Citation data can be used to evaluate the editorial policies and procedures of scientific journals. Here we investigate citation counts for the three different publication tracks of the Proceedings of the National Academy of Sciences of the United States of America (PNAS). This analysis explores the consequences of differences in editor and referee selection, while controlling for the prestige of the journal in which the papers appear.We find that papers authored and “Contributed” by NAS members (Track III) are on average cited less often than papers that are “Communicated” for others by NAS members (Track I) or submitted directly via the standard peer review process (Track II). However, we also find that the variance in the citation count of Contributed papers, and to a lesser extent Communicated papers, is larger than for direct submissions. Therefore when examining the 10% most-cited papers from each track, Contributed papers receive the most citations, followed by Communicated papers, while Direct submissions receive the least citations.Our findings suggest that PNAS “Contributed” papers, in which NAS–member authors select their own reviewers, balance an overall lower impact with an increased probability of publishing exceptional papers. This analysis demonstrates that different editorial procedures are associated with different levels of impact, even within the same prominent journal, and raises interesting questions about the most appropriate metrics for judging an editorial policy's success. Citation data play an important role in the evaluation of scientific research. Citation counts can be used to characterize individual studies and researchers, scientific disciplines, journals, institutions, and entire nations The majority of papers published in PNAS are submitted directly to the journal and follow the standard peer review process. The editorial board appoints an editor for each Direct submission, who then solicits reviewers. During the review process the authors are blinded to the identities of both the editor and the referees. PNAS refers to this publication method as “Track II”. In addition to the direct submission track, members of the National Academy of Sciences (NAS) are allowed to “Communicate” up to two papers per year for other authors. Here, authors send their paper to the NAS member, who then procures reviews from at least two other researchers and submits the paper and reviews to the PNAS editorial board for approval. As with Direct submissions, authors of Communicated papers are at least in theory blinded to the identity of their reviewers, but not to the identity of the editor. PNAS refers to this publication method as “Track I”. Lastly, NAS members are allowed to “Contribute” as many of their own papers per year as they wish. Here, NAS members choose their own referees, collect at least two reviews, and submit their paper along with the reviews to the PNAS editorial board. Peer review is no longer blind, as the authoring NAS member selects his or her own reviewers. PNAS refers to this publication method as “Track III”. For more information on the different PNAS publication tracks, see To empirically investigate the impact of papers published via each track, we inspect 2695 papers published between June 1, 2004 and April 26, 2005, covering PNAS Volume 101 Issue 22 through Volume 102 Issue 17. For each paper, we examine Thomson Reuters Web of Science citation data as of October 2006 and May 2009, as well as page-view counts as of October 2006. We also note the track through which each paper was published, the topic classification of each paper, the date of publication, and whether each article was published as open access and/or as part of a special feature. For all subsequent analysis, we log10-transform citation counts, with the addition of a constant value 1 to all citation entries so as not to exclude un-cited papers. Because citation rates come from a long-tailed distribution with the most cited papers attracting many more citations than the average paper, this transformation brings the citation count distributions much closer to normal , and marginally greater variation in Communicated papers relative to Direct submissions . To explore the consequences of this larger variance, we now compare the citation counts of the 10% least and most cited papers from each Track.Among the 10% least cited papers in each track, we see similar results to the analysis comparing citation counts among all papers . There iAmong the 10% most cited papers in each track, however, we find the opposite pattern . The topWe now demonstrate that the relationships shown in Our additional analyses reproduce the effects demonstrated by our comparison of means above. For full regression tables, see Similarly, considering only the 10% least cited papers from each track, there is no significant difference between Communicated papers and Direct submissions , while Contributed papers are cited significantly less than Direct submissions . These regression coefficients indicate that when only considering the least cited papers, Contributed papers receive approximately 20% fewer citations than Direct submissions in the 2006 citation count, and approximately 30% fewer citations in the 2009 citation count.Considering only the 10% most cited papers from each track, however, Communicated papers are cited significantly more than Direct submissions in 2009 but not 2006 , and Contributed papers are cited significantly more than Direct submissions in both 2006 and 2009 . These regression coefficients indicate that Contributed papers receive approximately 10% more citations than Direct submissions when considering only the most cited papers.In addition to citation counts, the number of page-views received by each article provide another measure of impact. However, examining page-view counts as of October 2006 shows no significant variation across Tracks when controlling for open access publication, inclusion in a special feature, days since publication and topic classification. More current page-view statistics may shed greater light on differences between Tracks if differences in page-views are amplified over time. This data, however, was not available to us. Among the 10% most viewed papers in each track, page-views follow the same pattern as citation counts, with Communicated and Contributed papers receiving significantly more page-views than Direct submissions. See more citations than the 10% most cited Direct submissions. Thus the Contributed track seems to yield less influential papers on average, but is more likely produce truly exceptional papers. In addition, we find no significant difference in overall citation count between papers submitted directly to PNAS and papers Communicated for others by NAS members, while the 10% most cited Communicated papers also receive significantly more citations than the 10% most cited Direct submissions.The analysis presented here clearly demonstrates variation in impact among papers published using different review processes at PNAS. We find that overall, papers authored by NAS member and Contributed to PNAS are cited significantly less than papers which are Direct submissions. Strikingly, however, we find that the 10% most cited Contributed papers receive significantly What might be responsible for these differences in impact between the different publication tracks at PNAS? It is possible for NAS members to Communicate or Contribute papers in fields outside their area of expertise, and to soften the challenges of the peer review process through their choice of referees. The anonymity of referees and reviewers is also decreased for Communicated and Contributed papers relative to Direct submissions. These factors could result in lower quality papers being submitted and published through these alternative publication avenues. Our analysis suggests that this may in fact happen for Contributed papers, but does not support the suggestion that papers Communicated by NAS members are on average of lower quality than Direct submissions. While it is still possible that more low-quality papers are submitted through the Communication track, our results suggest that the PNAS Editorial Board successfully screens out such papers.In contrast to these potential dangers associated with Communicated and Contributed papers, it is also possible that these alternative publishing procedures may facilitate the publication of time-sensitive and groundbreaking work which is of high quality but might suffer under the standard review process. Our analysis supports this hypothesis, by showing that the most cited Contributed and Communicated papers are more influential than the top Direct submissions. The benefit of facilitating publication of extremely high-impact Contributed papers could be argued to out-weigh the potential cost of allowing more low quality papers to also be published.Together, these two observations about Contributed papers raise interesting questions about the optimal publication procedures, as well as the appropriate metric for assessing optimality. Editors at some journals may seek to maximize the overall impact of the papers they publish, while others may be willing to accept lower median impact in exchange for increased impact among the most influential papers. Our results suggest that making publication easier for researchers with established track records may have the latter effect. Which strategy is most appropriate or effective remains an open question, and one which may not have a normative answer. Further empirical and theoretical work exploring these questions is needed.One additional factor which may influence citation counts is whether a given paper was highlighted in a PNAS press release. We were unable to explore this issue because of lack of data regarding which papers received press releases. The effect of press releases, and popular press coverage more generally, on citation counts is an open question which deserves further study The empirical analysis presented here clearly demonstrates systematic variation in citation counts across publication tracks at PNAS. Differences in the submission and review process between Direct submissions, Communicated papers and Contributed papers seem to have significant effects on the influence of papers published in PNAS. As well as being of interest to decision makers at, and readers of, PNAS, these results have potentially important implications for those making decisions about submission and review procedures at other journals, and raise interesting questions about how editors should evaluate a journal's success. Quantitative comparisons of impact across similar journals with different publication processes represent an important opportunity for exploring publication dynamics which lie at the heart of the modern scientific enterprise.Text S1Supplementary file containing additional analysis and figures.(0.47 MB DOC)Click here for additional data file.


The matrix metalloprotease MMP-9 localizes to tumour-associated macrophages in human ovarian cancer but little is known of its regulation. Co-culture of human ovarian cancer cells (PEO-1) and a monocytic cell line (THP-1) led to production of 92-kDa proMMP-9. PEO-1-conditioned medium (CM) also stimulated THP-1 cells or isolated peripheral blood monocytes to produce proMMP-9. Expression of TIMP-1, however, remained unaffected. There was evidence that tumour necrosis factor alpha  was involved in tumour-stimulated monocytic proMMP-9 production. Antibody to TNF-alpha inhibited proMMP-9 production, and synthesis of TNF-alpha mRNA and protein preceded proMMP-9 release. In addition, the synthetic matrix metalloprotease inhibitor (MMPI) BB-2116, which blocks TNF-alpha shedding, inhibited proMMP-9 release in the co-cultures and from CM-stimulated monocytic cells. Further experiments suggested that the stimulating factor present in CM was not TNF-alpha, but acted synergistically with autocrine monocyte-derived TNF-alpha to release monocytic proMMP-9. Thus, ovarian cancer cells can stimulate monocytic cells in vitro to make proMMP-9 without affecting the expression of its inhibitor TIMP-1. This induction is mediated via a soluble factor  that requires synergistic action of autocrine or paracrine TNF-alpha.


