Tumor necrosis factor (TNF) is a critical cytokine in the immune response whose transcriptional activation is controlled by a proximal promoter region that is highly conserved in mammals and, in particular, primates. Specific single nucleotide polymorphisms (SNPs) upstream of the proximal human TNF promoter have been identified, which are markers of human ancestry.Using a comparative genomics approach we show that certain fixed genetic differences in the TNF promoter serve as markers of primate speciation. We also demonstrate that distinct alleles of most human TNF promoter SNPs are identical to fixed nucleotides in primate TNF promoters. Furthermore, we identify fixed genetic differences within the proximal TNF promoters of Asian apes that do not occur in African ape or human TNF promoters. Strikingly, protein-DNA binding assays and gene reporter assays comparing these Asian ape TNF promoters to African ape and human TNF promoters demonstrate that, unlike the fixed differences that we define that are associated with primate phylogeny, these Asian ape-specific fixed differences impair transcription factor binding at an Sp1 site and decrease TNF transcription induced by bacterial stimulation of macrophages.Here, we have presented the broadest interspecies comparison of a regulatory region of an innate immune response gene to date. We have characterized nucleotide positions in Asian ape TNF promoters that underlie functional changes in cell type- and stimulus-specific activation of the TNF gene. We have also identified ancestral TNF promoter nucleotide states in the primate lineage that correspond to human SNP alleles. These findings may reflect evolution of Asian and African apes under a distinct set of infectious disease pressures involving the innate immune response and TNF. The gene encoding tumor necrosis factor (TNF), a cytokine that plays a critical role in lymphocyte biology and in the innate and adaptive immune response, is transcribed rapidly and to high levels in response to multiple stimuli. This process is directed by the formation of inducer-specific nucleoprotein complexes, known as enhanceosomes, over a region of approximately 200 base pairs upstream of the start site of transcription The human TNF gene is located within the most polymorphic region of the human genome, the major histocompatibility complex (MHC) region of chromosome 6, which includes the human leukocyte antigen (HLA) genes Since TNF promoter SNPs were first described, multiple laboratories have studied their association with susceptibility to autoimmune and infectious diseases. Although statistical associations with a variety of diseases have been found in some studies, there has been no consistent functional role demonstrated for any TNF promoter SNP in the regulation of the TNF gene itself Pan paniscus), chimpanzee (Pan troglodytes), gorilla (Gorilla), and orangutan (Pongo)], all four genera of gibbon (Hylobatidae), and multiple species of Old World monkeys (OWM) and New World monkeys (NWM)  .Using a comparative genomics approach, we found a total of 332 fixed differences from the consensus human TNF promoter sequence in the primate species examined. We show that 41 of these fixed genetic differences serve as markers of primate species and subspecies. Strikingly, the monoallelic or biallelic variants carried in multiple human TNF promoter SNPs match divergent sequences in the primate lineage. In fact, the rare alleles found in two human SNPs (−1030 T/C and −375 G/A) are identical to TNF promoter sequences found at the same position in the New World monkey clade, which diverged from the common ancestor of other primates roughly 35 million years ago.Mycobacterium tuberculosis (MTb) stimulation, but that this site is not required for human TNF gene regulation in T cells stimulated via antigen receptor engagement or calcium influx We also identified fixed differences unique to TNF promoters from the Asian apes in an Sp1 site that lies −173 to −163 nt relative to the start site of transcription of the human TNF promoter. We have previously shown that this Sp1 site is involved in the regulation of the human TNF gene in macrophages in response to stimulation with lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria, or to Remarkably, here we show that these Asian ape-specific fixed genetic differences impair binding of Sp1 and Sp3 proteins to the site and decrease the relative level of TNF gene transcription in response to LPS and MTb as compared to the human or African ape TNF promoters. By contrast, these changes have no impact on the regulation of the gene in T cells by calcium influx, consistent with previous studies of the effects of specific mutations in this site in stimulus-specific regulation of the human TNF gene promoter. Thus, we provide evidence for adaptive changes in an innate immune response gene correlated with the current geographic distribution of higher primates. Furthermore, we demonstrate that fixed nucleotide differences and polymorphisms in the TNF promoter serve as markers of primate ancestry and biodiversity.Pan paniscus); the chimpanzee (Pan troglodytes), including the four subspecies of chimpanzee; the two species of gorilla, the western (Gorilla gorilla) and the eastern (Gorilla beringei) gorilla; the two species of orangutan, the Bornean (Pongo pygmaeus) and the Sumatran (Pongo abelii) orangutan; and seven gibbon species representing all four genera of gibbon . We also obtained samples from several species of African and Asian OWM and NWM  and NWM  clades, similar to the OWM and NWM groups observed in the analysis of genetic distances based on the promoter or open reading frame of the CC chemokine receptor 5 (CCR5) gene Papio, Macaca, Cercocebus, Saimiri, and Aotus genera Papio-Macaca and Saimiri-Aotus clades in a Kimura 2-parameter analysis of genetic distances Approximately 1.2 kb of the TNF promoter was sequenced for each sample, and the sequences corresponding to positions −1153 to +69 nt relative to the start site of transcription in the human TNF promoter were aligned  and analIn general, the majority of gene sequences previously analyzed in hominids supports a chimpanzee-human clade, although a smaller proportion of the analyzed genes is consistent with a chimpanzee-gorilla or human-gorilla clade Chlorocebus pygerythrus) and the −98 T/A transversion in the white-headed capuchin  in these primate TNF promoters relative to the human sequence . The fixFunctional elements in mammalian gene promoters can be identified by high conservation between species distantly related on the evolutionary scale, particularly for genes with conserved function across different taxa Pongo pygmaeus from Pongo abelii: the cytosine residues present at positions −879 and −861 in Pongo pygmaeus and humans are a thymine and a single nucleotide deletion, respectively, in Pongo abelii. In gorillas, five fixed differences at positions −1112, −770, −521, −518, and −444 distinguish three subspecies within the genus Gorilla: the western lowland gorilla (G. g. gorilla) is marked by −770 T/C and −518 C/T; the eastern lowland gorilla (G. beringei graueri), by −770 T/C; and the mountain gorilla (G. b. beringei), by −1112 G/T, −521 C/T, −518 C/T, and −444 C/A. We note that we only have one representative of G. beringei graueri and G. b. beringei because they are critically endangered species; thus, we formally cannot rule out that these sequence differences represent SNPs. A number of SNPs were identified for Pan troglodytes, but no fixed differences occurred among the four chimpanzee subspecies , indicating that the fixation of the adenine at position −862 correlates with the divergence of the Cebidae from common primate ancestors of the NWM approximately 16–20 million years ago As shown in Symphalangus syndactylus. Four clades of gibbons, recently designated as genera and distinguished by distinct diploid chromosome number, are well established: Hylobates, Hoolock, Symphalangus, and NomascusHylobates diverged most recently, comparative genomics analysis of nuclear and mitochondrial DNA sequences has yielded conflicting models of the phylogenetic relationships among the genera  bear a cytosine, the rare allele in humans, as a fixed difference at position −1030 in the TNF promoter, while Pan pansicus and Pan troglodytes bear an adenine at position −1030. Thus, the Pan-specific cytosine to adenine transversion at position −1030 appears to coincide with the divergence between the human-chimpanzee clade and gorilla clade approximately 6–9 million years ago In the case of the −1030 T/C human SNP, all primates except for the Hominini tribe  involved a CpG dinucleotide Finally, one human TNF promoter SNP, −856 C/T, is also polymorphic in Fixed differences within or near the Sp1 site of the human TNF promoter (5′-CCCCGCCCCC-3′) at −172 to −163 bp relative to the start site of transcription are observed in representatives of the Asian apes (Pongidae and Hylobatidae) and NWM, but not in African great apes and OWM . This siPongo pygmaeus, Pongo abelii, and Nomascus l. leucogenys strongly interferes with the constitutive binding of Sp1 and Sp3 in T cells and monocytic cells as compared to the binding of these proteins to the human Sp1 site sequence. By contrast, the single −161 C/T transition found in Hylobates lar, Hoolock leuconedys, and Symphalangus syndactylus and the single −164 C/T transition found in Hylobates moloch, Hylobates agilis, and Hylobates pileatus, which are outside of the predicted core of the Sp1 site To test this hypothesis, we first assayed the ability of these divergent Asian ape Sp1 sites to bind Sp1 proteins by performing electrophoretic mobility shift assays (EMSAs) with nuclear extracts from murine and human T cell lines and a monocytic murine cell line. For this analysis we used oligonucleotides matching the upstream Sp1 site of the two orangutan species and seven gibbon species that represented all four genera of gibbon as probes and compared their binding to that of an oligonucleotide matching the human upstream Sp1 site . Using sPongo, Hylobates moloch, and Nomascus l. leucogenys. We also prepared a TNF-luciferase reporter from Gorilla, which has an Sp1 site that is identical to the human sequence but which varies from the human proximal promoter sequence at position −9, and compared the activation of all of these reporters to an isogenic human TNF reporter construct Pongo and Nomascus TNF promoters was impaired up to 60% relative to the human promoter in MTb-infected or LPS-treated monocytes but was not affected in T cells treated with ionomycin  revealed no fixed differences between Several TNF promoter SNPs are present in extended haplotypes that mark human ethnic populations and human ancestry A number of scenarios could explain why specific alleles of human TNF promoter SNPs are identical to fixed nucleotides at corresponding positions in primate TNF promoters. Moreover, different scenarios could apply to specific nucleotide positions. First, the variant SNP allele could represent the ancestral state by descent. The allele could have reached fixation in the human population due to genetic drift, or it might have appeared through recent positive selection. Second, the variant SNP allele could represent recent homoplasy, either through selection due to certain constraints on promoter sequence patterns or due to linkage with another genetic region, such as HLA.Regardless of whether these variant human alleles are identical to divergent sequences in the primate lineage by descent or are identical by state, all of the nucleotide positions of these SNPs fall outside the core