-TNF mice  injection. In contrast to WT mice TNF was undetectable in the sera of both TNF-KO and mem-TNF mice (not shown). While bone marrow derived macrophages (BMDM) secrete high TNF levels in response to mice Fig , the latmice Fig . Therefomice Fig  and dendMtb given by intranasal administration. Within 4–5 weeks of infection TNF-KO mice displayed rapid weight loss (not shown), impeded locomotor activity and succumbed to infection rapidly , but not in WT and only slightly in mem-TNF mice Fig . Lungs o6 CFU counts in the lungs . Therefore, acute Mtb infection is controlled in the presence of membrane TNF only.We then determined the mycobacterial load in the lungs. At 30 days WT mice displayed a 4 log increase of viable mycobacteria reaching 10ungs Fig , which smice Fig . Mtb disMtb infection. At 30 days post infection the lungs of mem-TNF mice displayed well-defined granulomatous lesions that were characterised by foamy epitheloid like macrophages with surrounding and interspersed and perivascular lymphocytic infiltration, similar to the granuloma structures formed in WT mice , in line with the severe pathology in these mice, while the sole presence of mem-TNF prevented the augmented lymphocyte recruitment as shown in mem-TNF mice , which is at variance with the recent data from Saunders et al. [First we attempted to confer resistance in irradiated TNF KO mice to acute s et al. .Mtb infection of TNF-KO mice. Lethally irradiated TNF-KO mice reconstituted with bone marrow cells from mem-TNF mice controlled Mtb infection as demonstrated by survival over three months, while TNF-KO mice succumbed to acute necrotic pneumonia within 40 days  mutated in the TACE cleavage site . The expA critical role of TNF for the effective control and resolution of mycobacterial infection has been demonstrated previously ,10,24, wThe mechanism how membrane TNF confers protection may be due to cell-to-cell contacts of T cells, macrophages and other cells and needs further investigations. Several biological functions of membrane TNF have been reported previously and a preferential TNFR2 signalling has been suggested in vitro , while iMembrane TNF is sufficient for the development of granulomas. In the absence of TNF there was no formation of well-defined granulomas ,36. NeutInterestingly, lymphocyte recruitment was lower and comparable in mem-TNF mice and WT mice Fig.  unlike iFinally, the question arises why mem-TNF mice succumb in the chronic phase of infection. It may be hypothesized that membrane expressed TNF activates macrophages in the absence of soluble TNF to a certain level, but the killing may not be as effective as in WT macrophages. Secreted TNF and hence distal signalling is likely required in the chronic phase where direct cell contact between T cells and macrophages may be more difficult to achieve in the chronic inflammatory and fibrotic tissue and therefore soluble TNF may be more efficient to maintain the activation of the innate host defence.Mtb infection. Bone marrow cell reconstitution, but not lymphocyte transfer from mem-TNF mice confer resistance to infection in TNF-KO mice. Long-term infection control, however, with chronic inflammation disrupting TNF mediated cell-cell signalling, requires additionally soluble TNF.Membrane expressed TNF allows cell-cell signalling and control of acute The data are of clinical significance, as neutralising therapies used to treat patients with rheumatoid arthritis may reactivate latent TB infection . TNF bloMtb M. tuberculosis,CFU colony forming unit,mem-TNF membrane bound TNFThe author(s) declare that they have no competing interests.CF and NA were driving the project by designing the protocol and conducting the infectious protocol together with ID and MJ. SG was advising on the genetic mouse model, PCR control and cell transfer experiments. VQ with VY conducted the in vitro cell culture experiments with FACS analysis. BR with MJ was responsible for the overall design and control of the studies.


Alcohol abuse has long been known to adversely affect innate and adaptive immune responses and pre-dispose to infections. One cellular mechanism responsible for this effect is alcohol-induced suppression of TNF-α (TNF) by mononuclear phagocytes. We have previously shown that alcohol in part inhibits TNF-α processing by TNF converting enzyme (TACE) in human monocytes. We hypothesized that the chain length of the alcohol is critical for post-transcriptional suppression of TNF secretion.Due to the complex transcriptional and post-transcriptional regulation of TNF in macrophages, to specifically study TNF processing at the cell membrane we performed transient transfections of A549 cells with the TNF cDNA driven by the heterologous CMV promoter. TNF/TACE interactions at the cell surface were assessed using fluorescent resonance energy transfer (FRET) microscopy.The single carbon alcohol, methanol suppressed neither TNF secretion nor FRET efficiency between TNF and TACE. However, 2, 3, and 4 carbon alcohols were potent suppressors of TNF processing and FRET efficiency. The effect of ethanol, a 2-carbon alcohol was reversible.These data show that inhibition of TNF-α processing by acute ethanol is a direct affect of ethanol on the cell membrane and is reversible upon cessation or metabolism. Alcohol is a potent immunosuppressive drug and has been widely recognized for many centuries as an important risk factor for the development of infections. Among infection, pneumonia has been highly linked to alcohol abuse. In a recent case-controlled study in Spain, of 50 control subjects and 50 patients with community-acquired pneumonia, high alcohol intake was identified as a risk factor for pneumonia .The cellular mechanisms by which alcohol results in immunosupression have recently been reviewed . One proBased on these data we hypothesized that this was a direct affect of ethanol and therefore reversible when ethanol was no longer present in the cell and secondary to the ability of alcohol to intercalate in the cell membrane. If this latter condition were true, than the suppression of TNF/TACE interactions should be related to the hydrophobicity and the chain length of the alcohol. To examine these two hypotheses we used florescence resonance energy transfer (FRET) to assess TNF/TACE interactions in A549 cells as previously described  using clKpnI and BamHI and inserted in-frame into p3xFLAG-CMV-14 protein expression vector  to form a vector: p3xFLAG-TNF-α-CMV-14 which can express a FLAG-tagged human TNF-α, driven by the CMV promoter.The full-length coding sequence of human TNF-α was