TNF-α gene  and the presence of XFG in a Caucasian population.TNF-α has been suggested to participate in the pathogenesis of exfoliation glaucoma (XFG). The purpose of the present study was to investigate a hypothesized association between two common functional polymorphisms in the promoter region of the TNF-α –308 G>A and TNF-α –238 G>A polymorphisms were determined by polymerase chain reaction (restriction fragment length polymorphism).The present case-control study comprised 408 participants (204 patients with XFG and 204 control subjects). Control subjects were matched for age and sex. Genotypes of the TNF-α –308 G-allele was associated with an insignificant odds ratio of 0.98  while the presence of the TNF-α –238 G-allele was associated with an insignificant odds ratio of 0.64 .No significant differences regarding genotype distribution or allelic frequencies were found between patients and control subjects (p>0.025). The presence of the TNF-α –308 G>A and the TNF-α –238 G>A polymorphisms are unlikely to be major risk factors for XFG in an European population of Caucasian descent.Our data suggest that both the  Uncontr191160) is a ubiquitous cytokine involved in various physiologic as well as pathologic processes like inflammation, immunoregulation, proliferation, and apoptosis [Tumor necrosis factor-alpha  while the second is characterized by a G to A substitution at position −238 . In vitro studies of these polymorphisms have been shown to increase TNF-α production after lipopolysaccharide stimulation [Two common functional polymorphisms in the promoter region of mulation . Other smulation -29.TNF-α −308 GG genotype among 110 Turkish patients with XFG. This finding, however, has not yet been confirmed in other populations, which is mandatory to draw firm conclusions on the role of TNF-α polymorphisms. Our study was set to investigate a hypothesized association between the TNF-α –308 G>A and TNF-α –238 G>A polymorphisms and the presence of XFG.Only recently, Tekeli and coworkers  found anTwo hundred and four unrelated patients with XFG and 204 control subjects were enrolled in the present retrospective case-control study. All study participants were Caucasians from the same geographical area in the southern part of Austria. Participants were seen at the Department of Ophthalmology, Medical University Graz  between May 2003 and September 2008 and gave informed written consent before enrollment. The study was conducted in accordance with the National Gene Technology Act of Austria and the guidelines of the local ethics committee.Patients with XFG underwent slit lamp biomicroscopy in mydriasis, testing for best corrected visual acuity, Goldmann applanation tonometry, gonioscopy, pachymetry, and standard automated perimetry  or in cases of profoundly decreased visual acuity, Goldmann perimetry. Control subjects underwent slit lamp biomicroscopy in mydriasis, Goldmann applanation tonometry, and testing for best corrected visual acuity. Optic discs in all participants were assessed by glaucoma specialists .XFG was defined by the presence of typical exfoliation material on the anterior lens capsule, an intraocular pressure before initiation of a pressure lowering therapy of at least 22 mmHg, an open anterior chamber angle, optic disc changes characteristic for glaucoma , visual field defects characteristic for glaucoma , and absence of conditions leading to secondary glaucoma.Control subjects showed biomicroscopically no evidence of exfoliation material on the anterior capsule of the lens and no morphological damage indicative for open-angle or angle closure glaucoma. Control subjects were admitted to our department for cataract surgery.2, 1 µl DNA sample, and 2 U Taq polymerase . Annealing temperature was 62 °C. The PCR products were digested at 37 °C with NcoI to detect the SNP in the −308 gene allele and MspI to detect the polymorphism of the −238 nucleotide. The PCR product was then subjected to 3% agarose-gel electrophoresis. “No target” controls were included in each PCR batch to ensure that reagents had not been contaminated.Venous blood was collected in 5 ml EDTA tubes. DNA was isolated using QIAamp DNA blood mini-kit  and stored at −20 °C. All polymerase chain reactions (PCRs) were run under conditions previously described . Primer 2 test. The odds ratio (OR) and 95% confidence interval (95% CI) were calculated by logistic regression. According to the Bonferroni correction, the criterion for statistical significance was set at p≤0.025. Hardy–Weinberg equilibrium has been calculated using HW Diagnostics-Version 1.beta . Statistical analysis was done using the SPSS statistical package .Descriptive statistics were used to calculate frequencies and percentages of discrete variables. Continuous data are given as mean±standard deviation (SD). Means were compared using the Mann–Whitney test. Proportions of groups were compared by the χThe present study comprised of 204 patients with XFG  and 204 control subjects, all matched for age and gender. The mean age of patients with XFG was 76.1±7.0 years (range: 60.6–91.7 years), and the mean age for control subjects was 75.3±6.9 years (range: 60.3–93.1 years).TNF-α –308 G>A and TNF-α −238 G>A polymorphisms in patients with XFG and control subjects. No significant differences in either genotype distribution or allelic frequencies of the TNF-α –308 G>A and the TNF-α −238 G>A polymorphisms were found between patients with XFG and control subjects. The presence of the TNF-α –308 G-allele was associated with an insignificant odds ratio of 0.98  for XFG. An insignificant odds ratio of 0.64  for XFG was also found among carriers of the TNF-α –238 G-allele. The present study had a statistical power of 0.80 to detect an odds ratio ≥2.09 for the TNF-α –308 G/G genotype and an odds ratio ≥4.19 for the TNF-α −238 G/G genotype in patients with XFG.The observed genotype distributions did not deviate from those predicted by the Hardy–Weinberg equilibrium, and for control subjects, they were similar to those reported for Caucasian populations . Table 1MMP-3, MMP-9, TIMP-1, and TIMP-3 were upregulated by TNF-α [TNF-α has been shown to affect activity and synthesis of MMPs and TIMPs, which play a major role in the turnover of the ECM in the trabecular meshwork. Treatment of rat cardiac fibroblasts with TNF-α exhibited an increased activity of MMP-2, MMP-9, and MMP-13, while stimulation of human smooth muscle cells led to increased activity of MMP-1, MMP-3, and MMP-9 ,24. Furtby TNF-α .TNF-α –308 G>A polymorphism suggested that this polymorphism may exert protection against XFG. In their study that included 110 patients with XFG, 6.4% of the patients with XFG were found to have the heterozygote TNF-α –308 GA genotype compared to 16.4% of the control subjects. In the present study, no significant differences were found in the genotype distribution or allelic frequency of the TNF-α –308 G>A polymorphism as was the case for the TNF-α −238 G>A polymorphism. An insignificant odds ratio for XFG was found in both carriers of the TNF-α −308 G-allele and of the TNF-α −238 G-allele, suggesting that these polymorphisms themselves are not major risk factors for XFG. Possible explanations for these conflicting results may include small sample size as well as varying genotype distributions among different populations.Various studies provided evidence that MMPs and TIMPs may be involved in the pathogenesis of XFG. Schlötzer-Schrehardt and coworkers found a decreased ratio of MMP-2 to TIMP-2 in the aqueous humor of patients with XFG, which has been corroborated by Määttä and coworkers ,19. Ho aA limitation of the present study is the sample size, whereby minor genetic effects may be undetected. Furthermore, it can not be ruled out that other, as yet unknown functional polymorphisms in the TNF-α gene influence susceptibility to XFG.TNF-α –308 G>A polymorphism has also been recently implicated in the pathogenesis of primary open-angle glaucoma. An increased prevalence of the TNF-α −308 A-allele was reported in patients with primary open-angle glaucoma in a Chinese study, albeit this result could not be replicated in subsequent studies [The  studies -37.TNF-α –308 G>A nor the TNF-α −238 G>A polymorphism is a major risk factor among Caucasian patients with XFG.In conclusion, based on the results of the present study, neither the


Administration of either tumour necrosis factor alpha  or 16,16-dimethylprostaglandin E2 (PGE2) to female NMRI mice caused a decrease in body weight accompanied by a reduction in both food and water intake and a decrease in carcass water content. A single injection of TNF-alpha caused an enhanced production of PGE2 by spleen cells from treated animals, that was significant within 1 h of treatment, and persisted until at least 6 h. These results suggest that the anorectic effect of TNF-alpha may be mediated by a prostaglandin intermediate. Indomethacin (10 mg kg-1) administered 2 h before TNF-alpha (7.5 x 10(7) U kg-1) caused a significant reduction in the extent of weight loss and inhibited PgE2 production. Administration of indomethacin 0.5-1.5 h before the TNF-alpha had no significant effect on loss of body weight, but still inhibited PgE2 production. Also PgE2 production was still enhanced in response to TNF-alpha administered chronically, despite the inability of prolonged TNF-alpha administration to produce continued loss of body weight. These results suggest that prostaglandins are not involved in the anorectic effect of TNF-alpha.