region of the TNF promoter known to be critical for TNF gene regulation. This observation is consistent with the accumulation of mutations under neutral evolution, which in turn may reflect linkage with other genes that have been positively selected Pan paniscus, Pan troglodytes, and Gorilla). Fixed genetic differences are present in Asian apes (5′-CCCCACCCC-3′ in both Pongo species and 5′-CCCCACCTC-3′ in Nomascus l. leucogenys) that impair binding of Sp1 proteins to the site and the transcriptional activity of the TNF gene in a cell- and inducer- specific fashion. This recapitulates the regulation of the human TNF promoter Most notably, in addition to these fixed genetic differences outside of the proximal TNF promoter, we have identified fixed genetic differences in primate TNF promoters with direct impact upon TNF gene transcription. These occur within a region corresponding to the upstream Sp1 site (−173 to −163 relative to the start site of transcription) of the human TNF promoter (5′-CCCCGCCCCC-3′), which is completely conserved in the African apes (Hylobates genus (see Trachypithecus francoisi) and the red-shanked douc (Pygathrix nemaeus)], the upstream Sp1 site is identical to the human sequence  of monocytic cells, rather than activation of T cells, became fixed in the gibbon-orangutan and the African great ape clades.For orangutans and gibbons, the considerable geographic flux of the Malay Archipelago region of southeast Asia over millions of years, alternately isolating and reuniting local animal populations in the course of their adaptation and speciation Homo neanderthalensisGorilla beringei beringei, Gorilla beringei graueri, and Pongo abelii are among the 25 most endangered primate species in the world As genomic sequence data is acquired for different primate species, including data for extinct species such as Pan troglodytes troglodytes, AF102683; P. t. schweinfurthii, AF102687; P. t. verus, AF315499; P. t. vellerosus, AF315498) on a phylogenetic tree using maximum likelihood methods with PHYLIP 3.65 package Cell lines and samples of blood or DNA from representative individuals were obtained for the primate species and subspecies as described in Genomic DNA was isolated from whole blood using the QIAamp DNA Blood kit (Qiagen). The TNF promoter was amplified using the following primer sets: for human, apes, and OWM, TNF-F1 (5′-AGT GAG AAC TTC CCA GTC TAT CTA AG-3′) and TNF-R1 (5′-CCG TGG GTC AGT ATG TGA GA-3′) Using PCR, we amplified primate TNF promoters from −200 to +87 nt relative to the transcription start site and the NheI-BglII restriction fragment was subcloned into pGL3 Basic (Promega) and sequenced. Constructs for transient transfection were thus isogenic to the human −200 TNF-Luc reporter 2, in RPMI 1640 (Gibco) supplemented with penicillin, streptomycin, 10% fetal bovine serum, and 2mM L-glutamine. Transfections were performed using DEAE-Dextran as described 2 in Dulbecco's MEM media (Gibco) suplemented with penicillin, streptomycin, 10% fetal bovine serum. Transfections were performed using FuGene6 (Roche) using a 3∶2 ratio (pGL3: pR-TK Renilla) according to the manufacturer's protocol. Eight hours after transfection, cells were treated with 2 µg/ml of LPS  or 5 µg/ml of H37Rv whole sonicate of M. tuberculosis  and harvested 16 hours later. Luciferase assays were performed according to the manufacturer's instructions  using a Dynex luminometer, with Renilla luciferace (pR-TK) as an internal control.The Jurkat T cell line (ATCC) and T cell hybridoma 68-41 were maintained at 37°C, 5% COM. tuberculosis as described above. Nuclear extracts were prepared, and electrophoretic mobility shift assays were performed, as previously described Gorilla, 5′-AAA TCC CCG CCC CCG CGA TGG-3′ and 5′-CCA TCG CGG GGG CGG GGA TTT-3′; Pongo, 5′-AAA TCC CCA CCC CCG TGA TGG-3′ and 5′-CCA TCA CGG GGG TGG GGA TTT-3′; Hylobates moloch, 5′-AAA TCC CCG CCC CCG TGA TGG-3′ and 5′-CCA TCA CGG GGG CGG GGA TTT-3′; Hoolock leuconedys, 5′-AAA TCC CCG CCC TCG CGA TGG-3′ and 5′-CCA TCG CGA GGG CGG GGA TTT-3′; Nomascus l. leucogenys, 5′-AAA TCC CCA CCC TCG CGA TGG-3′ and 5′-CCA TCG CGA GGG TGG GGA TTT-3′.Nuclear extracts were prepared from Jurkat and 68-41 cell lines after stimulation for 2 hours with ionomycin, and from J774A.1 cells after stimulation for 2 hours with LPS or The sequences were aligned with the CLUSTALW program, version 1.7 Figure S1P. t. troglodytes is also shown. On the human TNF promoter sequence, positions of human SNPs and every hundredth base pair position are in boldface, and every tenth base pair position is underlined.Complete alignment of the TNF promoter sequences from primate species used in this study. Numbering corresponds to the human TNF promoter sequence, −1153 to +69 nt relative to the start site of transcription, shown at the top. Species and subspecies are indicated, along with nucleotide changes, conserved positions (.), and deletions (−). All alterations from the human sequence represent fixed differences, except positions in which SNPs were detected in 40% or more of the individuals examined, which are denoted R (A or G), Y (C or T), M (A or C), or S (C or G). The −1027 to −1029 deletion found as a SNP in (0.15 MB DOC)Click here for additional data file.Table S1Total fixed differences among the primate TNF promoters. Positions of single nucleotide changes and discrete insertions or deletions, relative to the consensus human TNF promoter sequence, observed in this study are shown.(0.04 MB PDF)Click here for additional data file.Table S2SNPs in primate TNF promoters. For each of the indicated species or subspecies, the position, sequence change, and frequency  of each SNP detected in the TNF promoter is shown. SNPs in which the wild type allele corresponds to the sequence found in the other subspecies of a given species are shown, while the mutant alleles that correspond to the human sequence are noted with an asterisk.(0.07 MB PDF)Click here for additional data file.Table S3Cebus capucinus and Saimiri sciureus; the Atelinae subfamily, Ateles geoffroyi and Lagothrix lagotricha. See Fixed differences in the TNF promoter that mark primate clades. Total fixed genetic differences in the TNF promoter that are unique to and that are completely conserved within the indicated primate taxa are shown. A. For each position, the sequence found within and outside of the indicated clades is shown. B. For each position, the sequence found in humans and in the indicated genus or clade is shown. The Cebidae family includes (0.03 MB PDF)Click here for additional data file.


In response to invasion by microbial pathogens, host defense mechanisms get activated by both the innate and adaptive arms of the immune responses. TNF (tumor necrosis factor) is a potent proinflammatory cytokine expressed by activated macrophages and lymphocytes that induces diverse cellular responses that can vary from apoptosis to the expression of genes involved in both early inflammatory and acquired immune responses. A wide spectrum of microbes has acquired elegant mechanisms to overcome or deflect the host responses mediated by TNF. For example, modulatory proteins encoded by multiple families of viruses can block TNF and TNF-mediated responses at multiple levels, such as the inhibition of the TNF ligand or its receptors, or by modulating key transduction molecules of the TNF signaling pathway. Bacteria, on the other hand, tend to modify TNF-mediated responses specifically by regulating components of the TNF signaling pathway. Investigation of these diverse strategies employed by viral and bacterial pathogens has significantly advanced our understanding of both host TNF responses and microbial pathogenesis. This review summarizes the diverse microbial strategies to regulate TNF and how such insights into TNF modulation could benefit the treatment of inflammatory or autoimmune diseases. Metazoans have developed a variety of reactive mechanisms to control invading pathogens. On the other hand, microbial invaders such as viruses, bacteria, and intracellular parasites have co-evolved with their hosts to counteract the innate and adaptive responses mounted by the host. Of the many host pathways activated by pathogen invasion, pro-inflammatory cytokines play particularly significant roles in orchestrating both the early and late host responses. TNF is one such pleiotropic pro-inflammatory cytokine that plays an important role in diverse host responses such as septic shock, induction of other cytokines, cell proliferation, differentiation, necrosis, and apoptosis. TNF is expressed as either a membrane-bound or secreted ligand mainly by activated macrophages, lymphocytes, natural killer cells, and epithelial cells. Three classes of TNFs have been identified: TNFα , lymphotoxin-α (LT-α), and LT-β, all of which are bioactive as trimers. A TNF protein superfamily that exhibits 15%–20% identity to each other now comprises at least 20 members .The TNFR superfamily members fall into three major groups, death domain (DD)-containing receptors, decoy receptors, and TNF receptor-associated factor (TRAF) binding receptors . DD-contTNF can induce either an NF-κB-mediated survival (and proinflammatory) pathway or an apoptotic response depending on the cellular context . TNFR1 iTNFR2 does not contain a cytoplasmic death domain and cannot directly engage the apoptotic machinery, and thus its precise involvement in TNF-mediated cell death is controversial. It can enhance the cell death signal of TNFR1, possibly through TRAF2 degradation and enhanced recruitment of FADD and RIP to TNFR1 . TNFR2 aThe TNF-induced NF-κB–mediated survival pathway can be activated independently by either TNFR1 or TNFR2 . In respListeria monocytogens, Mycobacterium tuberculosis, M. avium, Salmonella typhimurium, intracellular parasites such as Leishmania major or Trypanosoma cruzi, and viruses such as herpes simplex virus (HSV-1), mouse cytomegalovirus (MCMV), or lymphocytic choriomeningitis virus (LCMV)[TNF orchestrates powerful anti-microbial responses by a variety of mechanisms, including the direct killing of infected cells (cytolysis), induction of apoptosis, inhibition of intracellular pathogen replication, and upregulation of other diverse host responses. Using TNF or TNFRs-deficient mice, it has been demonstrated that they are essential for survival of infections with bacterial pathogens such as us (LCMV),4,13. TNus (LCMV),20. TablThe first-identified TNF inhibition strategy deployed by viruses was revealed by the discovery of encoded soluble TNFRs homologs which function by binding and sequestering extracellular TNF. Among these vTNFRs are the T2-like family members encoded by Leporipoxviruses and the cytokine response modifier (Crm) family members encoded by Orthopoxviruses ,22. The Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1) mimics a constitutively activated TNFR member, CD40. LMP1 is essential for EBV conversion of infected B lymphocytes into perpetually proliferating lymphoblasts and is expressed in EBV-associated lymphoproliferative disease, Hodgkin disease, and nasopharyngeal carcinoma . Two motSome viruses exploit TNFR family members as receptors to enter into cells. HSV-1 glycoprotein D (gD) interacts with HVEM (herpes virus entry mediator) to enter into resting T cells, monocytes, and immature dendritic cells . A TNFR A new class of TNF binding protein has been recently identified from Tanapox virus, a member of the Yatapoxvirus genus of poxviruses . This vTThe adenovirus early transcription region 3 (E3) encodes at least seven proteins, five of which block the acquired or innate immune response. Three