prepared by polymerase chain reaction (PCR) from its cDNA coded in pcDV1 (ATCC#39894) with COOH-terminal deletion using following primers: sense, 5'-AAGCTTGGTACCACCACTATGAGCACTGAAAGCATGATC-3'; antisense, 5'-TGACTAGGATCCCAGGGCAATGATTCCAAAGTAGAC-3'. The PCR product was digested with A549 cells were maintained in D-MEM/F12 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine.2 humidified incubator for 24 hours to get the greatest protein expression (data not shown).Due to the complex transcriptional and post-transcriptional regulation of TNF in macrophages, to specifically study TNF processing by TACE at the cell membrane we performed transient transfections of A549 cells (which express TACE) with the TNF cDNA. A549 cells were transfected with p3xFLAG- TNF-α-CMV-14 using Lipofectamine™2000 reagent (Invitrogen) as recommended by the manufacturer. Transfected cells were incubated in the 37°C, 5% COTwenty-four hours post-transfection, A549 cells were split into 24-well plates, adhered for 3 hours, and randomized to treatments for 1 or 2 hours with fresh medium or medium with 50 mM or 100 mM alcohols of different one to four carbon chain lengths, methanol, ethanol, 1-propanol, and n-butanol. A subgroup of cells randomized to the 2 hour incubation time had media changed to remove the alcohol after 1 hour and cultured for an additional 1 hour washout period. Culture supernatants were then harvested for secreted TNF-α determinations by ELISA and cells were removed from culture plate by incubating with 2 mM EDTA and lysed with 1%NP-40 in phosphate-buffered saline (PBS) containing 1 mM EDTA and proteases inhibitor cocktail (Roche) to measure cell-associated TNF-α as previously described . In certThe levels of the cell associated form of TNF-α in cell lysates and the secreted from in culture supernatant were measured by ELISA. Before testing, both cell lysates and culture media were spun by centrifugation for 15 min at 13,000 rpm in a microfuge. ELISA was performed using kit from R&D Systems  and following the manufacturer's protocol. Cell-associated TNF values were normalized to total protein using a Pierce Protein assay . Concentrations of EtOH up to 100 mM had no inhibitory effect on the TNF ELISA.Transfected A549 cells were adhered to cover slips in culture. After alcohol exposure for 1 or 2 hour or after the washout period, the coverslips were washed in cold PBS to remove excess media and fixed in a 4% solution of 10% methanol-free formaldehyde  in PBS for 15 minutes at room temperature. After thoroughly washing the fixative off with cold PBS, the cells were blocked with 5% rabbit serum in 1% bovine serum albumin (BSA) in PBS for 15 minutes. Subsequently, the blocker was replaced with the first primary antibody, mouse anti-human TACE clone M222 , diluted in 1% BSA at 10 ug/mL. The coverslips were thoroughly washed in three PBS changes after the 45-minute primary antibody incubation. The antibody was indirectly labeled with a rabbit anti-mouse, Cy-3 conjugated Fab fragment  at 6.5 ug/mL in 1% BSA. Next, cells were washed and blocked with 5% mouse serum for 10 minutes to saturate any open antigen binding sites within the first primary antibody. The blocker was replaced with the second, primary antibody mouse anti-human, FITC-conjugated, TNF-α clone Mab11  at a 10 ug/mL dilution in 1% BSA for 30 minutes. All antibody dilutions remained constant throughout experiments and were titrated to be detected at their highest saturation point, hence maximizing to an optimal donor to acceptor ratio. Finally, the coverslips were washed, mounted cell-side down in PBS on slides with spacers, and secured with nail polish.Human A549 cells mounted on coverslips were imaged using a digital fluorescence microscopy system. The system included a 12-bit, chilled charged-coupled device (CCD) Sensicam QE with a 1376 × 1040 resolution and 65% quantum efficiency at 550 nm  on an automated Leica DMRXA microscope  using a 1.4 NA, 63 × Plan-apochromat objective. FITC and Cy3 were detected using appropriate filters  . Fluorescence was excited with a 75 W Xenon arc lamp. The samples were photobleached with 100 W Mercury source. All components were controlled by Slidebook software , which was used for capture, nearest neighbor deconvolution, and FRET analysis. Image acquisition was adjusted in milliseconds to achieve the maximum CCD camera range. As part of detecting bleed-through, typical exposure times were used to confirm no visualization of FITC-labeled samples with the Cy3 filter cube and vice versa.All experiments were based on FRET measured by acceptor photo bleaching recovery using FITC as the donor and Cy3 as the acceptor. The Cy3 was determined to be photolabile enough to undergo the pre-measured 3-minute photo-bleaching step for a subsequent <2% initial intensity measurement. The donor had a high enough quantum yield and stability to not fade significantly. All experiments included a donor-only and acceptor-only sample to verify minimal crossover between them, hence no significant energy transfer. Data were collected for 20 different fields from a single cover slip. Each fluorescent channel was collected at six consecutive z-planes measuring 0.3 um each. The software allowed stack auto-alignment between exposures to compensate for thermally induced shifts. The FRET experiment imaging began with an initial image stack of the FITC-labeled protein (in the presence of the Cy3-labeled antibody) obtained with the FITC filter set immediately followed by an image stack captured with the Cy3 filter cube. Next, the sample was exposed to constant illumination for 3 minutes to photo bleach. An image stack of the Cy3 fluorescence after photo bleaching was then obtained followed by another FITC fluorescence image stack using the FITC filter set. The exposure times were unchanged between pre and post-photo bleaching steps. Post-FRET calculation images were deconvolved using a nearest neighbor algorithm for presentation purposes only.Images indicating FRET between the labeled antibodies against TNF and TACE were calculated based in the increase in donor fluorescence after acceptor photobleaching by using the following formula:post photo bleach - FITC pre photo bleach / FITC post photo bleachE = FITC Here, E represents FRET efficiency after performing a background subtraction caused by scattered light, auto fluorescence, and dark current of the CCD camera. Fluorescence intensities were reported in arbitrary units and on a pixel-to-pixel basis. This was achieved by digitally performing the pre-photo bleaching channel subtraction from the