These TNF-α resistant HNSCC lines expressed TNF receptor I, and exhibited constitutive and TNF-α-inducible activation of NF-κB as demonstrated by nuclear localization of NF-κB p65 by immunohistochemistry. UM-SCC-9 I11 cells which stably expressed an inhibitor of NF-κB, IκBαm, were susceptible to TNF-α-induced growth inhibition. DNA cell cycle analysis revealed that TNF-α induced growth inhibition was associated with accumulation of cells with sub-G0/G1 DNA content. Cell death was demonstrated by trypan blue staining, and was blocked by caspase inhibitor. We conclude that HNSCC that exhibit constitutive and TNF-α-inducible activation of transcription factor NF-κB are resistant to TNF-α, and that inhibition of NF-κB sensitizes HNSCC to TNF-α caspase-mediated cytotoxicity. The demonstration of the role of activation of NF-κB in resistance of HNSCC to TNF-α may be helpful in the identification of potential targets for pharmacological, molecular and immune therapy of HNSCC. © 2000 Cancer Research CampaignTumour necrosis factor-α (TNF-α) is a cytokine that can induce cell death of different cancers via a cellular cascade of proteases, the caspases. However, TNF-α has been detected in tumour and serum of patients with head and neck squamous cell carcinoma (HNSCC), and tumour cell lines derived from this environment often exhibit resistance to TNF-α-induced cell death. Cell death mediated by TNF-α and caspases may be inhibited by cytoprotective genes regulated by transcription factor nuclear factor-κB (NF-κB). We recently showed that NF-κB is constitutively activated in HNSCC, and that inhibition of NF-κB by expression of a nondegradable mutant inhibitor of NF-κB, IκBαM, markedly decreased survival and growth of HNSCC cells in vivo. In the present study, we examined the TNF-α sensitivity and response of HNSCC with constitutively active NF-κB, and of HNSCC cells in which NF-κB is inhibited by stable expression of a dominant negative mutant inhibitor, IκBαM. Human lines UM-SCC-9, 11B and 38, previously shown to exhibit constitutive activation of NF-κB, were found to be highly resistant to growth inhibition by up to 10


Rapid outgrowth of EM virus occurs during the first few weeks of infection. However, the rate of escape plateaued soon after, revealing a prolonged persistence of WT viremia not detectable by standard cloning and sequencing methods. The rate of escape of KP9 correlated with levels of vaccine-primed KP9-specific CD8+ T cells present at that time. Similarly, when non-KP9 responder  macaques were infected with SHIVmn229 stock containing a mixture of EM and WT virus, rapid reversion to WT was observed over the first 2 weeks following infection. However, the rate of reversion to WT slowed dramatically over the first month of infection. The serial quantitation of escape mutant viruses evolving during SIV infection shows that rapid dynamics of immune escape and reversion can be observed in early infection, particularly when CD8 T cells are primed by vaccination. However, these early rapid rates of escape and reversion are transient and followed by a significant slowing in these rates later during infection, highlighting that the rate of escape is significantly influenced by the timing of its occurrence.CD8+ cytotoxic T lymphocytes (CTL) can be effective at controlling HIV-1 in humans and SIV in macaques, but their utility is partly offset by mutational escape. The kinetics of CTL escape and reversion of escape mutant viruses upon transmission to MHC-mismatched hosts can help us understand CTL-mediated viral control and the fitness cost extracted by immune escape mutation. Traditional methods for following CTL escape and reversion are, however, insensitive to minor viral quasispecies. We developed sensitive quantitative real-time PCR assays to track the viral load of SIV Gag Immune escape from AIDS virus–specific cellular immunity is common. The driving forces behind how quickly cellular immunity forces escape are poorly understood. We developed a novel assay for a common immune escape variant of SIV in macaques. This allowed us to sensitively track the rates of immune escape even when levels of escape mutant or wild-type virus were low. We found that prior immunization of macaques resulted in very rapid immune escape during acute infection. However, when escape starts to occur later, during chronic infection, the rate of immune escape is much more gradual. Thus, both prior vaccination and timing influence the rates of immune escape and provide a fuller picture of the effectiveness of T cell immunity to HIV. CD8+ cytotoxic T lymphocyte (CTL) responses during acute HIV-1 and simian immunodeficiency virus (SIV) infection in humans and macaques, respectively, correlate with effective control of acute viremia . CTL resMane-A*10 negative pigtail macaques infected with EM challenge stock SHIVmn229, suggesting a significant fitness cost of the K165R mutation ,25mn229 Our findings have important implications when trying to infer immune selection pressure from escape rates. For example, it has been suggested that the CTL response in humans is less effective than in macaques, because of the faster escape rates observed in macaques . HoweverUnderstanding when and at what rate escape occurs at different epitopes is complex. Escape may occur late because the mutations required are rare, or because the EM virus is only weakly selected once it occurs. The strength of immune control and the fitness costs of escape mutation have been suggested as criteria for selection of optimal vaccine epitopes . HoweverAlthough we describe in this report novel sensitive qRT-PCR assays to detect minor viral quasispecies, several limitations of this assay should be noted. First, although our assay can specifically discriminate WT from EM viruses, only the EM virus that the assay was designed for can be distinguished, and other mutants that may arise will not be detected, as it is the LNA-Forward primer that drives the specificity of this qRT-PCR. Additional qRT-PCRs could be designed for rarer escape mutations if needed. For example, we have rarely detected a typically transient mutation with KP9 at position 9 (P172S) during the evolution of the canonical K165R escape ,26; undeIn summary, we have developed qRT-PCRs to measure the kinetics of CTL escape and reversion. We show that both the timing of escape and reversion and prior vaccination influence the dynamics of EM viremia. These findings facilitate a better understanding of the evolution and fitness of escape mutant viruses occurring during HIV-1 and SIV infection.


TNF serum also has interferon activity, and as TNF and interferons have a number of properties in common their relationship has been investigated further. TNF was assayed by cytotoxicity TNF appears not to be an interferon, on the following bases:1. TNF activity could be separated from the Type I interferon of TNF serum by passage through a Cibacron blue-agarose column or by sequential salt precipitation, ion-exchange chromatography and gel filtration.2. Preparations of Type I interferon induced by poly I, poly C or virus lacked TNF activity.3. Though it was not possible to compare TNF with rabbit Type II interferon (as methods used to induce Type II interferon in other species were unsuccessful in the rabbit) rabbit TNF has a number of properties which distinguish it from the Type II interferons of other species.4. Rabbit TNF inhibited the growth of a human melanoma cell line, and also had effects on certain mouse and rabbit cell lines, whereas the anti-cellular effects of interferons are reported to be species-specific.


Five groups of ten female guinea-pigs were passively sensitized against ovalbumin (OA) (n = 9) or control guinea-pig serum (n = 1). 24 h later, they received mepyramine  and 30 min later inhaled aerosols of: (A) OA ; (B) saline ; (C) LPS ; and (D) the control animal was treated as in (C) (n = 1). Their tracheas were cannulated under pentobarbital anaesthesia and bronchial alveolar lavage (BAL) was performed with 2 × 5 ml PBS containing BSA (1%) (n = 1 group), or BSA (1%) and aprotinin (1000 KIU/ml) (n = 4 groups), at 30, 60, 90 or 120 min post-inhalations. BAL fluids recovered were centrifuged, the supernatants recovered and frozen until assayed for tumour necrosis factor-α (TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6). No TNF-α could be detected unless aprotinin was present in the lavaging solution. BAL fluid from OA-sensitized and control animals that had inhaled LPS contained high levels of TNF-α that peaked at 90 min. BAL fluid from OA sensitized animals that inhaled OA aerosols contained no detectable TNF-α at 30 min, but it was found in increasing amounts at 60, 90 and 120 min; TNF-α was not detected in fluid from any of the animals that inhaled saline. As BAL fluids were toxic to the cells used in the assays, neither IL-1 nor IL-6 could be measured. We conclude that the monokine TNF-α is released into BAL fluid following anaphylactic challenge of passively sensitized guinea-pigs. The presence of the antiprotease, aprotinin, in the lavaging solution is essential for the detection and measurement of TNF-α in BAL fluid.


Tumour necrosis factor alpha  is a cytokine with a variety of immunological properties. The identification of two receptors for this molecule, i.e. the 75 kDa and the 55 kDa TNF receptors (TNF-R), recently clarified the mechanisms through which this cytokine provides its wide range of immunomodulatory activities. In this study we have investigated the expression and the functional properties of these receptors on tumour-infiltrating lymphocytes (TILs) recovered from 17 patients with solid cancers . To this end, TIL lines and freshly isolated TILs were evaluated for (a) the expression and the functional role of TNF receptors following culture in the presence of interleukin 2 (IL-2) and (b) the production of TNF-alpha following culture with IL-2 and the role of this cytokine in IL-2-driven TIL proliferation. Flow cytometry analysis demonstrated that TILs bear the 75 kDa TNF-R. Moreover, TIL lines express detectable messages for TNF-alpha and release this cytokine. Functional in vitro studies have shown that anti-TNF-alpha, as well as anti-75 kDa TNF-R antibodies, are able to inhibit the IL-2-induced TIL proliferation. These data demonstrate that TILs are equipped with a fully functional TNF-R system and suggest a putative role for this receptor and its ligand in the activation and expression of TILs following immunotherapy with IL-2.