of these, Ad E3–14.7K, Ad E3–10.4K, and Ad E3–14.5K, impose inhibitory effects on the TNF pathway . Two of The EBV-encoded immediate-early gene product BZLF1  prevents cellular responses to TNF, including TNF-induced cell death . During Small DNA viruses, such as human papillomaviruses (HPVs) are the major cause of cervical cancer (>90%) and a significant number of head and neck cancers . They inHuman and murine CMV have also developed mechanisms to evade a TNF-induced antiviral state by dysregulating TNFRs. HCMV infection of THP1 cells reduced the level of TNFR1 on the cell surface by accumulating the receptor pool in the trans-Golgi network . Time coThe hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a multifunctional phosphoprotein that utilizes multiple mechanisms to inhibit both extrinsic and intrinsic apoptotic stimuli . Using NActivation of caspases and release of cytochrome c from the mitochondria generally lead to the induction of apoptosis and cell death. A number of viral proteins have been shown to inhibit caspases or intervening at the mitochondrial checkpoint to prevent TNF-mediated apoptosis. Proteins that inhibit caspase activation include vIAP from ASFV, CrmA from poxvirus, vICA from HCMV, vFLIPs from several γ-herpesviruses, and its ortholog MC159 from MCV –69. One NF-κB is a critical regulator of the immediate early pathogen response, playing an important role in promoting inflammation, and in the regulation of cell proliferation, activation and survival . NF-κB tThe Tax transactivator oncoprotein of human T cell leukemia virus type I (HTLV-I) persistently activates NF-κB signaling pathways, resulting in the deregulation of cellular gene expression and immortalization of HTLV-I-infected T cells . Tax intViruses also can inhibit NF-κB activation by different methods, which can result in increased sensitivity to TNF-induced apoptosis. KSHV-encoded viral interferon regulatory factor 3 (vIRF3) inhibits the activation of NF-κB induced by TNF . In 293TInduction of apoptosis in HBV-infected hepatocytes is mediated by the viral X protein (HBx). HBx-dependent activation of p38MAP kinase and JNK pathways leads to the activation of both TNFR1/TNF and Fas/FasL . HBx alsBacterial mechanisms of inhibiting TNF-mediated responses differ significantly from that of viruses. To date, no bacterial or parasite protein has been reported that directly binds and inhibits TNF, TNFR, or their associated factors. Instead, they rely on indirect mechanisms to modulate TNF signaling, for example by reducing the synthesis of inflammatory mediators, cytokines, and the modulation of signaling pathways such as NF-κB or MAPK . Table 2Yersinia, and known as Yops (Yersinia outer proteins). Among the six Yop effector proteins, YopP/YopJ is involved in the inhibition of NF-κB and MAPK pathways, which results in the downregulation of cytokines such as TNF, chemokines, and adhesion molecules. This then inhibits the recruitment and activation of macrophages and natural killer cells to the site of infection [Yersinia enterocolitica also induces apoptosis in murine dendritic cells and inhibits TNF production [Yersinia pestis, Low calcium response V (LcrV) or V antigen and YopB inhibit production of TNF from murine peritoneal macrophages by inhibiting the transcription factor NF-κB after LPS treatment [Bacterial pathogens generally mediate their interactions with host cells via preformed soluble small-molecule or peptide effectors secreted by type III secretion system, a multicomponent translocation apparatus that spans the bacterial cell wall from the interior of the bacterium into the external environment . The besnfection . YopJ isnfection ,97. Receoduction . Two othreatment .Salmonella typhimurium, also demonstrates potent inhibitory action towards the NF-κB pathway but does not seem to affect MAPK activation [S. typhimurium, is also involved in host modulation involving the MAPK pathway by inhibiting Raf activation, which ultimately attenuates the secretion of TNF from infection-activated macrophages [An ortholog of YopJ, AvrA from tivation . Unlike rophages .Escherichia coli K1 outer membrane protein A (OmpA) in infected monocytes suppresses the production of cytokines such as TNF by inhibiting IκB phosphorylation and blocking the translocation of NF-κB to the nucleus [E. coli strains. The Shiga toxin-producing E. coli (STEC), enterohemorrhagic E. coli, and enteropathogenic E. coli all interfere with NF-κB–activation initiated by TNF. EspB (E. coli–secreted protein B), a component of the type III secretion system, is also involved in the inhibition of NF-κB activation and proinflammatory cytokine production [ nucleus . OmpA al nucleus . Suppresoduction .Neisseria gonorrhoeae, increases the transcription of several host antiapoptotic genes, including bfl-1, cox-2, and c-IAP-2, by the activation of NF-κB, and thus protects human urethral epithelial cells from apoptosis [Helicobacter pylori, the main causative agent of chronic active type B gastritis, enhances the expression of TNFR1 and Fas and induces apoptosis of gastric epithelial cells [H. pylori are required for NF-κB activation, which enhances the production of TNF and other pro-apoptotic cytokines [Listeria monocytogenes virulence protein Listeriolysin O (LLO) and Outer membrane protein (OMP) from Bartonella henselae both activate NF-κB and induce antiapoptotic signaling, which prolongs bacterial survival [Some bacterial proteins activate NF-κB, which inhibits TNF-mediated apoptosis by upregulation of antiapoptotic genes, or induces TNF production to enhance apoptosis. The outer membrane protein, porin, from poptosis . Infectial cells . Proteinytokines . Listerisurvival ,109.Staphylococcus aureus, a pathogen associated with pneumonia and sepsis. Activation of TNFR1 by protein A induces TNF-like responses which are associated with the pathogenesis of staphylococcal pneumonia [Neisseria gonorrhoeae is capable of inhibiting the TNF-mediated apoptosis of the human myelo-monocytic cell line U937 [Bacteria can also use host TNFRs to mediate pathogenicity. A recent study has demonstrated that TNFR1 is a receptor for protein A from neumonia . Extraceine U937 . This prTrypanosoma cruzi, Toxoplasma gondii, Theileria parva, and other pathogens such as Chlamydia pneumoniae, Plasmodium falciparum, and Rickettsia rickettsii all activate NF-κB and inhibit apoptosis to enhance parasite replication [Francisella tularensis, inhibits TNF secretion from the murine macrophage like cell line J774A.1 by blocking the degradation of IκB and inhibiting NF-κB [Trichinella spiralis can induce expression of TNF, TNFR1, TRADD, caspase-3, caspase-8, TRAF-2, and RIP in infected muscle cells, resulting in induction of either apoptosis or the transformation of muscle cells to nurse cells [Like virus and bacteria, parasites can also modulate NF-κB function and regulate host immune responses. They can either activate NF-κB by degrading the IκB or inhibit NF-κB activation by blocking the degradation of IκB. Protozoan parasites such as lication –117. Reclication –120. Degng NF-κB . It has se cells .Although TNF plays a major role in growth regulation, cell differentiation, and response to microbial infections, its inappropriate overexpression has been implicated in the pathogenesis of a wide spectrum of human disorders, such as autoimmunity , allergy, septic shock, allograft rejection, and insulin resistance. TNF derived from mast cells also plays a crucial role in initiation of inflammation, particularly in the case of rheumatoid arthritis . TNF mayMany approaches have been investigated to inhibit TNF activity for the treatment of various inflammatory/autoimmune diseases . The currently commercially available TNF antagonists are infliximab , etanercept (a soluble fusion protein combining two p75 TNFRs with an Fc fragment of human IgG1), and adalimumab . Although they have shown to be partially effective in clinical trails, still more needs to be learned in terms of the biology of TNF. These current inhibitors need to be delivered at high doses, and some adverse events have been reported, so that the long-term safety of all these molecules is not thoroughly understood . The inv


Particulate air pollution has been associated with lung and cardiovascular disease, for which lung inflammation may be a driving mechanism. The pro-inflammatory cytokine, tumor necrosis factor (TNF) has been suggested to have a key-role in particle-induced inflammation.Tnf-/- mice after exposure to diesel exhaust particles (DEPs). Mice were exposed to either a single or multiple doses of DEP by inhalation. We measured the mRNA level of the cytokines Tnf and interleukin-6 (Il-6) and the chemokines, monocyte chemoattractant protein (Mcp-1), macrophage inflammatory protein-2 (Mip-2) and keratinocyte derived chemokine (Kc) in the lung tissue at different time points after exposure.We studied the time course of gene expression of inflammatory markers in the lungs of wild type mice and Tnf mRNA expression levels increased late after DEP-inhalation, whereas the expression levels of Il-6, Mcp-1 and Kc increased early. The expression of Mip-2 was independent of TNF if the dose was above a certain level. The expression levels of the cytokines Kc, Mcp-1 and Il-6, were increased in the absence of TNF.Tnf is not important in early DEP induced inflammation and rather exerts negative influence on Mcp-1 and Kc mRNA levels. This suggests that other signalling pathways are important, a candidate being one involving Mcp-1.Our data demonstrate that  When deposited in the lung, ultra-fine particles, i.e. particles with a diameter less than 100 nm, are probably more dangerous than large particles. The smaller the particles are, the greater surface area they have per mass unit: This implies that they have a greater chemical activity, can carry more PAH and other adsorbed chemicals, and they react more strongly with the lung tissue, thereby causing more inflammation than larger particles [Il-6) has been reported to be increased in particle-induced inflammation [Several cytokines have been suggested to be involved in particle-induced inflammation. Focus has primarily been directed to the cytokines tumor necrosis factor (TNF) and macrophage inflammatory protein , which have been suggested to play an important role in particle-induced inflammation in the lung . MIP-2 aammation ,8.Tnf -/- and Tnf +/+ mice [Tnf and other inflammatory markers after exposure and 2) by investigating whether Tnf deficiency induces a change in the time course of inflammation induced by DEP. We evaluated the inflammatory response at the mRNA level, because cytokine expression seems to be regulated at the transcriptional level and by mRNA stability. Furthermore mRNA can conveniently be quantified in tissues. It has been shown that DEP up-regulates several genes involved in the inflammatory response, at both the mRNA and protein level in rats [We recently reported from an inhalation study, that TNF was not required for DEP-induced inflammation when we evaluated the inflammatory effects after four repeated inhalation exposures in +/+ mice . The aim in rats .Tnf, Il-6, Mip-2, Kc and Mcp-1) in the lung tissue from BALBcJ (study 1) and C57xCBA mice (study 2) exposed to a single dose of DEP by inhalation at different time points. Study 1 consists of experiments with mice that we have reported of previously [To evaluate the time course of particle-induced inflammation, we measured the expression of five cytokines  1 day later , whereas the expression level of Tnf was the same as in the air-exposed mice in the time-period from 1–6 hours after exposure  