post-photo bleaching channel (see above formula) and thresholding the difference, labeled as "mean FRET intensity". FRET efficiency data was determined by analyzing at least 50 cells per coverslip.All data are presented as mean ± SEM. Significance was estimated using ANOVA followed by Tukey's Multiple Comparison Procedure with p < 0.05 being considered significant.In pilot experiments, we determined the hourly rate of TNF secretion in transfected A549 cells and found that the rate of secretion of TNF-α was 1400–1550 pg/ml from hour 27–28 and hour 28–29 post transfection , block TNF secretion by posttranslational mechanism by inhibiting protein: protein interactions between TNF and TACE in the cell membrane. There have been many studies to better understand the mechanisms by which alcohol contributes to immunosuppression. Many types of infection, including bacterial pneumonia and, more recently, Hepatitis C have been shown to be adversely affected by concomitant alcohol abuse -18. In aOne possible mechanism underlying the immunosuppressive effects of acute alcohol exposure is its effects on innate immunity. In human volunteers and experimental animal models, alcohol dose-dependently suppresses neutrophil recruitment in response to chemotactic stimuli. One mechanism operative in the lung is suppression of the pro-inflammatory cytokine, TNF, which is required for induction of CXC-chemokines critical for neutrophil recruitment ,21, as wIn addition to the known affects of ethanol on TNF-α transcription ,22, anotUsing protein assays and FRET microscopy, we demonstrated that acute alcohol exposure resulted in dose dependent suppression of TACE-mediated processing of TNF. Moreover the suppression of TNF secretion was recoverable within one hour of removing the ethanol. Methanol which contains a single carbon was ineffective in suppressing TNF in A549 transfectants whereas both 1-propanol (3-carbon) and n-butanol (4-carbon) were more potent in suppressing in TNF secretion. Furthermore, methanol was a weak inhibitor of TNF/TACE interactions as measured by FRET efficiency whereas both ethanol and 1-propanol potently and reversibly inhibited FRET efficiency. Taken together these data show that alcohol disrupts TNF/TACE interactions by an activity which is reversible and related to alcohol-chain length. These data support a model by which ethanol, at clinically relevant concentrations, disrupts the cell membrane in a reversible fashion, resulting in inhibition of interactions between TNF-α and TACE.The mechanism of the reduction TNF/TACE interactions at present is unclear. The chain-length data suggest the possibility of changes in membrane fluidity  induced Alcohol chain length is clearly a factor in ethanol's ability to inhibit TNF secretion as well as TNF/TACE interactions as assessed by FRET. Moreover the effect of EtOH on this process is reversible. Taken together these data strongly support a model that alcohol disrupts TNF/TACE interaction in the cell membrane.The author(s) declare that they have no competing interests.KS performed the transient transfections and TNF ELISA, X-J. Z. assisted with the transfections, LM did the FRET analysis, P.O., SN, and JKK assisted in experimental design and drafting the manuscript.


Moreover, blocking PKC or MAPK effectively reduced Pb+LPS-induced TNF-α expression and liver injury. In summary, monocytes/macrophages were the cells primarily responsible for producing, through the PKC/MAPK pathway, the excess Pb-increased/LPS-induced TNF-α that caused liver injury.Although lead and lipopolysaccharide (LPS), both important environmental pollutants, activate cells through different receptors and participate in distinct upstream signaling pathways, Pb increases the amount of LPS-induced tumor necrosis factor-α (TNF-α). We examined the cells responsible for the excess production of Pb-increased LPS-induced TNF-α and liver injury, and the roles of protein kinase C (PKC) and p42/44 mitogen-activated protein kinase (MAPK) in the induction of TNF-α. Peritoneal injection of Pb alone (100 μmol/kg) or a low dose of LPS (5 mg/kg) did not affect serum TNF-α or liver functions in A/J mice. In contrast, coexposure to these noneffective doses of Pb plus LPS (Pb+LPS) strongly induced TNF-α expression and resulted in profound liver injury. Direct inhibition of TNF-α or functional inactivation of monocytes/macrophages significantly decreased the level of Pb+LPS-induced serum TNF-α and concurrently ameliorated liver injury. Pb+LPS coexposure stimulated the phosphorylation of p42/44 MAPK and the expression of TNF-α in CD14 Tumor necrosis factor-α (TNF-α) regulates a variety of biologic functions, including organ development, immune homeostasis, and malignance. The body subtly regulates the expression kinetics and dose of TNF-α to ensure its proper effect because TNF-α has opposite biologic effects in different circumstances . On the in vivo lipopolysaccharide (LPS)-induced model of liver damage of mice, used to determine TNF-α–mediated organ failure, is both sensitive and convenient. LPS, a component of the outer membrane of gram-negative bacteria, plays a major role in inducing septic shock and is a potent inducer of TNF-α in vivo and in vitro , TNF-α inhibitor pentoxifylline (PTX), and macrophage cytotoxic agent GdCl3–6H2O (gadolinium chloride hexahydrate) from Sigma Chemical . Lead acetate was purchased from Merck . PKC inhibitor chelerythrine chloride (C21H18NO4Cl) was obtained from Calbiochem , and p42/44 MAPK inhibitor U0126 was purchased from Promega .We obtained LPS  supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin (100 U/mL). Cells were then seeded at 2 × 105/well in a 96-well plate, incubated at 37°C for 4 hr, and washed with phosphate-buffered saline (PBS) to remove unattached cells. Attached cells, taken as macrophages and confirmed with F4/80 stain, were used for sequential experiments. RAW264.7 cells, a mouse macrophage cell line , were cultured in DMEM supplemented with 10% FBS. A/J mice weighing 20–25 g were obtained from and housed in the laboratory animal center of our institution. Animals were housed individually in a room with a 12/12-hr light/dark cycle and central air-conditioning , and were fed with standard food ad libitum. The animal experiment procedures were reviewed and implemented through the Institutional Animal Care and Use Committee process of National Cheng Kung University.Peritoneal macrophages were isolated by flushing the peritoneal cavity of A/J mice with 10 mL of sterile ice-cold phosphate buffer. Peritoneal lavage fluid was centrifuged at 200g, 