Drosophila melanogaster, both nutrition and stress can alter the activity of the pathway. Here, we test experimentally the hypothesis that a widespread endosymbiont of arthropods, Wolbachia pipientis, can alter the degree to which mutations in genes encoding IIS components affect IIS and its resultant phenotypes. Wolbachia infection, which is widespread in D. melanogaster in nature and has been estimated to infect 30 per cent of strains in the Bloomington stock centre, can affect broad aspects of insect physiology, particularly traits associated with reproduction. We measured a range of IIS-related phenotypes in flies ubiquitously mutant for IIS in the presence and absence of Wolbachia. We show that removal of Wolbachia further reduces IIS and hence enhances the mutant phenotypes, suggesting that Wolbachia normally acts to increase insulin signalling. This effect of Wolbachia infection on IIS could have an evolutionary explanation, and has some implications for studies of IIS in Drosophila and other organisms that harbour endosymbionts.Insulin/IGF-like signalling (IIS) is an evolutionarily conserved pathway that has diverse functions in multi-cellular organisms. Mutations that reduce IIS can have pleiotropic effects on growth, development, metabolic homeostasis, fecundity, stress resistance and lifespan. IIS is also modified by extrinsic factors. For instance, in the fruitfly  Natural variation in some ecologically significant traits has also been found to be associated with IIS. For example, allelic variants of the PI3K are implicated in the latitudinal variation of reproductive diapause in Drosophila melanogaster  pathway is ubiquitous in multi-cellular animals and may have been involved in the evolution of multi-cellularity itself , adult lC. elegans and Drosophila, genetically upregulating JNK signalling leads to decreased IIS, increased resistance to oxidative stress and extension of lifespan  pathway regulates IIS, linking responses to environmental stresses with metabolic homeostasis  resulted in moderate dwarfism, reduced fecundity and extension of female lifespan, all typical phenotypes of reduced IIS. In the absence of Wolbachia, the previously moderate effects of InRDN expression were accentuated, resulting in flies that showed severe IIS-related phenotypes including extreme dwarfism, sterility, increased fat levels and shortened lifespan. The absence of Wolbachia in control genotypes resulted in a decrease in fecundity and adult weight compared with infected control genotypes, but no effect on other IIS-related phenotypes. These data suggest that Wolbachia can increase IIS in infected flies.In the course of specifying and standardizing the 2.(a)A1409K (chr. II) was obtained from the Bloomington Drosophila Stock Centre . The UAS-InRDN transgene causes an amino acid substitution in the kinase domain (R1409A) of the Drosophila insulin receptor (dInR), resulting in its dominant negative activity  (Fly Base ID FBti0013991) was obtained from the BDSC. UAS-PTEN was provided by Prof. Ernst Hafen , and w1118;;S1,106-GeneSwitch (chr. III) is described in whiteDahomey (wDah) background stock is described in wDah (Wolbachia-infected) to standardize genetic background, beginning with an initial cross between wDah females and transgenic males, followed by five subsequent backcrosses between transgenic females and wDah males. All stocks were maintained and experiments performed on standard sugar/yeast food as described in −1 yeast was used for GeneSwitch experiments using Mifepristone .yw;UAS-dInR(b)wsp gene . To create genetically identical Wolbachia-uninfected stocks (Wol−), the backcrossed Wol+ GAL4, UAS and wDah strains were treated with 30 µg ml−1 of tetracycline contained in standard food for two generations and retested by PCR to confirm that Wolbachia had been removed (a). All stocks were then cultured under normal conditions for at least five generations to allow full recovery from the effects of tetracycline treatment and to restore intestinal flora Procedures for lifespan studies were as described in (d)Females for measuring body weight were prepared as for lifespan experiments and weighed individually when 7 days old. For fecundity, the number of eggs laid per vial of 10 females per day in lifespan experiments was counted.(e)Triacylglyceride (TAG) content quantification was performed as described in (f)mageJ.Lipid droplets were stained with Oil Red O Anti-phospho-Drosophila Akt (no. 4054) and Akt (no. 9272) antibodies were purchased from New England BioLab . Anti-dFOXO is described in (h)W-test on studentized residuals (Wolbachia status) analyses of variance (ANOVA) were performed and planned comparisons of means were made using the Tukey HSD test or Student's t, as appropriate. Data are presented as means of raw values ± s.e.m., and asterisk denotes significant difference from controls.Statistical analyses were performed using JMP (v. 7) software (SAS Institute). Lifespan data were subjected to log-rank tests and are presented as survival curves. Weight, fecundity, TAG levels and fat-cell number data were tested for normality using the Shapiro–Wilk esiduals . Two-way3.(a)Wolbachia infection status affects IIS, we characterized typical IIS-related phenotypes in Wolbachia-infected and -uninfected IIS mutant flies and their controls in parallel. IIS mutant flies were created by expression of a dominant negative form of the insulin receptor (UAS-InRDN) under the control of the ubiquitous and constitutive daughterless-GAL4 (daGAL4) driver.To determine how w control genotypes, irrespective of Wolbachia status . However, removal of Wolbachia greatly accentuated the reduction in weight and fecundity of the daGAL4/InRDN genotype and moderately reduced these phenotypes in control genotypes .Reduced growth and fecundity are typical phenotypes of lowered IIS and, as expected, daGAL4/InRDN females weighed significantly less and laid fewer eggs than their daGAL/+, InRDN/+ and a) and adult fat body morphology (b) compared to Wol+ controls. In contrast, Wol− daGAL4/InRDN females had significantly higher TAG levels  and alterations in fat body morphology (figure 2b) compared to all other genotypes. Unlike the morphologically normal abdominal fat tissue of Wolbachia-infected daGAL4/InRDN and control flies (figure 2b(i–iii)), this tissue was misshapen and shrunken in Wol− daGAL4/InRDN flies (figure 2b(vi)), similar to that seen under starvation conditions .Increases in TAG levels and alterations in adult fat tissue morphology are often associated with reduced IIS a and adurphology b comparec; a), whereas Wol− daGAL4/InRDN females were very short-lived . For control genotypes, Wolbachia infection status had no effect on lifespan . This effect of Wolbachia removal to drastically shorten the lifespan of daGAL4/UAS-InRDN females is consistent with an accentuation of the inhibitory effect of InRDN expression, resulting in a reduction in IIS to levels detrimental to survival. The effect of over-expression of InRDN on lifespan of males in the Wol+ strain was also measured, and a slight reduction in median lifespan was found . This detrimental effect of InRDN expression to male lifespan appeared to be accentuated in the absence of Wolbachia as only very few Wol− daGAL4/UAS-InRDN males enclosed, and those that did died within a few days. This sex difference in the effect on survival is typical for IIS mutants in Drosophila , suggesting that the downregulation of IIS owing to daGAL4-driven expression InRDN was stronger in the absence of Wolbachia than in its presence. Active AKT phosphorylates dFOXO, and this phosphorylation retards the migration of dFOXO on SDS–PAGE (d).The previous data suggested that  protein . We meas protein d, suggesSDS–PAGE d.3; 3 to PIP2 .Interestingly, the enhanced IIS-related phenotypes of the Wol− daGAL4/InRDN females resembled those due to a strong downregulation of the pathway obtained using daGAL-driven expression of UAS-PTEN. The essential function of the phosphoinositide phosphatase PTEN is to antagonize the effects of PI3K by dephosphorylation of the second messenger phosphatidylinositol-3,4,5-trisphosphate Wolbachia, described above, suggested a dose dependency of IIS downregulation. To begin to determine if these effects are general, we used the RU486 GeneSwitch-inducible GAL/UAS system (1106-GS driver has previously been shown to be sufficient to extend lifespan (The quantitatively different effects of InRDN expression on IIS-related phenotypes in the presence and absence of 1106-GS driver under the same induction conditions as in Wolbachia (a). However, removal of Wolbachia from S1106-GS/UAS-InRDN female flies resulted in extension of lifespan and revealed a dose-dependent effect of expression of InRDN (b). At 200 µM, RU486 inducer Wol− S1106-GS/UAS-InRDN females showed a 6.5 per cent increase in median lifespan (χ2 p = 1.16×10−7) and at 300 µM RU486 a 13 per cent increase (χ2 p = 3.65×10−15) compared with the same genotype at 0 µM RU486. There was no effect of RU486 on survival of driver control female flies . S1106-GS/InRDN expression had no effect on fecundity or lipid levels , confirming the previous study modulating IIS in this tissue .Expression of InRDN using the Solbachia a. Howeveof InRDN b. At 200Wolbachia from Wol+ normal-lived S1106-GS/InRDN females acted to increase the inhibitory effect of InRDN in this tissue to a threshold sufficient for lifespan extension. The data further suggest that ubiquitous expression of InRDN by daGAL4 may have affected fecundity, growth and fat phenotypes via expression during development and/or in other tissues.These data suggest that removal of 5.Drosophila can be enhanced by removal of Wolbachia, suggesting that one of the normal consequences of Wolbachia infection is enhanced IIS. The data are consistent with a general effect on IIS, and not a specific interaction with mutants such as the daGAL4/InRDN genotype, because removal of Wolbachia from control strains had similar, although weaker, effects. More work will be needed both to determine the generality of these kinds of effects and exactly how Wolbachia modifies IIS-related phenotypes.The data presented here demonstrate that mutant IIS and its consequent phenotypes in Wolbachia interacts with IIS may have its origin in the way that these endosymbionts are transmitted. Their habitat is inside the cytoplasm of cells, and they are transmitted maternally, via infection of eggs (Wolbachia are thought to persist in host populations by providing females with a reproductive advantage via mechanisms that can include male killing, feminization of genetic males and cytoplasmic incompatibility (see D. melanogaster, an effect of Wolbachia infection is to confer reproductive advantage through cytoplasmic incompatibility (Wolbachia-infected females can successfully mate with infected or uninfected males, but uninfected females are incompatible with infected males. Thus, Wolbachia infection can spread and persist in the host population, although other mechanisms must also be involved (Evolutionary considerations suggest that the mechanisms by which Wolbachia, part of the response to selection for an optimal regime of host egg production through the life history may thus be attributable to evolution of the effects of the symbiont on host reproduction. However, this alone would not explain why Wolbachia would act as seen in this study, solely to promote high host fecundity. A possible explanation could come from events during adaptation to life in the laboratory. The life history of Drosophila evolves so as to optimize fitness under the prevailing culture regime (Wolbachia, part of the adaptation to laboratory conditions may be achieved by the evolution of the symbiont so as to alter host physiology in ways that promote an optimal regime of egg production through copious, early female reproduction. One prediction of the hypothesis is that these effects of Wolbachia would be expected to be particularly obvious in strains that have undergone laboratory adaptation, in traits promoting fitness through the regime of fecundity and in females. Alternatively, interactions of IIS with the Wolbachia infection itself could play a role.In evolutionary terms, the interests of the female host and of the symbiont in host egg production are identical. Both host and symbiont are selected to maximize host fitness through this route. In populations of flies that are infected with e regime . The usuWolbachia has itself been found to result in a decline of mitochondrial activity and increase in mitochondrial DNA density, which may increase insulin insensitivity regardless of the removal of the endosymbionts (Wolbachia-free flies had no effect on lifespan when tested several generations after treatment (The tetracycline treatment used for the removal of ymbionts . Howeverpkb3 mutant was due to its reduced AKT activity level (The variability of the phenotypes owing to IIS reduction in the fly is probably due to the complex nature of the pathway itself, as well as the developmental stage and tissue affected by the genetic manipulation in question. However, the dose dependency of IIS downregulation identified here supports the idea that some of the variability could be related to differing strengths of IIS downregulation. Over-expression of the FOXO transcription factor in the fly eye imaginal disks induced cell death (Wolbachia effect has implications for studies of IIS in Drosophila. It is desirable to ensure that the Wolbachia status of all strains being compared is the same, to rule out any possibility that traits apparently attributable to altered IIS are in fact owing to differences in Wolbachia status. However, provided this stricture is observed, alteration in Wolbachia status could be a convenient way of altering the intensity of IIS. The initial state of IIS as well as the stage and tissue specificity of the IIS manipulation may determine how a change in Wolbachia status affects IIS phenotypes.Despite the uncertainties as to the mechanism by which IIS is modified, the 6.Wolbachia acts to enhance IIS, thus modifying the extent to which IIS mutants affect signalling and the consequent pleiotropic phenotypes of the pathway. Furthermore, for some of these phenotypes, such as lifespan, the phenotype peaks at an intermediate level of IIS downregulation.The data presented here show that the effects of mutant IIS are dose-dependent, and that