and 1 day  after exposure, whereas no change in expression levels was observed in the mice exposed to 20 mg/m3 at any time point   and 1 day (p < 0.05) after exposure (Table Mcp-1 was increased in the BALBcJ mice exposed to 80 mg/m3 both 1 hour (7-fold) and 1 day (9-fold) after exposure , whereas no increase in expression level was seen at any time point in mice exposed to 20 mg/m3  or multiple doses of 20 mg/m3 SRM2975 (study 4). Study 3 is a new animal experiment, whereas we have reported of the animal experiments in study 4 before [In a second set of experiments, we investigated the specific effect of TNF. This was done by comparing the inflammatory response in Tnf, Il-6, Mip-2, Kc and Mcp-1 in the lungs of the Tnf+/+ and Tnf-/- mice at 1 hour, 1 and 2 days after end of exposure to a single dose of 20 mg /m3 SRM2975. In addition, we assessed the recruitment of inflammatory cells into the lung lumen. We chose to evaluate the time course after a single dose of 20 mg/m3 SRM2975 in Tnf+/+ and Tnf-/- mice on the background of our previous results, that showed that Il-6 expression was significantly increased in BALB/CJ mice exposed to a single dose of 20 mg/m3 SRM1650 [Tnf proficient and deficient mice are summarized in table We studied the time course of inflammation by measuring the expression levels of the cytokines  SRM1650 . The resTnf+/+ mice the fraction of neutrophils in the BAL cells was strongly increased 1  and 2 days after exposure   after exposure compared to the air exposed mice. The Tnf-/- mice did not respond to the DEP exposure with a higher Mip-2 expression.In the Tnf deficient and proficient mice to repeated DEP doses by measuring the expression levels of Mip-2, Kc and Mcp-1 in the lung tissue from mice exposed to four bouts of inhalation of 20 mg/m3 SRM2975 . The expression level of Kc was significantly increased in the Tnf -/- mice (p < 0.01), but there was no increase of expression level in the Tnf+/+ mice. The Mcp-1 expression levels were increased 5-fold in the Tnf deficient mice (p < 0.01), and 1.5-fold in the Tnf+/+ mice although the latter was not statistically significant. In our recent publication [Tnf and Il-6 mRNAs in the lung tissue and determined the fraction of neutrophils in the BAL cells. Results from this and the present study are summarized in table We studied the inflammatory response in 75 Table . In bothTnf+/+ mice and Tnf-/- mice to DEP by inhalation, and showed that TNF is not required for DEP-induced inflammation [Mip-2, independently of Tnf status, whereas enhanced Kc expression preceded Mip-2 expression only in Tnf-/- mice, which suggests that Kc may compensate for the deficiency of Tnf, or that TNF exerts negative feedback on KC signalling. The DEP-induced expression of Mcp-1 and Il-6 occurred in the absence of Tnf. Our results indicate that other signalling pathways, perhaps involving MCP-1, are important for early DEP induced neutrophilic inflammation.It has been suggested that TNF is a key-player in particle-induced inflammation , and thaammation . In the Il-6, Mip-2, Kc and Mcp-1, in the early and late phase of the DEP-induced inflammatory process. The results from the BALBcJ [Il-6 is expressed early in inflammation (1–6 hours after exposure) and that the expression of Il-6 has declined a day after exposure, although it is also elevated after repeated exposures.Thus, we evaluated other signalling patways with the expression of four additional cytokines, e BALBcJ  and the Mip-2 and Kc are strongly coupled to the influx of neutrophils in the rodent lung [3) independently of mouse strain or type of DEP. Mip-2 levels increased further 1 day after exposure and this also included the low dose of SRM2975, whereas a further increase in Mcp-1 mRNA levels only occurred after SRM1650 in BALBcJ mice and not with SRM2975. The mRNA levels of Kc were maximal at 1 hour after exposure to SRM1650 and even showed a decrease 3 hours after exposure to 20 mg/m3, although there was a slight further increase 1 day after exposure to 80 mg/m3 SRM2974. Only the Kc level was significantly increased 1 hour after the low dose of 20 mg/m3 SRM1650 in BALBcJ mice. Accordingly, Il-6, Mip-2, Mcp-1 and Kc are cytokines induced in the early phase of DEP-induced inflammation, while Tnf is expressed late at about a day after exposure. Mip-2 may have a more important role in the later phase as judged by the further increased expression 1 day after exposure. The results may be consistent with that DEP activates Toll like receptor 4 (TLR4) signalling because the Tnf response occurs late in this pathway. In fact, there are indications that air pollution particles stimulate the TLR4 pathway [de novo synthesis, secretion, and autocrine signalling by TNF over the TNF receptors [Mcp-1 and Mip-2, but no change in Tnf, was found in BAL cells isolated from DEP (SRM2975) exposed rats [The chemokines ent lung , whereasent lung . All of  pathway -12. Bacteceptors ,14. Moresed rats . HoweverIl-6 was upregulated upon repeated DEP exposure both in Tnf+/+ and Tnf-/- mice [Il-6 expression level was higher 1 hour after exposure in both Tnf+/+ mice and Tnf-/- mice after a single dose exposure.As we have previously reported, -/- mice . This mi-/- mice . AlthougTnf+/+ and Tnf-/- mice responded with an increase in Mip-2 response in the lungs. The Mip-2 induction was similar in Tnf+/+ and Tnf-/- mice. This is surprising because MIP-2 was believed to be an inflammatory marker downstream of TNF [Mip-2 was increased in the Tnf+/+ mice 1 day after exposure and borderline significant 2 days after exposure. The Mip-2 expression tended to correlate with the influx of neutrophils, because the influx of neutrophils was statistically significant 1 day and 2 days after exposure. Similarly, TNF receptor 1 deficient mice had an intact neutrophil infiltration after pulmonary exposure to silica [Mip-2 mRNA levels was seen in the single dose exposed Tnf -/- mice at any time: Possibly, there is a threshold for the induction of Mip-2 in the Tnf -/- mice, because the repeated exposure in the Tnf deficient mice resulted in a Mip-2 response. In the repeated dose experiment, the expression of Kc was only increased in the Tnf -/- mice, which together with the results from the BALBcJ and C57xCBA mice suggests that TNF exerts an inhibitory effect on Kc expression.In the repeated dose TNF study, both the m of TNF . MIP-2 ho silica . No incrKc increased earlier than Mip-2. This is consistent with a recent report that CINC (KC) apparently functions as a priming signal to the neutrophils, which afterwards are attracted to the more potent but local chemoattractant MIP-2 in the lung [Mip-2 and Kc are increased in the absence of Tnf. Both Mcp-1 and Kc mRNA levels were induced to lesser extent by DEP in the Tnf+/+ than in the Tnf-/- mice. This might indicate TNF exerts negative feedback on these cytokines.MIP-2 and KC are both chemotactic for neutrophils and have been suggested to be redundant . MIP-2 hthe lung . Our resTnf-/- mice had intact LPS induction of Mcp-1 expression levels [Recently, it was reported that primary embryonic fibroblasts from n levels . Knock on levels -11. ThisIn one of our experiments we used the SRM1650 material which is particulate matter collected from a heavy-duty diesel engine. Because this is not available any more, we used SRM2975 in the rest. SRM2975 is a DEP preparation collected from a diesel-powered industrial forklift. The DEP materials differ in their chemical composition. E.g., SRM 1650 contain a higher amount of metals and PAHs than SRM 2975 ,22. AlthThese experiments were performed using several different mouse strains. There were minor differences in expression patterns, which may be ascribed to strain or DEP material effects. The overall picture was consistent across mouse strains, indicating that the observed effects are not strain specific.Mip-2, that was independent of Tnf status, if the dose was above a certain level. Kc expression seemed to precede the Mip-2 expression and Kc was only expressed at a higher level in the DEP-exposed Tnf-/- mice, which suggests that KC may compensate for the deficiency of TNF, or that TNF exerts negative feedback on KC signalling. The DEP-induced expression of Mcp-1 and Il-6 was increased in the absence of Tnf. Our results indicate that TNF signalling is not important for early DEP induced neutrophilic inflammation. Attention should be focused on other pathways, a possible candidate being one involving MCP-1.In summary, we have shown that TNF is not important in early DEP-induced inflammation. Exposure to DEP by inhalation caused an increased expression level of Tnf-/- mice  were obtained from JAX Mice (USA). C57 BL/6J mice were used as Tnf+/+ mice. C57 BL/6J and BALBcJ mice were purchased from Taconic Europe, Denmark. C57xCBA mice that express luciferase under the control of NFκB [Tnf-/- and Tnf+/+ mice were studied at 9–11 weeks of age. Both male and female C57xCBA mice were studied at 7–8 weeks of age. Female BALBcJ mice were studied at 8 weeks of age. of NFκB  were obtDEP were Standard Reference Material 2975 and 1650 from the National Institute of Standards and Technology (NIST). SRM1650 are combustion particles from a heavy duty diesel engine. SRM2975 are DEP collected from a diesel-powered industrial forklift. A detailed description of the particles can be viewed at .3 SRM1650, 2) a single dose exposure of C57xCBA mice to 80 mg/m3 SRM2975, 3) a single dose exposure of Tnf-/- mice and Tnf+/+ mice to 20 mg/m3 SRM2975, and 4) four repeated exposures of Tnf-/- mice and Tnf+/+ mice to 20 mg/m3 SRM2975. In all four parts, the mice were exposed by inhalation for 90 minutes either one single time  or repeated on each of four consecutive days (study 4) in an 18 l nose-only exposure chamber.The study consists of four parts: 1) a single exposure of BALBcJ mice to 20 or 80 mg/m3 or 80 mg/m3 SRM1650 and the endpoints were analyzed 1 hour, 3 hours or 1 day (22 hours) after exposure.1) The design of the single dose exposure of BALBcJ mice has been described elsewhere . In summ3 SRM2975. Endpoints were analyzed at different time points.2) C57xCBA mice were exposed to a single dose of 80 mg/m3 SRM 2975 at similar conditions as the repeated exposure. Endpoints were analyzed 1 hour, 1 and 2 days after end of last exposure. Study 2 and our previous studies show that the levels of expression of inflammatory cytokines are unchanged over time in the air-exposed mice [3) In the third study, the mice were exposed a single time to 20 mg/msed mice . On thatTnf-/- mice and Tnf+/+ mice by inhalation to 20 mg/m3 DEP or filtered air for 90 min on each of four consecutive days. One hour after the last inhalation the mice were killed and organs were snap frozen in liquid nitrogen and stored at -80°C. Endpoints were analyzed.4) The design of the repeated exposure has been described in detail elsewhere . In brie5/cm3 and 3.0·106/cm3, respectively. Particle numbers in the multiple-dose TNF study and the BALBcJ study are described elsewhere [3 and 80 mg/m3, respectively.The particles were aerosolized by a microfeeder with dispersion nozzle . The number of particles was measured by a condensation particle counter (TSI model 3022A). The numbers of particles in the single dose TNF study and C57xCBA study were about 9.1·10lsewhere . The parAfter exposure, tissue and BAL cells from the mice were for all studies prepared as described previously ,8.