10 min, 4°C). Serum samples were stored at 70°C. To determine the serum concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), serum was spotted to slides  and evaluated using a slide analyzer .The blood of mice was collected from the inferior vena cava under ethyl ether anesthesia, drawn using venipuncture into serum separation tubes, allowed to clot for 10 min at room temperature, and then centrifuged  , measuring absorbance at 450 nm and extrapolating from a standard curve with a sensitivity limit of 32.5 pg/mL.Induction of TNF-α in mouse whole blood was performed as described previously . BrieflyTo identify the TNF-α–secreting cells, Pb or LPS was added to whole blood with monensin . Red blood cells were lysed using hypotonic shock, and leukocytes were subjected to surface CD14 labeling using phycoerythrin-conjugated CD14 antibody (eBioscience). Cells were fixed and per-meabilized using a commercial kit  and stained for intracellular TNF-α using fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse TNF-α Ab (PharMingen). To analyze the phosphorylation status of p42/44 MAPK in peritoneal macrophages, exudate cells were fixed using 2% formaldehyde; they were then resuspended in methanol at a concentration of 90%. Cells were incubated with the primary phospho-p42/44 MAPK antibody  for 30 min at room temperature. After being washed in PBS containing 0.5% FBS, secondary antibody hybridization was carried out using goat anti-rabbit IgG . Macrophage-specific marker F4/80 was first stained with biotin-conjugated anti-F4/80 antibody and then with phycoerythrin-conjugated streptavidin secondary antibody (eBioscience). Flow cytometric analysis was then performed . Data were analyzed using the CellQuest  and WinMDI 2.8 software packages .2, 0.2 mM EDTA, 0.1% Triton X-100, 20 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, 0.5 mM dithiothreitol, 100 μg/mL phenylmethylsulfonyl fluoride, and 2 μg/mL leupeptin. Approximately 10–30 μg of protein was separated using electrophoresis in a 10% sodium dodecyl sulfate–polyacrylamide gel. After electrophoresis, the protein was electro-transferred onto polyvinylidene fluoride membranes . Membranes were probed with antibodies specific for phospho-p42/44 MAPK or p42/44 MAPK (New England Biolabs). After being probed with a horseradish peroxidase–conjugated secondary antibody, protein signals were visualized using enhanced chemiluminescence reagents  combined with Kodak X-ML film exposure .RAW264.7 cells were cultured in 0.01% FBS/DMEM for 30 min and sequentially stimulated with Pb (10 μM), LPS (1 ng/mL), Pb (10 μM) plus LPS (Pb+LPS) (1 ng/mL), or saline (control). After stimulation, cells were washed with cold PBS and then solubilized with ice-cold buffer containing 25 mM HEPES (pH 7.5), 300 mM NaCl, 1.5 mM MgClLiver tissue obtained from mice was fixed in 3.7% buffered formalin and embedded in paraffin. Sections (5 μm) were routinely stained with hematoxylin and eosin Y stain.t-test. Statistical significance was set at p < 0.05.Data are expressed as mean ± SE. We used one-way or two-way analysis of variance and Student’s We evaluated the role of TNF-α in Pb-increased LPS-induced liver injury. A/J mice were intraperitoneally given Pb (100 μmol/kg) , LPS 5 , Pb+LPS in vivo . We then checked blood cells using an in vitro whole-blood culture model. LPS (5 μg/mL) increased the expression of mean TNF-α in whole-blood culture to 75 pg/mL  in 1.5 hr. Although Pb (1 μM) itself did not induce detectable TNF-α, it significantly increased the TNF-α–inducing effect of LPS, which then reached approximately 125 pg/mL  were separated from single negative cells  24 hr before Pb+LPS treatment. Mouse peritoneal macrophages that received GdCl3 were drastically reduced, and few cells expressed a high level of F4/80 (F4/80high), representing peritoneal macrophages in peritoneal exudate (data not shown). GdCl3 decreased the level of serum TNF-α in mice that had received Pb+LPS . We further characterized the phosphorylation status of p42/44 MAPK in peritoneal macrophages, which showed high F4/80 expression . An incrin vivo, mice were treated with either C21H18NO4Cl (5 mg/kg) 30 min or U0126 (25 μmol/kg) 10 min before being exposed to Pb, LPS, or Pb+LPS. Both C21H18NO4Cl and U0126 effectively decreased the serum TNF-α induced by Pb+LPS  significantly increased the expression of TNF-α induced by low doses of LPS (0.1 and 1 ng/mL) in mouse F4/80rophages . In addirophages . This Pb21H18NO4Cl, a PKC inhibitor, also reduced Pb+LPS-associated TNF-α expression (Using MAPK and PKC inhibitors, we demonstrated that p42/44 MAPK and PKC were involved in the Pb+LPS-induced TNF-α expression of peritoneal macrophages and RAW264.7 cells. At doses between 7.5 and 30 μM, U0126, a p42/44 MAPK inhibitor, significantly suppressed the Pb+LPS-induced expression of TNF-α. ,E. At a pression .Monocytes/macrophages are the primary secretors of TNF-α during inflammation and infection . We demo+ production nor activated phosphorylation of MAPK in hepatocytes other than endothelial cells. Previous studies on the cellular source of TNF-α in animal exposed to Pb or LPS were not conclusive. Pb might increase the transcription of TNF-α mRNA in hepatocytes (in vivo have been identified as CD14+ (+ cells in the whole-blood culture and in mouse F4/80+ peritoneal macrophages, which indicates that the TNF-α was produced by cells outside the liver. The increase in TNF-α caused by Pb in the serum of LPS-treated mice was approximately 10-fold. However, the increase of TNF-α was only 2-fold in peritoneal macrophages. This indicates that other cells and mechanisms might contribute to the Pb-increased LPS-induced TNF-α production in vivo and need further investigation.Pb and low doses of LPS neither directly stimulated TNF-αatocytes , and LPSatocytes . Howeveratocytes . In our as CD14+ , but Kupas CD14+ . We alsoA second line of evidence excluding hepatocytes as the source of TNF-α comes from studies of cell signaling. PKC and p42/44 MAPK are downstream signals of Pb stimulation in neurons , bone-maAs mentioned above, both Pb and a high dose of LPS activated PKC and MAPK, which stimulated TNF-α expression . In our In conclusion, the PKC/MAPK pathway leading to TNF-α expression played a key role in Pb+LPS-induced liver injury in mice. Our results also indicate that immune cells are very sensitive to environmental pollution and emphasize the synergistic effect of multiple pollutants in disease progression. Specifically, monocytes/macrophages may serve as watchful janitors in response to Pb+LPS, even when it injures their host.