Prevotella (P.) intermedia lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in differentiated THP-1 cells, a human monocytic cell line.In addition to regulating body weight, leptin is also recognized for its role in the regulation of immune function and inflammation. The purpose of this study was to investigate the effect of leptin on P. intermedia ATCC 25611 was prepared by the standard hot phenol-water method. THP-1 cells were incubated in the medium supplemented with phorbol myristate acetate to induce differentiation into macrophage-like cells. The amount of TNF-α and interleukin-8 secreted into the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). TNF-α and Ob-R mRNA expression levels were determined by semi-quantitative reverse transcription-polymerase chain reaction analysis.LPS from P. intermedia LPS-induced TNF-α production in a dose-dependent manner. Leptin modulated P. intermedia LPS-induced TNF-α expression predominantly at the transcriptional level. Effect of leptin on P. intermedia LPS-induced TNF-α production was not mediated by the leptin receptor.Leptin enhanced P. intermedia LPS-induced TNF-α production may be important in the establishment of chronic lesion accompanied by osseous tissue destruction observed in inflammatory periodontal disease.The ability of leptin to enhance  Prevotella (P.) intermedia is a major periodontal pathogen that is dominant in the periodontal pockets of patients with adult periodontitis [Periodontal disease is a chronic inflammatory process accompanied by destruction of surrounding connective tissue and alveolar bone, and sometimes loss of teeth . The pridontitis . This badontitis  and pregdontitis .P. intermedia. It has the ability to trigger a number of host cells, especially mononuclear phagocytes, to produce and release a wide variety of pharmacologically active mediators, including interleukin (IL)-1β, IL-6, IL-8, and, most importantly, tumor necrosis factor alpha (TNF-α) [Lipopolysaccharide (LPS) is a major constituent of the outer membrane of gram-negative bacteria, including  (TNF-α) .ob gene, is synthesized mainly by adipocytes and plays a crucial role in the homeostasis of body weight by regulating food intake and energy expenditure, through its action on the hypothalamus [Leptin, the 16-kDa nonglycosylated protein encoded by the thalamus . In addithalamus , hematopthalamus , reproduthalamus , immune thalamus , and mosthalamus .P. intermedia LPS-induced TNF-α production in differentiated THP-1 cells, a human monocytic cell line.Leptin has been classified as a cytokine because it shows tertiary structure similar to the class of long-chain helical cytokines that includes IL-6, IL-11, and leukemia inhibitory factor . The bioP. intermedia ATCC 25611 was used throughout. It was grown anaerobically on the surface of enriched Trypticase soy agar containing 5% (v/v) sheep blood, or in GAM broth  supplemented with 1 µg/mL menadione and 5 µg/mL hemin. Culture purity was assessed by gram staining and plating on solid medium.P. intermedia ATCC 25611 cells by the standard hot phenol-water method [LPS was prepared from lyophilized r method , and subr method , was les2/95% air at 37℃. The cells (5 × 105 cells/ml/well in 24-well culture plates) were incubated in the medium supplemented with 50 ng/mL of phorbol myristate acetate to induce differentiation into macrophage-like cells. The cells were allowed to differentiate and adhere to plastic for 72 hours and washed three times with medium. Various concentrations of P. intermedia LPS and leptin  were then added and the cells were cultured for the indicated times, after which culture supernatants were collected and assayed for TNF-α.The human monocytic cell line THP-1  was grown routinely in Nunc flasks in RPMI 1640 medium supplemented with 10% (v/v) heat inactivated FBS, 50 µM 2-mercaptoethanol, 1 mM sodium pyruvate, 25 mM HEPES, 100 U/mL of penicillin, and 100 µg/mL of streptomycin in a humidified chamber with 5% COThe amount of TNF-α and IL-8 secreted into the culture medium was determined by enzyme-linked immunosorbent assay (ELISA) using a commercially available kit  according to protocols recommended by the manufacturer. The sensitivity of the assay was 7.8 pg/mL, according to the manufacturer.6 cells per dish, and treated with various concentrations of P. intermedia LPS and leptin  for the indicated periods of time. Following incubation, they were washed twice with phosphate buffered saline and collected by centrifugation. Total RNA was isolated with an RNeasy Mini Kit , according to the manufacturer's instructions. Synthesis of cDNA from the extracted RNA and subsequent amplification of the cDNA by RT-PCR were carried out with an AccuPower RT/PCR Premix kit  and thermal cycler . β-actin served as internal control. The number of cycles that ensured nonsaturating PCR conditions was established in preliminary experiments. Primer sequences and RT-PCR conditions are listed in Cells were plated in 100-mm tissue culture dishes, at a density of 5 × 10t-test with P<0.05 considered statistically significant. Data are expressed as means±SD of three independent experiments.Statistical analysis was performed using Student's paired P. intermedia LPS-induced TNF-α production was enhanced by leptin in a dose-dependent manner with a significant effect already seen at 0.01 µg/mL compared with LPS alone  and short (Ob-Ra) leptin receptors were expressed in untreated and leptin-treated cells, while upregulation of Ob-R by P. intermedia LPS stimulation or its potentiation by leptin was not confirmed (To verify whether the Ob-R is responsible for mediating the synergistic effect of leptin on onfirmed . As showP. intermedia LPS-induced TNF-α production in differentiated THP-1 cells, a human monocytic cell line, was studied. Macrophages are known to be the main producer of TNF-α and a dense infiltration of inflammatory cells, including macrophages, occurs in the gingival connective tissues of patients with periodontal disease [Because production of TNF-α has been recognized as a marker in a variety of human diseases associated with inflammation , the eff disease .Leptin levels are increased by inflammatory stimuli such as LPS and cytokines -25. And,P. intermedia LPS-induced TNF-α production in a dose-dependent manner. Leptin modulated P. intermedia LPS-induced TNF-α expression predominantly at the transcriptional level. There is evidence to suggest that TNF-α plays a central role in the pathogenesis of periodontal disease. TNF-α has been found at high levels in gingival crevicular fluids and in gingival tissues from periodontally diseased sites over those in healthy sites [P. intermedia LPS-induced TNF-α production may be important in the establishment of chronic lesion accompanied by osseous tissue destruction observed in inflammatory periodontal disease.The results of this study indicate that leptin potentiates hy sites ,31. Morehy sites ,33. Furthy sites . The abiP. intermedia LPS might be related to an increase in Ob-R expression, Ob-R mRNA expression levels were determined by semi-quantitative RT-PCR analysis. This study demonstrated the presence of the long and short leptin receptors in differentiated THP-1 cells. However, leptin receptors were not modulated by P. intermedia LPS or leptin. These results indicate that effect of leptin on P. intermedia LPS-induced TNF-α production is not mediated by the leptin receptor. The mechanism by which leptin potentiates P. intermedia LPS-induced TNF-α production remains to be elucidated.To verify whether the mechanism behind the synergistic effect of leptin with


Serum from rabbits with BCG/endotoxin-induced shock is growth inhibitory or cytotoxic to a range ot tumour cell lines. The active component, tumour-necrosis factor (TNF), has been purified 1000-fold by sequential salt precipitation, ion-exchange chromatography and gel-filtration. TNF had a mol. wt of 67,000 on gradient PAGE and 39,000 on Ultrogel AcA44 gel-filtration. The isoelectric point was pH 5.1-5.2. TNF was susceptible to the proteolytic enzyme pronase, but resistant to trypsin or papain. On isopycnic ultracentrifugation it had a buoyant density of 1.27, confirming that it is protein in nature, with little or no carbohydrate. This is also suggested by its failure to bind to a range of lectins.