® RNA Blood Mini Kit  as described by the manufacturer. cDNA was prepared from DNase treated RNA using TaqMan reverse transcription reagents  as described by manufacturer.RNA from the multiple dose experiment was prepared using NucleoSpin 96 RNA kit (Macherey-Nagel) as described previously . RNA froTnf, Mip-2, Kc, Il-6 and Mcp-1 gene expression was determined using real-time RT-PCR with 18S RNA as the reference gene. Each sample was run in triplicate on the ABI PRISM 7700 sequence detector . For TNF, IL6 and MCP-1, TaqMan pre-developed reaction kits  were used. Unfortunately, the TaqMan pre-developed reaction kit for determination of TNF, we previously used [Tnf are approximately 5-fold higher when using the new kit. For MIP-2 and KC, probes and primers were designed as described previously [2 concentration was adjusted to 3.0 mM. Target and 18S RNA levels were quantified in separate tubes. The target genes were normalized (ΔCt) to 18S RNA by subtracting the cycle threshold value of 18S RNA (Ctreference) from the Ct value of the gene of interest (Cttarget), i.e. ΔCt = Cttarget - Ctreference. The relative expression of the target gene was calculated by the comparative method 2-ΔCt[The eviously . Howevereviously . This wahod 2-ΔCt. The aveData from the study 1, 3 and 4 were analyzed by two-way analysis of variance. Data from study 2 were analyzed by one-way analysis of variance. To fulfil the criteria for normality and variance homogeneity some of the markers were logarithmically transformed once or twice. For some markers, the data were ranked before a nonparametric test, because it was not possible to transform the data in a way that satisfied the criteria for normality and variance homogeneity. To determine the statistical significance of biomarkers, pairs of interest were compared with Tukey's Studentized range test with control of type 1 experimental error. In study 3, one mouse was excluded as an outlier because the data points for this mouse were more than 2 standard deviations from the rest of the data for all endpoints except one. In study 2 the air-exposed mice were grouped. Statistical analysis was performed with the SAS 8.2 statistical program.The author(s) declare that they have no competing interests.ATS carried out the real-time RT analysis, performed the statistical analysis and drafted the manuscript. NRJ, JBO, SLK, LR and MD carried out the exposure of the mice. ATS, SL, UBV and HWA conceived of the study and participated in its design and coordination. HWA, UBV and SL helped to draft the manuscript. All authors read and approved the final manuscript.


Intratumoral induction of tumour necrosis factor (TNF) by administration of Bordetella pertussis vaccine (BPV) as compared with that by the agent OK-432 was investigated in mice. Two hours after such administration tumour tissues tested were resected from the mice, homogenised, and the TNF activities in the homogenate were assayed using a L-929 fibroblast assay. Intravenous injection of BPV into mice bearing the MM46 carcinoma resulted in a greater concentration of TNF in the tumour homogenate than in the serum. With OK-432, however, there was a greater concentration of TNF in the serum than in the tumour homogenates. A high level of intratumoral TNF induction by BPV was also observed in mice bearing Meth A fibrosarcoma or Lewis lung carcinoma. The therapeutic effect against the Meth A fibrosarcoma was in parallel with the intratumoral TNF activity. Intratumoral TNF activity is therefore believed to be a good index of therapeutic effect.


Fibrosis, the replacement of functional tissue with excessive fibrous tissue, can occur in all the main tissues and organ systems, resulting in various pathological disorders. Idiopathic Pulmonary Fibrosis is a prototype fibrotic disease involving abnormal wound healing in response to multiple sites of ongoing alveolar epithelial injury.To decipher the role of TNF and TNF-mediated inflammation in the development of fibrosis, we have utilized the bleomycin-induced animal model of Pulmonary Fibrosis and a series of genetically modified mice lacking components of TNF signaling. Transmembrane TNF expression is shown to be sufficient to elicit an inflammatory response, but inadequate for the transition to the fibrotic phase of the disease. Soluble TNF expression is shown to be crucial for lymphocyte recruitment, a prerequisite for TGF-b1 expression and the development of fibrotic lesions. Moreover, through a series of bone marrow transfers, the necessary TNF expression is shown to originate from the non-hematopoietic compartment further localized in apoptosing epithelial cells.These results suggest a primary detrimental role of soluble TNF in the pathologic cascade, separating it from the beneficial role of transmembrane TNF, and indicate the importance of assessing the efficacy of soluble TNF antagonists in the treatment of Idiopathic Pulmonary Fibrosis. Fibroproliferative diseases are among the leading causes of morbidity and mortality worldwide. Fibrosis, the replacement of functional tissue with excessive fibrous tissue, can occur in all the main tissues and organ systems resulting in various disorders including cardiac, cerebral and peripheral vascular disease, liver cirrhosis, progressive kidney disease and a number of interstitial lung diseases such as idiopathic pulmonary fibrosis (IPF). Despite their obvious etiological and clinical distinctions, most of these fibrotic diseases have in common a persistent inflammatory stimulus that sustains and/or stimulates the production of growth factors and fibrogenic cytokines, which together stimulate the deposition of connective-tissue elements that progressively remodel and destroy normal tissue architecture.IPF  is a chronic, interstitial, fibrotic lung disease, with a prevalence of 7–10∶100000 (increasing with age) and a mean survival of 3–4 years (decreasing with age). Clinically, IPF is characterized by progressive, exertional dyspnea and nonproductive cough, worsening of pulmonary function and radiographically evident interstitial infiltrates (honeycombing). Histologically, IPF is associated with the appearance of Usual Interstitial Pneumonitis (UIP), which is characterized by patchy subpleural and/or paraseptal interstitial fibrosis alternating with areas of mild inflammation and normal lung. The hallmark of IPF/UIP is the distinctive presence of fibroblastic foci and exuberant Extracellular Matrix (ECM) deposition, leading to thickening of alveolar septa and the collapse of normal lung architecture. Due to the lack of a more effective alternative, the fundamental therapeutic approach has been use of corticosteroids, alone or in combination with other immunosuppressive agents; however, this has little impact on long-term survival Although the etiology and pathogenesis of IPF remain poorly understood, a number of conditions and risk factors are weakly associated with the disease: cigarette smoking, occupational/environmental factors, latent viral infections, as well as age/gender/genetic predisposition. The occurrence of pulmonary fibrosis  as a side effect in humans receiving bleomycin (BLM) for cancer chemotherapy led to the development of a BLM-induced animal model of pulmonary fibrosis (BLM/PF). Despite the intrinsic limitations and the evolutionary distance, the BLM/PF animal model shares many clinical features with the human disease, and therefore provides valuable insights into the pathogenetic mechanisms that govern disease activation and perpetuation. Utilization of this model, as well as site-specific and/or temporal overexpression or ablation of candidate pathogenic genes, is responsible for most of our knowledge concerning IPF pathogenesis. In this context, current research suggests that the mechanisms driving IPF reflect abnormal, deregulated wound healing within the lung, involving increased activity and possibly exaggerated responses by a spectrum of proinflammatory and profibrogenic factors Tumor Necrosis Factor (TNF) is a pleiotropic cytokine expressed by many cell types in response to infection or injury. TNF affects multiple responses that extend well beyond its well-characterized pro-inflammatory properties to include diverse signals for cellular differentiation, proliferation and death; its functions can be both beneficial, as well as detrimental. Inappropriate production of TNF has been implicated in the pathogenesis of a variety of human diseases, including sepsis, cerebral malaria, diabetes, cancer, osteoporosis, allograft rejection, multiple sclerosis, rheumatoid arthritis, and inflammatory bowel diseases In order to decipher the role of TNF and TNF-mediated inflammation in the development of PF, we have systematically administered intravenous BLM to genetically modified mice of the same susceptible genetic background, lacking components of TNF signaling in the hematopoietic and/or the non-hematopoietic cellular compartments. Transmembrane (tm) TNF expression from non-hematopoietic cells was shown to be sufficient to elicit an inflammatory response to BLM, while soluble (sol) TNF to be necessary for appropriate lymphocyte recruitment, TGF-b1 expression and the development of fibrotic lesions.−/−tnf mice were completely protected from the development of disease, exhibiting no inflammation, lack of collagen accumulation and normal lung architecture  lacking components of TNF signaling. In order to compare multiple mouse lines, experimental procedures were standardized as described in detail in itecture tnf−/−. tm/tm tnf mice developed marked pulmonary inflammation, as assessed by BALF inflammatory cell counts and histopathology , later to be cleaved by TNF-α Converting Enzyme (TACE) to release the soluble 17 kDa TNF cytokine (solTNF) athology tnftm/tmathology tnftm/tm lesions . Therefo−/−tnf mice, that failed to develop any inflammatory response and subsequent fibrosis, was almost devoid of any inflammatory cells  that are devoid of T and B cells To analyze the TNF-induced inflammatory response, shown to be necessary for the development of PF, the inflammatory cells in BALF were analyzed qualitatively by cytospins. As evident from ry cells  and 3B,Cpromised , as oppopromised . Therefophocytes . In acco−/−tnf to WT) developed both inflammation and fibrosis with proper lymphocyte recruitment; in contrast, abolishing TNF expression from non-hematopoietic cells (WT to −/−tnf) resulted in complete disease protection . Both mouse lines developed both pulmonary inflammation and fibrosis with minor differences from WT mice  and TNFRII (p75), expressed in almost all cell types  WT mice , indicat−/−)tnfR were utilized as donors and/or recipients in bone marrow transfer experiments as above (Outlined in −/−tnfR) or hematopoietic (−/−tnfR to WT) compartments were capable of eliciting an inflammatory response to BLM  was always associated with lack of TGF-b1 expression . TGF-b1 IPF and related pulmonary fibrotic disorders affect millions of individuals worldwide, where the clinical outcome for the vast majority of the patients remains poor. Administration of corticosteroids and other general anti-inflammatory and immunosuppressant drugs fails to control the disease, leading physicians to alternate modes of treatment. Surprisingly, there have been only a few isolated case reports in the literature accessing the efficacy of anti-TNF treatment, perhaps due to the failure of corticosteroids as well as to the increased risk of infections associated with current anti-TNF biologics. Treatment with infliximab (chimeric anti-TNF antibody) of a rheumatoid arthritis patient with progressive pulmonary symptoms associated with radiographic fibrosis in order to control articular symptoms resulted in sustained improvement in dyspnea, cough and exercise tolerance in addition to improvement of joint symptoms in vivo studies with an engineered