The binding and biological activity of human TNF alpha on endothelial and tumour cells has been studied in the presence of monoclonal antibodies (MAbs). In particular, one monoclonal antibody to TNF alpha (MAb 32) has been identified which failed to inhibit binding and cytotoxicity of TNF alpha on WEHI-164 tumour cells but which was a potent inhibitor of TNF alpha-induced endothelial cell procoagulant activity on bovine aortic endothelial cells. The ability of MAb 32 to inhibit selectively the actions of TNF alpha on endothelial cells but not on tumour cells suggests a mechanism for enhancement of the anti-tumour action of TNF alpha in vivo when in complex with this antibody. Treatment of tumour bearing mice (WEHI-164 and Meth A fibrosarcoma) with TNF alpha-MAb 32 complex resulted in a 5- to 10-fold enhancement in the potency of the cytokine in comparison to free TNF alpha. Complexes between this cytokine and other MAbs generally resulted in either no effect or inhibition of TNF alpha activity in vivo and in vitro. Neither intact MAb 32 nor FAb' fragments of MAb 32 showed any tumour regressive activity in the absence of TNF alpha. The FAb' fragments were equipotent to the bivalent form of the antibody in enhancing TNF alpha activity. These data provide evidence that it is possible to segregate the individual biological activities of TNF alpha with concomitant enhancement of the tumour regressive activity of the cytokine in vivo.


We studied human TNF transgenic mice – an in vivo model of TNF-induced arthritis – to examine phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK) and p38MAPKα in the inflamed joints by means of immunoblot and immunohistochemistry. In addition, the effects of systemic blockade of TNF, IL-1 and receptor activator of nuclear factor-κB (RANK) ligand on the activation of MAPKs were assessed. In vivo, overexpression of TNF induced activation of p38MAPKα and ERK in the synovial membrane, whereas activation of JNK was less pronounced and rarely observed on immunohistochemical analysis. Activated p38MAPKα was predominantly found in synovial macrophages, whereas ERK activation was present in both synovial macrophages and fibroblasts. T and B lymphocytes did not exhibit major activation of any of the three MAPKs. Systemic blockade of TNF reduced activation of p38MAPKα and ERK, whereas inhibition of IL-1 only affected p38MAPKα and blockade of RANK ligand did not result in any decrease in MAPK activation in the synovial membrane. These data indicate that TNF preferentially activates p38MAPKα and ERK in synovial membrane exposed to TNF. This not only suggests that targeted inhibition of p38MAPKα and ERK is a feasible strategy for blocking TNF-mediated effects on joints, but it also shows that even currently available methods to block TNF effectively reduce activation of these two MAPKs.Tumour necrosis factor (TNF) is considered to be a major factor in chronic synovial inflammation and is an inducer of mitogen-activated protein kinase (MAPK) signalling. In the present study we investigated the ability of TNF to activate MAPKs in the synovial membrane  Chronic inflammation of the synovial membrane (synovitis) is a hallmark of rheumatoid arthritis (RA). This process is fueled by proinflammatory cytokines, which not only induce but also maintain synovitis and therefore play an important role in progressive joint destruction ,2. SeverThe effects of TNF-α are mediated via a complex network of signalling pathways. Apart from activation of nuclear factor-κB, many signals are transduced through mitogen-activated protein kinases (MAPKs), which include extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinases (JNK) and p38MAPKα . These min vivo. Despite the potential of TNF to activate all three MAPKs, the pathways that are of relevance to chronic destructive arthritis remain to be elucidated. However, if we are to design therapeutic tools that can effectively block TNF-mediated inflammatory responses, then we must define the major signalling targets of TNF in inflammatory joint disease in vivo. Interestingly, all three MAPK families – p38MAPKα, ERK and JNK – are activated in RA synovial membrane, and TNF-α has the potential to signal through all of them [It is currently unclear whether TNF equally activates all three MAPK families in arthritis or has a certain predilection toward activating one of the families in the synovial tissue  of them ,18. Therin vivo overexpression of TNF on MAPK signalling in synovial tissue. Mice transgenic for human TNF (hTNFtg mice), which develop a chronic inflammatory joint disease, were assessed for immunohistochemical evidence of activation of the three MAPK families . Moreover, we defined the cell types in the synovial membrane that exhibit MAPK activation and investigated the effects of anticytokine therapies on MAPK signalling.In the present study we investigated the effect of Heterozygous Tg197 human TNF-α transgenic (hTNFtg) mice (strain C57/Bl6), which develop a chronic inflammatory and destructive polyarthritis within 4–6 weeks after birth, were described previously ; and groLeft and right hind paws were fixed in 4.5% buffered formalin overnight and then decalcified in 14% EDTA  at 4°C until the bones were pliable. Serial paraffin sections (2 μm) of the right hind paw were used for the immunohistochemical analyses.For immunohistochemical detection of the phosphorylated forms of ERK, p38MAPKα and JNK, ethanol dehydrated tissue sections were treated with 3% hydrogen peroxide in methanol followed by digestion with proteinase K (25 mg proteinase K in 50 ml PBS) for 5 min at 37°C and blocking with PBS buffer containing 20% rabbit serum for 1 hour. Then, sections were incubated with monoclonal antibodies to the phosphorylated isoforms of ERK-1 and ERK-2 , p38MAPKα  and JNK  at room temperature for 1 hour and for 30 min with biotinylated goat anti-mouse immunoglobulin (Santa Cruz biotechnology). Antibody binding was detected using an ABC complex  and 3,3-diaminobenzidine  as chromogen, resulting in brown staining of antigen-expressing cells.Characterization of cells expressing ERK, p38MAPKα and JNK was performed by double staining using cell-type specific antibodies. After applying the protocol as described above, the slides were incubated with rat anti-mouse monoclonal antibodies against macrophages , T cells  and fibroblasts . Thereafter, the sections were incubated by an alkaline phosphatase conjugated rabbit anti-rat immunoglobulin (Dako) and the reaction was visualized using a rat alkaline phosphatase–antialkaline phosphatase complex (Dako), nitroblue tetrazolium (0.25 μg/ml) and 5-bromo-4-chloro-3-indolyl phosphate (0.125 μg/ml). In case of staining for B cells  the cell-specific antibody was applied first and detected by ABC/DAB with subsequent staining of ERK, p38MAPKα and JNK, and detection with a mouse-specific alkaline phosphatase–antialkaline phosphatase complex system (Dako). Expression of ERK, p38MAPKα and JNK was quantitatively assessed by counting both total numbers of synovial cells and the numbers of positively stained cells in each immunohistochemical staining using a magnification of 200× or by counting positively stained cells per high power field (magnification 400×).2, 1 mmol/l DTT, 1 mmol/l EDTA, 0.1 mmol/l EGTA and 20% glycerol, as well as protease and phosphatase inhibitors  using an Ultra-Turrax T50 homogenizer . Tissue extracts were then separated from debris and fat by centrifuging at 13,000 rpm for 15 min. Protein content was measured by Bradford assay and 200 μg tissue protein was subjected to electrophoresis on a 10% SDS polyacrylamide gel followed by transfer onto nitrocellulose membranes. After blocking, the membranes were