Over a period of 40 years, SNOMED has developed from a pathology-specific nomenclature (SNOP) into a logic-based health care terminology. In spite of its long existence and continuous evolvement, it is yet unknown to what extent SNOMED is used in clinical practice, and what benefits were achieved. The aim of this paper is to investigate this by providing an overview of published studies in which a version of SNOMED was studied or applied.This paper analyzes the use of SNOMED over time, as reflected in scientific publications, by means of Medline literature search in PubMed. The search included papers from 1966 until June 2006. For each selected paper the following characteristics were classified: version, medical domain, coding moment (during or after the care process), usage, and type of evaluation .250 papers were included in this research. The number of annual publications has increased, as has the number of domains in which SNOMED is being used. Theoretical studies mainly concern comparison of SNOMED to other terminological systems and SNOMED as an illustration of a terminological systems' theory. Few studies are available on the usage of SNOMED in clinical practice, largely involving coding information and retrieval/aggregation based on SNOMED codes.The clinical application of SNOMED is broadening beyond pathology. The majority of studies concern proving the value of SNOMED in theory. Fewer studies are available on the usage of SNOMED in clinical practice. Literature gives no indication of the use of SNOMED for direct care purposes such as decision support. Terminological systems (TS) are often mentioned to be an important prerequisite for making electronic medical records a success. Terminological systems provide terms denoting concepts and their relations from a specific domain and can be used to describe information in a structured and standardized way .Well-known examples are MeSH   for cate• enabling a consistent way of indexing, storing, retrieving and aggregating clinical data across specialties and sites of care,• enabling structuring and computerizing the medical record, thereby reducing the variability in the way data is captured, encoded and used for clinical care of patients and research,• enabling automated reasoning, e.g. decision support.Whereas some of these benefits require the formal rigor of SNOMED CT, others can be obtained with earlier versions of SNOMED.It is yet unknown to what extent terminological systems such as the SNOMED family are used in clinical practice. Furthermore, scientific evidence is lacking that benefits such as the ones mentioned above were achieved in clinical practice.The aim of this paper is to provide an overview of published studies in which one of the members of the SNOMED family was the main subject of study or plays a secondary role in a study, e.g. to select a patient population for epidemiologic research or to describe an information system in which a SNOMED version was used to code patients. By this overview we will describe the applications and development of the SNOMED family in the medical literature and we will detect the areas in which these applications have proven their value as well as the areas needing future research.A Medline literature search by PubMed was performed to select papers in which SNOMED plays any role. The search period was from 1966 to June 2006. We used ("SNOMED" OR "SNOP") as search query, thereby selecting all papers that have been MeSH-indexed by "SNOMED" and all papers that used "SNOMED" or "SNOP" as a text word in the title or the abstract. Only papers with an English abstract were included. From all papers found, two independent researchers manually excluded papers that were not on the Systematized Nomenclature of Pathology or on the Systematized Nomenclature of Medicine, e.g. when the author's name was Snop. Next, for each selected paper the following characteristics were classified based on the information contained in the abstract:Version ∘ Medical domain ∘ Coding moment ∘ Usage:∘ a. SNOMED is the primary object of study b. SNOMED is the secondary object of study Type of evaluation. If SNOMED was evaluated in the study, the aim/research question of evaluation was determined: does it work ; does it help (effect on care), what are the consequences (for the organization); unknown/not applicable.∘ All papers have been independently analysed by the two authors, different judgement has been discussed and resolved.The PubMed search resulted in 318 hits. Among these hits 64 papers were excluded because of lacking an English abstract and 4 papers were excluded because SNOP was used with a different meaning 2 or the author's name being Snop). The remaining 250 Medline indexed papers on SNOMED have been published in 80 different journals. The AMIA conference proceedings comprised the largest number of papers . Overall, 43% of the papers were published in medical informatics conference proceedings . The other 57% have been published in a wide variety of medical and medical informatics journals. A majority of the medical journals came from the field of pathology.Figure Review of the 250 included papers on the aspects "version", "medical domain", "coding moment", "usage", and "type of evaluation" provided the following results.Half of the papers described the use of "SNOMED" without any further specification of its version. Without specification it could not be determined from the abstracts whether this referred to the 1974 version of SNOMED or any later version. Among the other half, most papers described a study on SNOMED CT  of the papers did not clearly describe a specific medical domain of the study or SNOMED was used in the study for multiple disciplines or medicine in general. Among the 40% (n = 100) of the papers in which a specific medical domain had been described, pathology (n = 56), nursing n = 13) and cancer (n = 10) were most frequently mentioned  it was not clearly described whether concepts were coded using SNOMED during and/or for the purpose of the care process or post-coded for research purposes. In those papers in which this was clearly described, 62% addressed coding that occurred during and/or for the care process, e.g. coding pathology finding in patient reports. In the remainder (38%) coding was performed for research purposes, e.g. post-coding a set of patient cases to compare content coverage of several TSs.SNOMED was the main object of study in 163 papers. The other 87 papers described a study in which SNOMED plays a secondary role. As is shown in figure There was no difference in usage among various medical specialties. Figure In the earlier years, most studies describe the implementation of a pathology information system in which a SNOMED version is used to code and/or retrieve patients. In the most recent years the usage has become more diverse, with mapping to other systems and illustration of TS theory as the most prominent topics.Emerging subjects include the use of SNOMED as a standard for the electronic health record and content coverage studies.52% (n = 129) of the papers described some kind of evaluation of SNOMED. Out of these, 81% (n = 104) concerned a technical evaluation to find an answer to the question "does it work?", e.g. content coverage, automated classification, and 19% (n = 25) evaluated SNOMED on the aspect "does it help?" which mostly concerned the supportive role of SNOMED in retrieving or aggregating patient(group)s and making management reports.This review included 250 papers on members of the SNOMED family. A large number of medical domains and application have been described. Even though in many cases there is no description given of the clinical domain in which SNOMED is being used, the cases in which such a description is given show that the clinical application of SNOMED is broadening beyond pathology.It is generally known that searching medical bibliographies such as PubMed will not result in finding all published studies on a particular subject . As we sIn many cases it was hard to determine which version of SNOMED was actually used. Mention of the systems was often limited to "SNOMED", not making explicit whether this was the version as released in 1974 or any of its successors. One possible explanation for this is the fact that our study has been based on abstracts and not on full papers. However, checking the full paper of a small sample of abstracts in which the SNOMED version was not specified does not provide more information on the characteristics collected for each paper included.The two largest groups of papers on a SNOMED version concern the comparison of SNOMED to other TSs and papers in which a theory such as automatic coding, natural language processing and description logics is illustrated by (case studies with) SNOMED. These kinds of studies are particularly relevant for proving the value of SNOMED in theory. Fewer studies are available on the usage of SNOMED in clinical practice. The use of SNOMED as described in these papers largely involves coding information and retrieval/aggregation based on SNOMED codes. Only 2 rather old papers (1975 and 1992) gave some indication of the use of SNOMED to improve the care process or outcome of care. One stated that SNOP provides significant economy in data handling. One shoLiterature gives no indication of the use of SNOMED for direct care purposes such as decision support. As decision support relies on formal representation, which was introduced in SNOMED RT, studies investigating this aspect can now be performed.The authors declare that they have no competing interests.The authors have collaboratively performed the extraction of articles from Pubmed.Both authors have analyzed the retrieved articles, and independently determined the characteristics.Both authors have contributed substantially to the manuscript as well as producing the figures.