dominant-negative peptide acting as a selective inhibitor of soluble TNF Our results presented in this report reinforce the importance of assessing the efficacy of TNF antagonists in IPF treatment. More importantly, specific blockade of soluble TNF would be predicted to abolish the detrimental pro-fibrotic effects of TNF, while maintaining sufficient host defense against infections. Preliminary results from on-going −/−tnftm/tmtnf−/−tnfRI−/−tnfRII−/−rag-1The generation of 2 for RNA extraction while the other half was used for histopathology, where paraffin-embedded lung tissue samples were sectioned and stained with Haematoxylin/Eosin and/or Masson trichromy .PF was induced by a single tail vein injection of Bleomycin hydrogen chloride  to 6- to 8-wk-old mice as previously reported 7 bone marrow cells in 200 µl of HBSS were injected intravenously into lethally irradiated (980 rad) recipient mice (C57/Bl6), a dosage that never produced any signs of inflammation or fibrosis in control animals (as it can be seen in the control groups of Bone marrow from 8-wk-old mutant and wild-type mice (C57/Bl6) was obtained from femurs and tibia, essentially as previously described radiated 80 rad re2O2 followed by anti-goat IgG-HRP  for 30 min at RT. Bound peroxidase activity was detected by staining with diaminobenzidine . For the simultaneous in situ detection of apoptotic cells (TUNEL assay), sections were first processed as above and after color development with DAB, they were incubated with terminal deoxynucleotidyl transferase (TdT)  at 37oC for 1 h in a humidified chamber. Then, according to manufacturer's instructions, sections were incubated with the alkaline phosphatase-conjugated secondary Ab and developed using the Fast Blue kit . Prior to mounting, sections were counterstained with methyl green.Deparaffinized and rehydrated 5 µm-thick sections of paraffin embedded lung tissue samples were incubated with a goat polyclonal anti-mouse TNF Ab  overnight at 4°C. Sections were then incubated with 0.03% HFor the identification of TNF producing cells, lung sections processed as above were incubated with a goat polyclonal anti-mouse TNF Ab  for 1 h at RT. After washing, sections were incubated with a donkey anti-goat Alexa Fluor 555 secondary Ab  for 30 min at RT. Sections were then sequentially incubated with an anti- pro-SP-C Ab  at RT for 1 h, followed by a goat anti-rabbit Alexa Fluor 488 Ab . Finally, sections were mounted and photographed under a Nikon ECLIPSE E800 microscope .2 for 25 min into an airtight chamber containing 5–7 mice.Recombinant human TNF  was dissolved at a concentration of 200 ng/ml in normal saline, at a final volume of 3 ml. The solution was administered by a custom made nebulizer flowing at 4 L/min of OQuantification of TGF-b1 in BALF samples was performed with the Quantikine Immunoassay, according to manufacturer's instructions .BALF cell counts, collagen levels and ELISA values were analyzed for statistical significance with student's t-test (in pairwise comparisons), utilizing the statistical software package Sigmaplot .Figure S1Animal model of BLM-induced Pulmonary Fibrosis.(0.23 MB DOC)Click here for additional data file.Figure S2Schematic overview of bone marrow transplantation experiments.(1.62 MB TIF)Click here for additional data file.Figure S3solTNF but not tmTNF can support a non-degrading, profibrogenic environment.(0.19 MB TIF)Click here for additional data file.


PCR restriction fragment length polymorphism analysis was used to genotype patients for the A36G polymorphism in the TNF-RI gene and the T676G polymorphism in TNF-RII. Levels of sTNFRs were measured using ELISA. We also isolated T cells from peripheral blood of 58 patients with established RA with known TNF-R genotypes, and release of sTNFRs into the culture medium was measured in cells incubated with or without phytohaemagglutinin. Serum levels of the two sTNFRs (sTNF-RI and sTNF-RII) were positively correlated in both populations, and the level of each sTNFR was significantly higher in the patients with established disease (P < 0.0001). Multiple regression analyses corrected for age, sex and disease duration revealed a significant trend toward decreasing sTNF-RI and sTNF-RII levels across the TNF-RII genotypes (TT > TG > GG) of patients with established disease . A similar nonsignificant trend was seen for early disease. No relationship with the TNF-RI A36G polymorphism was observed. sTNFRs released by isolated T cells exhibited a similar trend toward decreasing levels according to TNF-RII genotype, although only the association with levels of sTNF-RII was significant. Strong correlations were found between levels of circulating sTNFRs and levels released by T cells in vitro. Our data indicate that the T676G polymorphism in TNF-RII is associated with levels of sTNFRs released from peripheral blood T cells, and with circulating levels of sTNFR in patients with RA.Levels of soluble tumour necrosis factor receptors (sTNFRs) are elevated in the circulation of patients with rheumatoid arthritis (RA). Although these receptors can act as natural inhibitors of tumour necrosis factor-α, levels of sTNFRs in RA appear to be insufficient to prevent tumour necrosis factor-α induced inflammation. The factors that regulate circulating levels of sTNFRs are unclear, but polymorphisms in the tumour necrosis factor receptor genes may play a role. We investigated the relationship between polymorphisms in the tumour necrosis factor receptor I (TNF-RI) and II (TNF-RII) genes and levels of sTNFRs in two groups of Caucasian RA patients: one with early (disease duration ≤2 years;  Tumour necrosis factor (TNF)-α is a pleiotropic cytokine that is important in the pathogenesis of rheumatoid arthritis (RA), in which it plays a role in cartilage degradation, bone resorption, adhesion molecule expression, leucocyte infiltration, enzyme production and cytokine synthesis  and the second had established disease  from each patient, was stored at -70°C until required for measurement of sTNFRs. Synovial fluid was also collected from 45 patients who presented with knee effusions at the time of blood collection. Fluids were centrifuged and separated from the resulting cell pellet, before storage at -70°C. The study was approved by the North Staffordshire local research ethics committee.n = 58). Cell isolation was by negative selection using a modified density gradient centrifugation technique that utilizes novel tetrameric antibody complexes . Isolated T cells (2 × 105 cells/200 μl) were cultured in RPMI 1640 synthetic culture medium supplemented with 10% heat-inactivated foetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin and 10% autologous serum, in 96-well cell culture plates. Cultures were incubated, with or without phytohaemagglutinin , at 37°C in a 5% carbon dioxide humidified air environment for 48 hours. Cell supernatants were then harvested and stored at -20°C until required for analysis of sTNFR levels.T cells were isolated from fresh peripheral blood samples (4 ml) from RA patients with established disease  collected in EDTA tubes were obtained from each patient and were stored at -20°C. After thawing at 37°C, the genomic DNA was isolated using a DNAce MegaBlood Kit procedure as directed by the manufacturer .The following primer sequences were used to amplify a 183 base pair fragment containing the SNP at nucleotide 36 in exon 1 of the TNF-RI gene : forwardA 242 base pair fragment containing the SNP at nucleotide 676 in exon 6 of the TNF-RII gene was amplified with the following primer sequences : forwardThe fragment of interest from each of the TNF receptor genes was amplified using an identical reaction mixture and conditions that were described previously . All ampSerum, synovial fluid and T cell supernatant levels of sTNF-RI and sTNF-RII were quantified using the respective Duoset ELISA Development Kit as directed by the manufacturer . For determination of sTNF-RI levels, sera, synovial fluids and T-cell supernatants were diluted 1:10, 1:50 and 1:3, respectively. For soluble TNF-RII, sera, synovial fluids and T-cell supernatants were diluted 1:20, 1:80 and 1:4, respectively. All samples were run in duplicate with the appropriate standards on 96-well microplates.P < 0.05 were considered statistically significant.The relationship between the two sTNFRs was assessed using Spearman's rank correlation, whereas differences in sTNFR levels between early and established RA were assessed using the Mann–Whitney U-Test. Multiple regression analysis was used to assess the relationship between each sTNFR and age (corrected for sex and disease duration), and between the TNF receptor genotypes and sTNFR levels . Where necessary the data were normalized by logarithmic transformation before analysis. All data were analyzed using the Number Cruncher Statistical Software package for Windows  s > 0.45; P < 0.0001). The levels of each sTNFR were also found to increase significantly with age in both patient groups (P < 0.0001), and this was independent of disease duration. Similar associations between sTNFR levels and age were seen in male and female patients (data not shown). Also, the median level of each sTNFR was significantly higher in patients with established disease than in those with early disease (P < 0.0001); this association remained after correction for age.Both sTNFRs were detected in the sera of all patients studied. Consistent with the findings reported by Cope and coworkers , levels The A36G SNP genotype frequencies in each population and the respective mean levels of both sTNFRs are shown in Table P for trend = 0.01) and sTNF-RII (P for trend = 0.03) in the order TT > TG > GG. An identical trend was seen for levels of sTNF-RI and sTNF-RII in patients with early disease, although these associations were not significant . In addition, the levels of sTNF-RI and sTNF-RII were significantly associated with TNF-RII genotype  when the two populations were combined and analyzed by multiple regression with correction for age, sex and disease duration.The genotype and sTNFR level data for the T676G polymorphism in patients with early and established disease are shown in Table Synovial fluids collected at the same time as sera were available in 45 patients. Mean levels of sTNF-RI and sTNF-RII in the synovial fluids were significantly higher  than in the paired sera, but there was no direct correlation between levels in the synovial fluid and serum. No association was found between synovial fluid sTNFR levels and the A36G TNF-RI or 676G TNF-RII genotypes (data not shown).We showed previously that polymorphisms in the TNF-RI and TNF-RII genes were not associated with radiographic or functional severity in a cross-sectional study of patients with RA . SimilarP = 0.1) or after 5 years of follow up . There was also no significant difference in HAQ score progression.In the present study we again found no association between TNF-RI or TNF-RII polymorphisms and cross-sectional measures of radiographic or functional severity in patients with early or established disease (data not shown). In a similar manner to that reported by Constantin and coworkers , we alsoAnalysis of other clinical parameters associated with disease severity  revealed no differences between TNF-RII genotypes (data not shown). However, in a separate study on anaemia in RA, involving many of these patients, we reported an association between carriage of the TNF-RII T allele and anaemia of chronic disease .P for trend = 0.049 and P for trend = 0.02; Table We investigated whether there was any association between polymorphism in the TNF receptor genes and