incubated by antibodies against the phosphorylated as well as total p38MAPKα, ERK and JNK .Hind paws from three wild-type and three hTNFtg mice aged 10 weeks were snap frozen in liqid nitrogen and mechanically homogenized at 4°C in buffer containing 20 mmol/l HEPES, 0.4 mol/l NaCl, 1.5 mmol/l MgClData are expressed as mean ± standard error of the mean. Expression of ERK, p38MAPKα and JNK in the different therapy groups and cell types was compared by means of Kruskal–Wallis test and Dunn's multiple comparison test.in vivo, we histologically assessed joints of hTNFtg mice and wild-type mice for phosphorylated forms of p38MAPKα, ERK and JNK. Synovial inflammatory tissue of hTNFtg mice exhibited widespread activation of p38MAPKα and ERK. Expression of phosphorylated forms of both p38MAPKα and ERK was abundant at sites of destructive synovial pannus but also in the synovial lining layer  lower proportion of T lymphocytes (7 ± 3%), B lymphocytes (9 ± 4%) and fibroblasts (11 ± 1%) exhibited activation of p38MAPKα  less frequent among T lymphocytes (13 ± 6%) and B lymphocytes  inhibited by TNF and IL-1 blockade, reducing the fraction of synovial cells exhibiting p38MAPKα activation by 53% and 55%, respectively  declare that they have no competing interests.BG carried out histological analyses and drafted the manuscript. SH participated in design and coordination of the study. BT carried out histological and statistical analyses. JZ participated in breeding of mice. JSS participated in the design of the study. GS conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.


One of the United States' national health objectives for 2010 is that 95% of children aged <6 years participate in fully operational population-based immunization information systems (IIS). Despite important progress, child participation in most IIS has increased slowly, in part due to limited economic knowledge about IIS operations. Should IIS need further improvement, characterizing costs and identifying factors that affect IIS efficiency become crucial.Data were collected from a national sampling frame of the 56 states/cities that received federal immunization grants under U.S. Public Health Service Act 317b and completed the federal 1999 Immunization Registry Annual Report. The sampling frame was stratified by IIS functional status, children's enrollment in the IIS, and whether the IIS had been developed as an independent system or was integrated into a larger system. These sites self-reported IIS developmental and operational program costs for calendar years 1998–2002 using a standardized data collection tool and underwent on-site interviews to verify reported data with information from the state/city financial management system and other financial records. A parametric cost-per-patient-record (CPR) model was estimated. The model assessed the impact of labor and non-labor resources used in development and operations tasks, as well as the impact of information technology, local providers' participation and compliance with federal IIS performance standards . Given the number of records minimizing CPR, the additional amount of resources needed to meet national health goals for the year 2010 was also calculated.Estimated CPR was as high as $10.30 and as low as $0.09 in operating IIS. About 20% of IIS had between 2.9 to 3.2 million records and showed CPR estimates of $0.09. Overall, CPR was highly sensitive to local providers' participation. To achieve the 2010 goals, additional aggregated costs were estimated to be $75.6 million nationwide.Efficiently increasing the number of records in IIS would require additional resources and careful consideration of various strategies to minimize CPR, such as boosting providers' participation. One United States national health objective for 2010 is to have at least 95% of U.S. children aged <6 years participate in fully operational population-based immunization registries or immunization information systems (IIS) [As of 2002, approximately 43% of children of U.S. children <6 years of age had two or more vaccinations recorded in an IIS [Using a nationally representative sample of IIS, this article evaluates economic determinants of IIS performance by estimating the influence of development and management costs of in-house and contracted labor and non-labor and resources used by IIS centralized systems. It also measures the effect of standards and other factors affecting the functioning of such systems, determines the threshold of patient records needed to minimize average cost per patient record (CPR), and identifies strategies to increase efficiency.Data were collected from a national sampling frame of the 56 states/cities that received federal immunization grants under Public Health Service Act §317b and completed the CDC/NIP 1999 Immunization Registry Annual Report – a self-administered questionnaire applied to immunization program managers. This questionnaire assessed the percentage of children <6 years of age with >2 immunizations enrolled in an IIS, the percentage of provider sites participating in the IIS, whether the IIS operated independently or integrated with other health information systems, and evaluated the functional status of the IIS according to 12 federal IIS performance standards  defined in more detail elsewhere [Table IIS were classified as 'low', 'medium', or 'high' in terms of children enrolled if the percentage of children enrolled was < 1.7%, >1.7% and < 53.5%, or > 53.5%. IIS were classified as 'low', 'medium', or 'high' in terms of IIS functionality score if the percentage of IIS functional standards achieved was < 37.1%, >37.1% and < 69.9%, or > 69.9%. These cut points were chosen to represent natural breaks in the data. Two IIS were randomly sampled from each stratum or all IIS sampled if ≤ 2 in a stratum with the exception of high functional score and low child enrollment where 3 were sampled because 2 states shared the same IIS. 17 IIS were under development and fell into a stratum of <37.1% functional score and <1.7% child enrollment; 4 IIS were randomly sampled from this group for participation in this study. The final sample consisted of 24 IIS. These IIS varied by duration of operation  and targeted age range (most IIS collected vaccination data on persons aged 0–18 years old).In August 1999, the IIS sites in the sample collected developmental and operational program costs for calendar years 1998 and 1999. From May to October 2003, researchers visited the selected sites with on-site interviews to collect IIS expenditures for calendar years 2000–2002 and verify data collected previously. Since the 1999 assessment, many IIS had upgraded to web-based systems, expanded their partnerships and marketing efforts to the private sector, and found additional funding sources. These data were combined with data from CY 1998–1999 to allow analysis over a five year period. All sites reported data from their financial management offices and internal records.Annual expenditures were collected as line items for in-house personnel (including fringe benefits), contractor full-time equivalents (FTEs), equipment, supplies, travel, contractual expenses, other expenses, and indirect costs. Object class definitions developed by the Office of Management and Budget (OMB) for Federal program costs were used to categorize expenditures .Data on IIS expenditures by source of funds were also collected. These included federal immunization grants under Public Health Service Act §317b; 'other' federal sources such as other CDC funds, Maternal and Child Health block grants, Vaccines for Children program funds, or funds from the Centers for Medicare and Medicaid Services; state and local government funds; private