Tumor necrosis factor alpha (TNF-α) was discovered more than a century ago, and its known roles have extended from within the immune system to include a neuro-inflammatory domain in the nervous system. Neuropathic pain is a recognized type of pathological pain where nociceptive responses persist beyond the resolution of damage to the nerve or its surrounding tissue. Very often, neuropathic pain is disproportionately enhanced in intensity  or altered in modality  in relation to the stimuli. At time of this writing, there is as yet no common consensus about the etiology of neuropathic pain - possible mechanisms can be categorized into peripheral sensitization and central sensitization of the nervous system in response to the nociceptive stimuli. Animal models of neuropathic pain based on various types of nerve injuries  have persistently implicated a pivotal role for TNF-α at both peripheral and central levels of sensitization. Despite a lack of success in clinical trials of anti-TNF-α therapy in alleviating the sciatic type of neuropathic pain, the intricate link of TNF-α with other neuro-inflammatory signaling systems  has indeed inspired a systems approach perspective for future drug development in treating neuropathic pain. Despite intense research over the last 30 years, debate is still ongoing regarding the nature of neuropathic pain, including controversy as to whether such pain is peripheral or central in origin, and as to whether its etiology is inflammatory or non-inflammatory. Increasing evidence has provided better understanding of the roles of both immune and pro-inflammatory mediators  in the mechanisms of both peripheral and central neuropathic pain -4. This per se, that is pivotal for the development of neuropathic pain. Clatworthy et al. [Neuropathic pain is characterized by disproportionate hypersensitivity to stimuli , abnormal pins-and-needles or electric-shock-like sensations (hyperpathia) and, finally, nociceptive responses to non-noxious stimuli . It is a pathological type of pain that persists despite resolution of the inciting damage to the nerve and the surrounding tissues. From a behavioral standpoint, nociception is an adaptive tool for better survival, while neuropathic pain is considered maladaptive. The prevalence of neuropathic pain ranges from 1% in UK  to 1.5% y et al.  further y et al. -34, brady et al. ,36, seroy et al. -39, ATP/y et al. -42, neury et al. -46, cytoy et al. -52, chemy et al. ,54, and y et al. ,55,56. Ty et al. -59 and sy et al. -63, therCytokines are low molecular weight glycoproteins that are secreted mainly, but not exclusively, by immunological cells such as T-cells, macrophages and neutrophils. Other cells that secrete cytokines include keratinocytes and dendritic cells of the skin  and SchwStreptococcus pyogenes and Serratia marcescens bacteria to treat tumors may in fact be the first discovery of tumor necrosis factor [+ current in cultured DRG cells from wild-type but not from TNFR1-knockout mice, and such current is abolished by a p38-MAPK inhibitor; implying that TNF acts via TNFR1 and activates TTX-R Na+ channels via the p38 MAPK system [In 1891, the success story of William Coley in using supernatant extract of heat-killed mixtures of s factor . It was s factor . TNF-α bs factor . TNFRs as factor . In the s factor -81; heres factor  and Schws factor ,83 by ims factor . Similars factor . Intra-ss factor ,86, and s factor . TNF-α eK system . FurtherK system . HoweverK system . In the + channels in nociceptive DRG neurons [+ ion conductance in a non-voltage-gated fashion [TNF-α plays a role in the peripheral mediation of neuropathic pain. Clinically, HIV therapy and chemotherapy produce peripheral neuropathy with massive release of TNF-α in serum  and TNF- neurons , TNF-α c fashion  leading In late 1990s, TNF-α was proposed to be one pro-inflammatory cytokine with a pivotal role in the "immune-to-brain" pathway of communication for pain, and in models of sickness response in general ,107. In et al [Back in 1991, it was shown that classic CCI leads to hypertrophy of astroglia in the dorsal horn of spinal cord as reflected by increased immunostaining of glial fibrillary acidic protein . Since tet al  demonstrOriginally identified in hippocampus as a substrate for memory storage and learning, the synaptic mechanisms of long term potentiation (LTP) in glutamergic neurons  have sin1 to P2X7) and 8 sub-types of P2Y receptors  [3, P2X4 and P2X7 receptors are thought to play a role; and in particular, P2X3 may act via the TTX-R voltage-gated sodium channel Nav 1.9 [Since the 1950s, release of ATP has been detected from nerve endings ,144 and , P2Y14) . Each su, P2Y14) . Within  Nav 1.9 .3 immuno-reactivity of the DRG neurons, depending on the type of nerve injury. When expression of P2X3 receptors in DRG is reduced using anti-sense oligonucleotides [3 receptors can reverse established neuropathic pain that re-emerges after cessation of the anti-sense treatment [3 receptors. Following a similar approach, Tsuda et al [4 receptor expression after chronic nerve injury, and showed that both pharmacological blockade and anti-sense oligonucleotide treatment abrogates the development of mechanical allodynia. Later studies have suggested that P2X4 receptor stimulation leads to secretion of brain-derived neurotrophic factor (BDNF) in spinal microglia, and that this BDNF is involved in mediating neuropathic pain [7 receptors are associated with TNF-α production in microglia through the p38-MAPK system [+ channels [Earlier studies using nerve injury models in rats revealed either an increase  or decreleotides  or siRNAleotides , developreatment , suggestda et al  demonstrhic pain ,159,160,hic pain . P2X7 reK system ,163, as K system . Indepenchannels . As an echannels ,165 and channels ,167. Forchannels . Studiesper se to induce astrocytic proliferation [Due to the unique trimeric structure shared between the TNF ligand and the TNF receptor (both belonging to the TNF/TNFR SPF), the transmembrane portion of TNF molecule (mTNF), besides being a ligand, is capable of acting as a receptor for a soluble form of TNF (sTNF) in a "reverse-signaling" manner , which tferation  and hencferation ,198.The role of TNF-α in chronic pain seems irrefutable in view of abundant data from various neuropathic animal models, and with the actual isolation of TNF-α from neuropathic nerves  and periTNF-α is undoubtedly a titan in the research of neuropathic pain, and is by no means the only one in the arena. It is a pivotal member of the cytokine mediator system that is intrinsic to the pathogenesis of neuropathic pain both at peripheral and central levels See Fig . Togethe5-HT: 5-hydroxytryptamine (Serotonin); ADO: Adenosine; ADP: Adenosine diphosphate; ATP: Adenosine triphosphate; BBB: Blood-brain barrier; BDNF: Brain-derived neurotrophic factor; CCI: Chronic constrictive injury; CCL2: Chemokine (C-C motif) ligand-2; CX3CL1: Chemokine (C-X3-C motif) ligand 1; CXCR4: CXC chemokine receptor-4; DRG: Dorsal root ganglion; CRDs: Cysteine rich domains; ELP: Elastin-like polypeptide; ERK: Extracellular signal-regulated kinases; HFS: High Frequency stimulation; HSV: Herpes simplex virus; IL-1: Interleukin-1; IL-1β: Interleukin-1 beta; IL-6: Interleukin-6; IL-8: Interleukin-8; IL-10: Interleukin-10; JNK: c-Jun N-terminal kinases; LPS: Lipopolysaccaride; LTP: Long Term Potentiation; MAPK: Mitogen activated protein kinase; NMDA: N-methyl-D-aspartic acid; NSAIDs: Non-steroidal anti-inflammatory drugs; siRNA: Small interfering RNA; RVM: Rostral ventromedial medulla; THD: TNF homology domain; mTNF: Transmembrane portion of TNF; sTNF: Soluble form of TNF; TNF-α: Tumor necrosis factor alpha; TNFR: Tumor necrosis factor receptor; sTNFR2: Soluble p55 TNF receptor; TNFR SFR: Tumor necrosis factor receptor super family receptor; TTX-R: Tetrodotoxin resistant.The authors declare that they have no competing interests.LL wrote the manuscript, CMC provided comments and proof-reading. Both authors have read and approved of the final version of the manuscript.