levels of sTNFRs released into the culture medium of unstimulated and stimulated T cells from RA patients. No association was found between the TNF-RI A36G polymorphism and levels of sTNFRs released (data not shown). However, a significant trend was found in levels of sTNF-RII released into culture medium by both unstimulated and stimulated T cells according to the TNF-RII genotype in the order TT > TG > GG . In multiple regression analyses of serum TNF receptor levels, which included levels released by T cells and monocytes as independent variables, we found that only levels released by T cells were associated with serum levels (unpublished observations).We also measured sTNFR release by isolated monocytes The association of the TNF-RII T676G polymorphism with circulating sTNF-RII levels is consistent with a previous study  that demThe T676G polymorphism in exon 6 of the TNF-RII gene occurs within the fourth cysteine-rich domain of the extracellular domain, close to a point where the proteolytic cleavage site for TACE is thought to lie . The polRecently, the TNF-RII 196R variant was shown to have a significantly lower ability to induce direct NF-κB signalling via TNF-RII and to enhance TNF-RI dependent TNF-α induced apoptosis . The dimThe mean serum levels of sTNF-RII were approximately three times greater than for sTNF-RI in each patient population. The levels of sTNF-RII in culture medium from unstimulated T cells were also about three times greater than those of sTNF-RI, although this increased to approximately five times greater after stimulation of T cells with PHA. This can be explained by increased release of sTNF-RII, but not of sTNF-RI, after PHA stimulation. This is similar to the situation previously observed in lipopolysaccharide stimulated monocytes and alveolar macrophages, in which sTNF-RII but not sTNF-RI release was enhanced after stimulation ,41. ThesPrevious studies in healthy individuals reported an age associated significant increase in serum levels of both sTNFRs ,44, wherP ≤ 0.007), which is consistent with the T allele association.The clinical relevance of our findings is unclear at present, but it has been shown that familial susceptibility to RA is associated with the TNF-RII G allele and particularly the GG genotype ,25, whicAlthough the differences in sTNFR serum levels between TNF-RII genotypes are not large, it is noteworthy that exactly the same trend was seen throughout for serum levels in early and established RA, and for unstimulated and stimulated T-cell cultures. Several studies have shown that circulating sTNFR levels and/or polymorphisms in the TNF-RII gene are associated with heart failure, hypertension, obesity and insulin resistance, and differences in serum levels of a similar magnitude to those found in this study were shown to be clinically relevant in these conditions -52. Our Our results indicate that there is an association between the T676G SNP in the TNF-RII gene and levels of sTNFRs released by T cells of RA patients. This finding is reinforced by an association between this polymorphism and circulating levels of sTNFRs in established RA. Although various inflammatory factors may influence the release of TNF receptors, our data indicate that genetic regulation involving the TNF-RII gene may play some role in determining circulating levels in RA.ELISA = enzyme-linked immunosorbent assay; HAQ = health assessment questionnaire; NF-κB = nuclear factor-κB; PCR = polymerase chain reaction; PHA = phytohaemagglutinin; RA = rheumatoid arthritis; SNP = single nucleotide polymorphism; sTNFR = soluble tumour necrosis factor receptor; TACE = TNF-α converting enzyme; TNF = tumour necrosis factor; TNF-RI = tumour necrosis factor receptor I; TNF-RII = tumour necrosis factor receptor II.The author(s) declare that they have no competing interests.JRG carried out genotyping, cell isolation, cell culture, and ELISA work, and wrote the first draft of the paper. PTD gave advice on patient selection, study design and interpretation of data. NBN carried out some genotyping and ELISA work. DLM conceived and oversaw the study, carried out statistical analyses and interpretation of data, and finalized the manuscript. The final manuscript was read and approved by all authors.


We suggest that CTLs kill a large number of infected cells (about 107) per day but are not responsible for the majority of infected cell death.Understanding the role of cytotoxic T lymphocytes (CTLs) in controlling HIV-1 infection is vital for vaccine design. However, it is difficult to assess the importance of CTLs in natural infection. Different human leukocyte antigen (HLA) class I alleles are associated with different rates of progression to AIDS, indicating that CTLs play a protective role. Yet virus clearance rates following antiretroviral therapy are not impaired in individuals with advanced HIV disease, suggesting that weakening of the CTL response is not the major underlying cause of disease progression and that CTLs do not have an important protective role. Here we reconcile these apparently conflicting studies. We estimate the selection pressure exerted by CTL responses that drive the emergence of immune escape variants, thereby directly quantifying the efficiency of HIV-1–specific CTLs in vivo. We estimate that only 2% of productively infected CD4 Although cytotoxic T lymphocytes (CTLs) kill a large number of HIV-infected cells every day, they may not be responsible for the majority of infected cell death. A productively infected cell is estimated to have a lifespan of about 1 d [+ cells in uninfected individuals [Half of all CD4out 1 d , consideviduals . It is nThere is good evidence that the CTL response contributes to the control of HIV-1 infection in vivo –11. HowThe rate at which an escape variant replaces the wild-type, “the rate of escape,” is determined by the balance between the efficiency of the CTL clone(s) evaded and the fitness cost of the mutations . The aim−1. The median rate of escape was 0.01 d−1, and taking the most optimistic interpretation, the median rate of escape was 0.04 d−1.We estimated the rate of escape of 21 reported CTL escape variants using longitudinal data from 12 HIV-1–infected individuals ,22–28 −1.It is possible that, as the proportion of cells infected with wild-type virus declines, the antigenic stimulus declines, causing a loss of epitope-specific CTLs. This would reduce the selection pressure for escape and so would lead to an underestimate of the escape rate that would be observed under continuing CTL pressure. In fact, in the majority of cases where CTL frequency was measured the magnitude of the response to the epitope of interest was reported to be fairly stable through time. However, there were cases where the CTL response did decline or else was not measured, so we quantified the impact of a declining CTL response on the escape-rate estimates. This was done by estimating the half-life of HIV-1–specific CTLs following highly active antiretroviral treatment (HAART) and then assuming that CTLs declined with the same half-life during outgrowth of the variant see. Even us−1. While these low fitness costs were surprising, they are consistent with data from in vitro competition assays [To see if this low rate of escape could be explained by a high fitness cost of CTL escape variants we quantified the rate of reversion of escape variants to wild-type on transmission to individuals who did not possess the human leukocyte antigen (HLA) allele necessary to bind the epitope of interest. We quantified the rate of reversion in seven datasets from five patients –21 Ta. The fit assays  and accu assays ,21,29,−1 (escape rate + reversion rate = 0.01 + 0.005 d−1) and was at most 0.06 d−1 (0.055 + 0.005 d−1). This implies that, using the best estimate, an infected cell that was subject only to lysis by CTLs specific for one epitope would live for about 50 days. A productively infected cell has a lifespan of about 1 d [Given that escape and reversion rates are consistent across individuals it is reasonable to consider the average rate of escape and reversion of a CTL escape variant. Putting these average estimates together we can estimate the rate of lysis of infected cells by a single CTL response. We found that the rate of lysis was about 0.02 dp = 0.004 Wilcoxon–Mann-Whitney two-tailed test,On comparing the rate of CTL lysis in individuals with primary and chronic infection we found that the rate of CTL lysis was significantly faster during primary infection than in chronic infection , CTLs may not be responsible for the majority of infected cell death. This conclusion, like all scientific conclusions, may change with the advent of new data but it is also consistent with reports of bystander apoptosis [+ cell death that cannot be directly attributed to HIV-1–specific CTL-mediated lysis.It would be fascinating to extrapolate these results on the rate of lysis by a single CTL response to estimate the total extent of CTL-mediated killing. However, to do this it is necessary to know how many of the CTL responses naturally present during HIV-1 infection are of sufficient strength to drive escape. In most but not all reports of CTL escape, the immune response selecting for the variant was immunodominant, suggesting that only a few oligoclonal CTL responses are of sufficient strength to drive escape ,30,31.ween 14  and 19 [ween 14 . Includioptosis  and chrooptosis ,36, whi−1 is considerably lower than in vitro estimates  for other virus infections which range from 1 d−1 for HTLV-I to 12 d−1 for acute LCMV [+ cell help may be important. Consistent with this, we found that the rate of CTL lysis was significantly faster during primary infection than in chronic infection, implying a significant weakening of single CTL responses with long-term infection.Our estimate of the rate of HIV-1–specific CTL lysis totalled across all CTL responses of 0.1 to 0.2 d, pote LCMV –42. Whyte LCMV ,16,43,−1 is equivalent to a difference of about 107 productively infected cells killed each day, every day, for several years. Such a large, cumulative difference could well alter the timing of disease progression.The conclusion that CTL may not be responsible for the majority of infected cell death unifies apparently contradictory results indicating that there is an association between HLA class I type and rate of progression to AIDS despite a lack of association between disease stage and infected cell clearance rate following therapy ,3. InfeThis work has implications for vaccines designed to induce a lytic CTL response. One of the aims of therapeutic, and possibly prophylactic vaccines, is to boost the chronic memory response, so it becomes important to understand why the HIV-1–specific CTL response weakens with time. Furthermore, if it is true that the CTL response kills a minority of infected cells but that this is sufficient to affect clinical outcome, this suggests that, unless the CTL response elicited by vaccines is several-fold more efficient than the natural response, vaccines relying on the lytic pathway are unlikely to prevent infection or to mediate complete viral clearance, but that they may well reduce viral load and lengthen the asymptomatic period.