sector funds; in-kind contributions; and funds from other sources, such as fees from IIS subscribers. Personnel and contractual cost line items included costs to develop and operate the IIS, to enter data at the central IIS, to evaluate the IIS, and for marketing efforts to recruit parents and health providers to participate in the IIS. Only costs to operate the central IIS within a geographic area were included . IIS costs to providers were excluded. For IIS that were a part of a larger, integrated information system , only the central IIS system's portion of these costs were collected, not the costs to develop and operate the entire integrated system.All IIS line item costs were separated into developmental and operational costs. Developmental costs were defined as costs incurred prior to any immunization data submission, storage, or access by an immunization provider. Software and hardware upgrade costs were also considered developmental costs, especially in regard to upgrading to a web-based system. Operational costs were defined as costs to maintain the current status of the IIS after immunization providers began to submit data or access IIS records.Annual mean IIS costs were estimated for labor, non-labor, development and operations by funding source and by number of patient records  in the IIS. Labor costs included personnel salaries and fringe benefits of persons who were city/state government employees as well as contractor full-time equivalents (FTEs) who worked as IIS employees. All other costs, including indirect costs, were included in non-labor costs. Cost data from 1998–2001 were adjusted to 2002 dollars using the Consumer Price Index . Descripi's IIS in period t was represented by:To examine factors that influenced average CPR in IIS and estimate prospective costs of achieving the Healthy People 2010 goals, a behavioral cost equation was estimated using weighted least squares with site-specific fixed effects. By allowing for site-specific heterogeneity, one could capture the effect on CPR of any unobserved site-specific characteristics. This greatly improved the precision of estimates. The equation for the CPR of site ACit is average cost for site i in period t, αi is the site-specific fixed effect for site i, wit is the wage of labor, rkit is expenditures on capital, Qit is total patient records in IIS, ξit is achievement of IIS functional standards, and the Xit are site characteristics. The regression error for site i in period t is eit. Each of the 24 sampled IIS were observed once per year for a period of five years. Therefore, the sample consisted of 120 data entries. In the final sample, each entry referred to the observation of a specific IIS in a given year, henceforth an IIS-year.where n → N.Average CPR was transformed by the natural log as were wages and expenditures to better approximate their relationship to average cost, which was log-normally distributed. In order to allow for the presumed U-shape of the average cost curve, output was specified in quadratic form. This fixed effects model was estimated using weighted least squares to account for stratified sampling with robust estimation of the covariance matrix. Standard errors were corrected using the finite population correction factor, since sample size approached population size, The cost of achieving the Healthy People 2010 goal for each sampled IIS was calculated using parameters retrieved from the average cost estimation, discounting at a rate of 3%. For simplicity, it was assumed that each IIS met all 12 functional standards  while other variables were evaluated at their 2002 values. It was assumed, again for simplicity, that the gap between current child participation and 95% child participation was closed at a linear rate of one-eighth the gap per year, while the IIS continued to maintain the average child population growth rate that it had exhibited in the previous five years. IIS that had already achieved or exceeded 95% child participation were assumed to continue to maintain current participation levels by growing with the child population.In order to illustrate the importance of vaccine provider participation in an IIS, the impact of simultaneously achieving 95% child participation and 95% provider site participation was also examined. The same assumptions used previously were employed. In addition to child participation, the gap between current provider site participation and 95% provider site participation would close at a linear rate of one-eighth the gap per year. Sampled IIS totals were weighted and the national cost for all 56 IIS to achieve the Healthy People 2010 goal was calculated with and without 95% provider site participation.Table Figure Table Funding sources affected average costs although receipt of CDC immunization grant funds did not significantly affect average costs. Receipt of other federal, local, and in-kind funds tended to be associated with increased average costs. Specifically, receipt of local government funds and in-kind contributions was associated with an increased CPR by $1.05 and $0.78 respectively while receipt of other Federal funds was associated with an increased CPR by $0.39. Conversely, receipt of state or private funds was associated with a reduced CPR by $0.41 and $0.24 respectively. Funds from other sources did not significantly affect average costs.Achievement of the 12 IIS functional standards also impacted the average CPR, though certain standards impacted more than others. Specifically, ensuring data accuracy and completeness increased average cost by $0.51 per record while IIS ability to receive and process vaccination data within one month of vaccine administration increased CPR by $0.41. These were followed in impact by the ability for a clinic to access IIS data during patient visits (-$0.31) and the ability to create an IIS record within six weeks of birth ($0.22). Average costs for IIS that met all 12 functional standards increased by $0.21 per record compared to IIS that did not meet all standards. IIS ability to identify persons needing vaccine reminder/recall notifications decreased the CPR by $0.16, and the ability to automatically produce immunization coverage reports and official immunization records decreased average costs by $0.11 and $0.17 respectively.Population characteristics and demographics are usually important determinants of average cost. While population density did not significantly influence CPR, as the proportion of IIS patient records belonging to children increased, average costs declined. A 1% increase in the proportion of child records was associated with 0.71 percent decline in average cost. This effect was both strong and highly significant.A 1% increase in the wages of development personnel and contractors was associated with declines in average cost of 0.05% and 0.02% respectively. Similarly, the elasticity of operations personnel wages with respect to average cost was – 0.12. Operations contractor wages did not significantly affect average cost. Expenditures on operating supplies, other purchases, and indirect costs  did not significantly affect average costs; nor did expenditures on travel or indirect overhead for development. For the remaining factors, the effect was positive and significant but uniformly inelastic.The results for patient records displayed in Table Figure Increased provider participation was associated with increased average costs up to a point, after which costs declined. Figure Results from these calculations indicated a cost of $75.6 million ($76.9 million with no discounting) in 2002 dollars over eight years for all 56 IIS to meet the Healthy People 2010 goal, assuming that all other parameters remain the