In the present study, we used tumor necrosis factor-R1 knock out mice (TNF-αR1KO) to understand the roles of TNF-α on epithelial function in models of carrageenan-induced acute lung inflammation. In order to elucidate whether the observed anti-inflammatory status is related to the inhibition of TNF-α, we also investigated the effect of etanercept, a TNF-α soluble receptor construct, on lung TJ function. Pharmacological and genetic TNF-α inhibition significantly reduced the degree of (1) TNF-α production in pleural exudates and in the lung tissues, (2) the inflammatory cell infiltration in the pleural cavity as well as in the lung tissues , (3) the alteration of ZO-1, Claudin-2, Claudin-4, Claudin-5 and β-catenin (immunohistochemistry) and (4) apoptosis . Taken together, our results demonstrate that inhibition of TNF-α reduces the tight junction permeability in the lung tissues associated with acute lung inflammation, suggesting a possible role of TNF-α on lung barrier dysfunction. An important consequence of acute lung injury is the disruption of the paracellular alveolar permeability barrier . The perMoreover, is well known that airway epithelial cells perform many important functions, serving as an interface between environmental stimuli and the lung parenchyma. Normally the lower airways are pristine, free of bacterial flora or inflammatory cells, and are well protected by several layers of defenses including antimicrobial peptides, mucin, and ciliary action. There is a brisk epithelial response to airway injury caused by many different mechanisms ,10. AcutAfter exposure to TNF-α, target cells may down-regulate their responsiveness to the cytokine by shedding the receptors into the circulation. A natural mechanism which has been hypothesized to counteract excessive concentrations of circulating TNF-α (and the subsequent enhanced surface receptor activation) is the release of soluble receptors. The two soluble receptor forms (sTNFR1 and sTNFR2) have longer half lives than TNF-α, and their concentration may reflect TNF-α activity .in vitro and has been examined in vivo for its effects in different animal model systems of inflammatory and autoimmune diseases [A primary role for TNF-α in inflammatory process  is suggested by several studies conducted upon cell lines, animal models and human beings -20. In idiseases .In addition, it has been demonstrated that TNF plays a role in the control of epithelial permeability -29 as wead libitum. Animal care was in compliance with regulations in Italy (D.M. 116192), Europe (O.J. of E.C. L 358/1 12/18/1986) and USA .Mice  with a targeted disruption of the TNF-αR1 (TNF-α R1KO) and wild-type controls (TNF-αWT) were purchased from Jackson Laboratories . The study was approved by the University of Messina Review Board for the care of animals. The animals were housed in a controlled environment and provided with standard rodent chow and water WT CAR group. WT mice were subjected to carrageenan-induced pleurisy (N = 10); (ii) CAR TNF-αR1KO group. TNF-αR1KO mice were subjected to subjected to carrageenan-induced pleurisy (N = 10); (iii) WT Sham group. WT mice were subjected to the surgical procedures as the above groups except instead of carrageenan 100 μl of saline solution were administered to the mice (N = 10); (iv) TNF-αR1KO Sham group. TNF-αR1KO mice were subjected to the surgical procedures as the above groups except that instead of carrageenan 100 μl of saline solution were administered to the mice (N = 10); (v) CAR WT+Etanercept group. Same as CAR WT group except for the administration of Etanercept  which was given at 2 h before the carrageenan injection (N = 10); (vi) WT Sham +Etanercept group. Same as the WT Sham group except for the administration of Etanercept  which was given at 2 h before saline injection (N = 10);Mice were randomly allocated into the following groups: (i) 2. The chest was carefully opened and the pleural cavity rinsed with 2 ml of saline solution containing heparin (5 U/ml) and indomethacin (10 μg/ml). The exudate and washing solution were removed by aspiration and the total volume measured. Any exudate, which was contaminated with blood, was discarded. The amount of exudate was calculated by subtracting the volume injected (2 ml) from the total volume recovered. The leukocytes in the exudate were suspended in phosphate-buffer saline  and counted with an optical microscope in a Burker's chamber after vital Trypan Blue staining.Carrageenan-induced pleurisy was induced as previously described . Mice weLung biopsies were taken 4 h and 24 h after carrageenan injection. Tissues biopsies were fixed for 1 week in 10 % (w/v) PBS-buffered formaldehyde solution at room temperature, dehydrated using graded ethanol and embedded in Paraplast . Lung sections were then deparaffinized with xylene, stained with hematoxylin and eosin. All sections were studied using light microscopy .TNF-α production was evaluated in the pleural exudate and lung tissues at 4 h and 24 h after the induction of pleurisy by carrageenan injection as previously described . The assClaudin-2, Claudin-4, Claudin-5, ZO-I and β-catenin detection, slices were treated with protease  (2 mg/ml) and for 10 min at 37°C. Detection of BCL-2 and Bax was carried out after boiling in citrate buffer, 0.01 M for 4 min. Sections were incubated overnight with polyclonal rabbit anti-claudin-2 Claudin-4, Claudin-5, ZO-I and β-catenin antibody . Sections were washed with PBS and incubated with secondary antibody. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex . The counter stain was developed with DAB (brown color) and nuclear fast red (red background). To verify the binding specificity, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations no positive staining was found in the sections indicating that the immunoreaction was positive in all the experiments carried out. Immunocytochemistry photographs (N = 5) were assessed by densitometry as previously described [At 4 and 24 h after carrageenan injection, tissues were fixed in 10% (w/v) PBS-buffered formaldehyde and 5 μm sections were prepared from paraffin embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. Non-specific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin , respectively. Sections were incubated overnight with 1) with anti-TNF-α antibody  or 3) with anti-Bax antibody  or 4) with anti-Bcl-2 antibody (Santa Cruz 1:100 in PBS v/v). After deparaffinization, for escribed  by usingMyeloperoxidase (MPO) activity, an indicator of polymorphonuclear leukocyte (PMN) accumulation, was determined as previously described . At the 2O2 for 5 min at room temperature and then washed with PBS. Sections were immersed in terminal deoxynucleotidyltransferase (TdT) buffer containing deoxynucleotidyl transferase and biotinylated dUTP in TdT buffer, incubated in a humid atmosphere at 37°C for 90 min, and then washed with PBS. The sections were incubated at room temperature for 30 min with anti-horseradish peroxidase-conjugated antibody, and the signals were visualized with diaminobenzidine.TUNEL assay was conducted by using a TUNEL detection kit according to the manufacturer's instructions . Briefly, sections were incubated with 15 μg/ml proteinase K for 15 min at room temperature and then washed with PBS. Endogenous peroxidase was inactivated by 3% HFrom each biopsy, at least 4 bronchiolar profiles were evaluated under a light microscope at a ×20 magnification for TUNEL-positive cells. The percentage is calculated as the number of positive cells/total number of bronchial epithelial cells.in vivo studies n represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative at least three experiments  performed on different experimental days on the tissues section collected from all the animals in each group. The results were analyzed by one-way ANOVA followed by a Bonferroni's post-hoc test for multiple comparisons. A p-value of less than 0.05 was considered significant.All values in the figures and text are expressed as mean ± standard error of the mean (s.e.m.) from 10 mice for each group. For the data not shown) and at 24 hours from sham WT mice -positive cells count Fig 4bb1see (TUNEL)-positive cells count Fig 4cc1see (TUNEL)-positive cells count Fig 4dsee (TUNEL)-positive cells count Fig 4esee (TUNEL)-positive cells count Fig 4fsee (TUNEL)-positive cells count Fig 4gsee (TUNEL)-positive cells count Fig To test whether functional TNF-α gene plays a role on apoptosis in acute lung inflammation, we measured TUNEL-like staining in the lung tissues at 4 h and 24 h after carrageenan administration. Almost no apoptotic cells were detected in the lung from sham-treated  and at 24 hours from sham WT mice  in the pleural cavity. When compared to the number of cells collected from the pleural space from sham WT mice and sham TNF-αR1KO mice, injection of carrageenan induced a significant time dependent increase in the number of PMNs Fig. . The numThe important presence of inflammatory cells in the pleural cavity appeared to be correlated with the influx of leukocytes into the lung tissue. Therefore, we investigate the role of functional TNF-α gene on the neutrophils infiltration by measurement of the activity of myeloperoxidase. Myeloperoxidase activity was significantly elevated (p < 0.001) at 4 h and 24 h after carrageenan injection in WT mice Fig. . In lungdata not shown see densitometry analysis Fig. 8asee densitometry analysis Fig. 8bsee densitometry analysis Fig. 8csee densitometry analysis Fig. 8dsee densitometry analysis Fig. 8esee densitometry analysis Fig. 8fsee densitometry analysis Fig. 8gsee densitometry analysis Fig. 8hsee densitometry analysis Fig. During the inflammatory process, tissue permeability is modified in part by changes in tight junctions ,36. Sincdata not shown see densitometry analysis Fig. 9a,brespectively see densitometry analysis Fig. 10a,brespectively see densitometry analysis Fig. 11a,brespectively see densitometry analysis Fig. 12a,brespectively, see densitometry analysis Fig. 9cdrespectively), Claudin-4 , Claudin-5  and β-catenin  was observed in the lung tissues sections of carrageenan-treated WT mice at 4 h , Claudin-4 , Claudin-5  and β-catenin  at 4 h , Claudin-4 , Claudin-5  and β-catenin  at 4 h , are conditions characterized by a diffuse, intense inflammatory process, by damage to both endothelial and epithelial cell barriers, resulting in marked extravasation of vascular fluid . The filRecently, we have demonstrated, using an experimental model of colitis, that the structure and function of TJs in the intestinal epithelial is dependent of the production of proinflammatory cytokines (e.g. TNF-α) . In partWhat is then the mechanism by which inhibition of TNF-α induced the structure and function of TJs alteration in acute inflammation caused by carrageenan?There is good evidence that TNF-α help to propagate the extension of a local or systemic inflammatory process ,47. We cMoreover various study have clearly point out that the immune system plays an important role in modulating epithelial permeability in different organs such us gut and lung ,49. The In the present study, we also demonstrate that carrageenan administration to mice induced an alteration of immunohistochemical localization signal for ZO-1, Claudin-2, Claudin-4, Claudin-5 and β-catenin at 4 h and 24 h after carrageenan. The genetic or pharmacological inhibition of TNF-α abolished the alteration of tight junction permeability. Thus, a key step in the pathogenesis of acute lung injury may be myosin light chain kinase activation by TNFα, leading to epithelial barrier dysfunction. There is extensive literature examining the consequences of specific proinflammatory cytokines, particularly tumor necrosis factor (TNF)-α, on the induction of apoptosis. An important biologic effect of TNF-α is the induction of apoptosis through its interaction with its receptor, TNFR. However, the same TNF-TNFR ligand-receptor interaction can also activate NF-κB . Activatin vivo that the pharmacological inhibition of TNF-α by Etanercept attenuates the development of TJ alteration in acute inflammation in mice.Further studies are needed to address this point. Thus, we propose the following cycle: Inflammation -> TNF-α-> endothelial/epithelial TJ alteration -> PMN infiltration -> more pro-inflammatory mediator release -> organ damage. The confirmation of this proposed feedback cycle, however, requires further investigation. In conclusion, we have demonstrated


TNF release was suppressed but IL-6 production was stimulated. On the other hand, serum levels of TNF were markedly increased over a period of 3 weeks. It was also demonstrated that the postvaccinal cytokine production disturbances may be corrected by pretreatment with a new synthetic hexapeptid (Imunofan®). It is possible that the imunofan treatment could prevent some postvaccinal complications.The effect of vaccination with diphtheria toxoid (AD-M) on TNF and IL-6 production has been studied in humans. In the present study it was demonstrated that immunization with AD-M resulted in changes of


GR number and affinity of MNC were determined by means of a whole cell DEX binding assay. MNC were incubated with DEX and LPS or A23187 in the absence or presence of RU486, a potent steroid antagonist. DEX caused a concentration dependent inhibition of TNF-∝, IL-6 and PGE2 production but had no effect on LTB4 production. RU486 significantly blocked the effect of DEX, but no correlations were found between the inhibition of mediator release and the Kd or receptor number.In order to find an explanation for corticosteroid resistance we assessed whether inhibition by dexamethasone (DEX) of the stimulated production of TNF-∝, IL-6, PGE