+ cells productively infected with wild-type virus(y) replicate at a ratea , are killed by CTLs recognising epitopes other than the escape epitope at a rateb and are killed by CTLs recognising the escape epitope at a ratec. CD4+ cells productively infected with a CTL escape variant(x) replicate at a ratea′  and are killed by CTLs recognising epitopes other than escape epitope at a rateb. Infected cell dynamics are therefore represented byInitially it was assumed that productively infected cell dynamics follow first-order kinetics, as detailed observation of both viral clearance and viral rebound have indicated that this is most appropriate. Later, we relaxed this assumption. Here we refer to the epitope that is no longer recognised due to mutation as the “escape epitope.” CD4k = a′ − b − (a − b − c) = c − a + a′.In a host able to mount the relevant CTL responses , the selective advantage of the escape variant  is the difference in growth rate between the escape variant and the wild-typeϕ = a − a′. Ifϕ is greater than zero then the mutation has a deleterious effect on viral replication (in the absence of a CTL response).Many escape mutations will carry a fitness cost that partially offsets the “benefit” to the virus of evading the CTL response. This fitness cost will be the difference in replication rate between the escape variant and the wild-type(c) and the fitness cost of the mutation(s)(ϕ = a − a′). The rate of escape(k = c − ϕ) was estimated from longitudinal escape data. Ifp(t) is the proportion of viral sequences that have escape mutations in the epitope of interest at timet then solvingThe rate of escape  is determined by the balance between the rate of lysis evadedg = y(0)/x(0) andk = c − ϕ.where(k = c − ϕ) estimated. The standard error of the escape rate was estimated using the asymptotic covariance matrix method.This model was fit to longitudinal escape data using nonlinear least-squares regression (Levenberg-Marquardt method) and the rate of escape of the variant(ϕ = a − a′). If the escape mutation carried a fitness cost then the virus will tend to revert to wild-type. If the mutation was cost neutral then it will tend to be stable over time. The proportion of viral sequences with an escape mutation will still be described byc = 0. By fitting the model  of a CTL escape variant was estimated from longitudinal escape data. The average fitness cost(ϕ = a − a′) of a CTL escape variant was estimated from reversion data. Putting these two estimates together we quantifiedc = k + ϕ the average rate of killing (per day) of productively infected cells by a CTL response against a single epitope. High levels of multiple infection and recombination [The average rate of escapeination  ensure t−1. This is an estimate of the cumulative impact of all CD8+ cells recognising that epitope. Many mathematical models express the killing rate of infected cells in units of killing per day per epitope-specific CD8+ cell we therefore re-expressed our estimate in these units to facilitate future development of these models.We have estimated that a single CTL response kills productively infected cells at a rate of 0.02 d+ cells able to bind the escape epitope–presenting allele complex was quantified by major histocompatibility class I tetramer; this figure was converted to the proportion of PBMCs able to bind the escape epitope–presenting allele complex assuming 15% of PBMCs are CD8+ . Using these 15 estimates of specific CD8+ cell frequency in PBMCs (median 0.1%) and an estimate of the number of PBMCs in the body of 1.4 × 1012 [−11 per day per epitope-specific CD8+ cell, considerably smaller than the CTL killing rate used in many models [In 13 of the escape datasets the proportion of peripheral blood mononuclear cells (PBMCs) responding to the escape epitope was measured by IFNγ ELIspot. In a further two datasets the proportion of CD8× 1012  we estim models ,47.The rate of escape was quantified in 21 datasets from 12 HIV-1–infected subjects . The rat(k) was obtained by fitting the model (k can often be obtained if some later data is discarded), but they provide an approximate upper bound on the possible rate of escape. The assumptions made to obtain an optimistic estimate are listed in Supporting Information. For the reversion data optimistic estimates of the rate of reversion were not made because the data were more straightforward—monotonic reversion of a single escape mutation—allowing little scope for alternative assumptions.The best estimate of the rate of escapee model  to the ek = 0.01 d−1) were very similar to those obtained from the full datasets. More importantly there were only ten such datasets (compared to 21 full datasets) despite there being a higher probability of observing them , suggesting that the majority of CTL escape is not happening on a much faster scale than that implied by our analysis of the full datasets.To make reliable estimates of the escape rate it was necessary to have at least two data points that were not zero (no escape variants detected) or one (no wild-type sequence detected). All datasets found that matched these criteria were analysed and the results reported here. However, this raised the possibility that faster rates of escape not captured by infrequent sampling protocols were omitted. To avoid this possible bias we also analysed all datasets reporting CTL escape regardless of the number of data points available. The escape rates estimated from these datasets . In five of the 21 datasets the escape variant was capable of eliciting a partial CTL response in vitro. There was a possibility that incomplete escape would lead to an underestimate of the rate of CTL lysis of infected cells. We took two approaches to quantifying the impact of incomplete escape. First, we simply removed the five cases where escape was incomplete. This yielded a median rate of lysis of 0.013 d−1. Alternatively, we estimated the “escapedness” of the variant in each of the 21 cases considered and adjusted our estimate of the rate of lysis by this factor. Escapedness was defined as the percentage of the CTL response escaped, so an escapedness of 100% means escape was complete. The median escapedness was 100%; the mean was approximately 85%. Clearly, adjusting by the median escapedness does not change our estimate of the rate of CTL lysis; adjusting by the mean changes it to (0.01 + 0.005)/0.85 = 0.02 d−1. Whichever approach is taken, it is clear that incomplete escape has very little impact on our estimate of the rate of CTL lysis (previously 0.015 d−1). Furthermore, the escape rate of the five cases where escape was incomplete was not significantly different from the escape rate of the remaining 16, where escape was between 90%–100% . One possible explanation for the absence of an association between a slow rate of escape and incomplete escape is that during the in vitro test for escapedness, when very high levels of peptide are added exogenously , it is possible to elicit a partial response to a variant that is a genuine complete escape variant in vivo [In five of the 21 datasets, sequences were obtained from proviral DNA rather than from viral RNA. There was no significant difference between the rate of escape calculated using DNA and RNA /x(0), butk is changed to−mt)/(mt) − ϕk = c(1 − e.where(m) was estimated using the rate of HIV-1–specific CTL decline following HAART. Four estimates of the rate of CTL decline have been made:m = 0.0002, 0.002, 0.003, and 0.015 d−1 [m = 0.005 d−1) and the maximum (m = 0.015 d−1). Following successful HAART viral load falls rapidly and dramatically, by two or more orders of magnitude in a few days [m = 0.005 d−1 we found that the median rate of escape was 0.055 d−1. Usingm = 0.015 d−1 we found that the median rate of escape was 0.086 d−1. In the latter case it was difficult to fit the model . We wished to know if between-individual differences in this lysis rate would be manifest in virus clearance estimates.Following antiretroviral treatment the rate of viral clearance is very consistent between individuals and there is no correlation with disease stage –4. This−1) and an individual with an average CTL response . This between-individual difference in CTL lysis would lead to a difference in infected cell clearance following HAART of 0.1 d−1. A between-individual difference of 0.1 d−1 is well within the range of variation observed  of the patient.As an extreme example we considered an individual with a very poor CTL response that is unable to kill any infected cells , despit.7 d−1 . Even th.7 d−1  of very 7 productively infected cells killed every day .−1, i.e., a tenth of the productively infected cell death observed. However, this neglected CTL responses below the “threshold of detection” of our method. It has been found [−1 . From−1. The nine to 14 inefficient responses were therefore estimated to have an efficiency of 0.002 + 0.005 = 0.007 d−1 or less. So the maximum rate of lysis of productively infected cells was 5 × 0.02 + 9 × 0.007 to 5 × 0.02 + 14 × 0.007 = 0.16 to 0.20 d−1; i.e., accounting for the extra inefficient CTL responses increased the proportion of productively infected cell death attributable to the CTL response to about 20%. In order for 50% of productively infected cell death to be attributable to the CTL response it would be necessary to have 60 CTL responses at the detection threshold (in addition to the five above the detection threshold). Even if we considered an extremely strong immune response in which all 14–19 responses were of sufficient strength to drive CTL escape, then the proportion of infected cells killed was still only between 28% and 38% . It is thought that CTL responses driving escape are of average strength or higher. If CTL responses driving escape were atypically weak  then this calculation would tend to underestimate the importance of CTL.We have quantified the rate of lysis of productively infected cells by a single CTL response . To make an order of magnitude approximation of the total extent of CTL lysis we needed to estimate how many CTL responses of sufficient strength to drive escape were naturally present during HIV-1 infection. In most, but not all reports of CTL escape, the immune response selecting for the variant was immunodominant, suggesting that only a few oligoclonal CTL responses were able to drive escape ,30,31.n found ,33 thatIn the basic model  and consln(1/p − 1) wherep is the frequency of the escape variant.ln(1/p − 1) is ill-defined forp = 0 orp = 1 so, where the frequency of the variant was0/n this was approximated by1/n + 1 and where the frequency wasn/n this was approximated byn/n + 1. The median rate of escape using this method was 0.05 d−1, which was very close to the median optimistic estimate (0.04 d−1). In cases where the CTL selection pressure for escape declines with time, either due to loss of the CTL response against the wild-type epitope or due to emergence of a new CTL response to the escape variant (when mutation has reduced T cell receptor recognition rather than MHC binding), it would be expected that initial rapid escape would be slowed or even reversed at later time points. This alternative method of calculating the optimistic estimate will reflect the most rapid period of escape and will not be influenced by later reduced selection pressure. Estimates made using this method can therefore be considered to be alternatives to the best estimates with correction for CTL decline or to the optimistic estimates.An alternative method of calculating the optimistic estimate was also performed for each of the 21 escape datasets. The rate of escape was calculated from the greatest rate of change in frequency of the variant (between two time points) in the dataset. This was done by fitting a straight line to the transformed datan = 14) was compared with the rate of CTL killing in chronic infection (n = 7) using the nonparametric exact Wilcoxon–Mann-Whitney two-tailed test.The rate of CTL killing in primary infection (Protocol S1(66 KB DOC).Click here for additional data file.