same. The cost to achieve the Healthy People 2010 goal as well as 95% provider participation amounted to $56.4 million ($57.7 million with no discounting) in 2002 dollars for all 56 IIS. Note that this figure only includes the cost to the central IIS and does not account for costs to providers.This study was designed to characterize the average costs of IIS and to estimate costs for IIS nationwide to be fully functional, population-based, and contain >95% of the children <6 years of age in their geographic areas. States developing IIS can estimate the costs in their plans to develop and operate an IIS based on their population size and anticipated IIS size. Based on results from the average cost model, it costs about $0.20 per patient record to achieve full functional status measured by the 12 IIS functional standards.In general, operations expenses tended to increase with IIS size while development expenses declined. This appeared to hold true within categories as well. An increase in expenditures was associated with an increase in IIS size, but IIS at or near efficient size had more diverse sources of funds. Funding sources might affect the operation of IIS but the effect on average costs is not highly significant.Past studies collected costs through 1997–1998, when many IIS had not yet migrated to web-based systems ,6-10. ThWhile the literature lacks examples of IIS costs at the national level, it also lacks studies that characterize the variety of funding sources used in IIS development and operations and the impact of economies of scale on CPR. Having the most recent data available, including funding sources, may account for the technological changes and new demands placed on the modern IIS. Timely cost data and investigating economies of scale are essential for planning national IIS goals through 2010.Provider participation is also essential for IIS to achieve their Healthy People 2010 goal. Up front investments by the IIS to integrate with electronic health records, patient management systems, and billing systems from the provider office could encourage provider participation by reducing data entry costs to the provider. Some of the IIS systems sampled devoted resources to provide computers and data entry services to providers and to integrating their system with other public health systems. These costs were included in the central IIS development and operations costs. While costly at first, the marginal cost of provider participation tended to decline as provider participation increased. As IIS size increased, CPR declined.This study is subject to several limitations. First, time has not been modeled explicitly in this analysis. The results assume a one time, lump-sum cost of achieving the functional standards and that the gap between 2002 child participation and 95% child participation will be closed linearly. In reality, it is likely that IIS will achieve functional standards over time and child participation may increase nonlinearly over time. The extent that actual IIS achievements of functional standards differ from our assumptions may affect the cost of those achievements. Although it is not clear whether the effect would be positive or negative, in general, technological improvements tend to lead to cost savings. Therefore, to the extent that achievement of functionality represents technology improvement, fully functional IIS should operate more efficiently than their less functional counterparts. Second, the data were self-reported using data from IIS financial systems and internal records. The quality of available data and the completeness of the state financial records varied between the sites. While estimates are comprehensive in terms of types of costs considered and in-person interviews were conducted, it is possible that some expenditures may not have been captured.Third, this is a study of costs incurred by the central IIS of federally funded IIS grantees over a five-year period. Resources did not permit the formal collection of costs incurred by provider practices or the costs incurred for daily operations of local IIS that export data to the central IIS. While all of the state IIS in the sample included costs of contracts with local health districts to obtain local vaccination data, these contracts did not fully support local IIS operations. Regarding costs to providers, costs for data entry were collected if the central IIS paid for these services. Two recent studies of IIS costs included the costs to providers to enter data and use the IIS for immunization assessment, immunization history reports, and other reports, but only included a small number of private practices in a small geographic area ,12; we cWhile further analysis is necessary to better understand development and operations of IIS, some implications for policy are clear. Increases in the scale of operations are necessary in order to take advantage of substantial economies of scale. While minimum efficient scale may not be a feasible achievement for IIS with smaller populations, any increase in scale up to 2,900,000 records should result in declining average cost and greater efficiency. IIS with smaller populations may consider partnering with other regions and states to improve their economies of scale. IIS policy makers should also consider improving functionality and the adoption of new information technology; this may lead to efficiency improvements that can reduce total costs. IIS are also encouraged to seek out diverse resources for long-term sustainability. Funding sources vary in accountability requirements and IIS with more diverse funding sources may face greater accountability resulting in more efficient practices. Finally, IIS should enhance electronic linkages with providers to encourage provider participation. Provider costs to use an IIS and/or submit data to an IIS can deter participation and it would be well worth the investment of IIS funds to assess the costs to participating providers and implement strategies to decrease these costs. Obtaining data electronically from electronic health records, managed care organizations, and other large vaccination data sources, could decrease CPR as well as the costs to providers to submit data. Also, for states facing hiring freezes or difficulties in recruiting technical staff, hiring contractors may be a cost-effective option; developmental contractor salaries were associated with a slight decrease in average costs and operational contractor wages did not significantly affect average costs.Although complementary resources and efforts will be needed from IIS nationwide, achievement of the 2010 national IIS objective is possible. Efficiently increasing the number of children in IIS, however, would not only require the identification of additional and sustainable resources but, for the most part, a careful consideration of various strategies that minimize IIS' average costs. Among such strategies, substantial increments in providers' participation could significantly decrease CPR.CDC-Centers for Disease Control and PreventionCPR-Cost per patient recordFTE-Full time equivalentsIIS-Immunization information systemsNIP-National Immunization Program OMB-Office of Management and BudgetUS-United StatesThe author(s) declare that they have no competing interests.DLB was responsible for instrument development, data collection, and manuscript preparation. N-AMM analyzed the data, interpreted the results and participated in manuscript preparation. IRO-S participated in data analysis and interpretation of results and manuscript preparation. GAU participated in the overall study design, instrument development, and data collection. All authors read and approved the final manuscript.