Absence Of Tumor Necrosis Factor Research Articles

Transcriptional and Chromatin Accessibility Profiling of Neural Stem Cells Differentiating into Astrocytes Reveal Dynamic Signatures Affected under Inflammatory Conditions

Astrocytes arise from multipotent neural stem cells (NSCs) and represent the most abundant cell type of the central nervous system (CNS), playing key roles in the developing and adult brain. Since the differentiation of NSCs towards a gliogenic fate is a precisely timed and regulated process, its perturbation gives rise to dysfunctional astrocytic phenotypes. Inflammation, which often underlies neurological disorders, including neurodevelopmental disorders and brain tumors, disrupts the accurate developmental process of NSCs. However, the specific consequences of an inflammatory environment on the epigenetic and transcriptional programs underlying NSCs' differentiation into astrocytes is unexplored. Here, we address this gap by profiling in mice glial precursors from neural tissue derived from early embryonic stages along their astrocytic differentiation trajectory in the presence or absence of tumor necrosis factor (TNF), a master pro-inflammatory cytokine. By using a combination of RNA- and ATAC-sequencing approaches, together with footprint and integrated gene regulatory network analyses, we here identify key differences during the differentiation of NSCs into astrocytes under physiological and inflammatory settings. In agreement with its role to turn cells resistant to inflammatory challenges, we detect Nrf2 as a master transcription factor supporting the astrocytic differentiation under TNF exposure. Further, under these conditions, we unravel additional transcriptional regulatory hubs, including Stat3, Smad3, Cebpb, and Nfkb2, highlighting the interplay among pathways underlying physiological astrocytic developmental processes and those involved in inflammatory responses, resulting in discrete astrocytic phenotypes. Overall, our study reports key transcriptional and epigenetic changes leading to the identification of molecular regulators of astrocytic differentiation. Furthermore, our analyses provide a valuable resource for understanding inflammation-induced astrocytic phenotypes that might contribute to the development and progression of CNS disorders with an inflammatory component.

Forces of RBC interaction with single endothelial cells in stationary conditions: Measurements with laser tweezers

Red blood cells (RBCs) are able to interact and communicate with endothelial cells (ECs). Under some pathological or even normal conditions, the adhesion of RBCs to the endothelium can be observed. Presently, the mechanisms and many aspects of the interaction between RBCs and ECs are not fully understood. In this work, we considered the interaction of single RBCs with single ECs in vitro aiming to quantitatively determine the force of this interaction using laser tweezers. Measurements were performed under different concentrations of proaggregant macromolecules and in the presence or absence of tumor necrosis factor (TNF-[Formula: see text]) activating the ECs. We have shown that the strength of interaction depends on the concentration of fibrinogen or dextran proaggregant macromolecules in the environment. A nonlinear increase in the force of cells interaction (from 0.4 pN to 21 pN) was observed along with an increase in the fibrinogen concentration (from 3[Formula: see text]mg/mL to 9[Formula: see text]mg/mL) in blood plasma, as well as with the addition of dextran macromolecules (from 10[Formula: see text]mg/mL to 60[Formula: see text]mg/mL). Dextran with a higher molecular mass (500[Formula: see text]kDa) enhances the adhesion of RBCs to ECs greater compared to the dextran with a lower molecular mass (70[Formula: see text]kDa). With the preliminary activation of ECs with TNF-[Formula: see text], the force of interaction increases. Also, the adhesion of echinocytes to EC compared to discocytes is significantly higher. These results may help to better understand the process of interaction between RBCs and ECs.

Bromodomain-containing protein 4 inhibition alleviates matrix degradation by enhancing autophagy and suppressing NLRP3 inflammasome activity in NP cells.

An imbalance between matrix synthesis and degradation is the hallmark of intervertebral disc degeneration while inflammatory cytokines contribute to the imbalance. Bromodomain and extra-terminal domain (BET) family is associated with the pathogenesis of inflammation, and inhibition of BRD4, a vital member of BET family, plays an anti-inflammatory role in many diseases. However, it remains elusive whether BRD4 plays a similar role in nucleus pulposus (NP) cells and participates in the pathogenesis of intervertebral disc degeneration. The present study aims to observe whether BRD4 inhibition regulates matrix metabolism by controlling autophagy and NLRP3 inflammasome activity. Besides, the relationship was investigated among nuclear factor κB (NF-κB) signaling, autophagy and NLRP3 inflammasome in NP cells. Here, real-time polymerase chain reaction, western blot analysis and adenoviral GFP-LC3 vector transduction in vitro were used, and it was revealed that BRD4 inhibition alleviated the matrix degradation and increased autophagy in the presence or absence of tumor necrosis factor α. Moreover, p65 knockdown or treatment with JQ1 and Bay11-7082 demonstrated that BRD4 inhibition attenuated NLRP3 inflammasome activity through NF-κB signaling, while autophagy inhibition by bafilomycin A1 promoted matrix degradation and NLRP3 inflammasome activity in NP cells. In addition, analysis of BRD4 messenger RNA expression in human NP tissues further verified the destructive function of BRD4. Simply, BRD4 inhibition alleviates matrix degradation by enhancing autophagy and suppressing NLRP3 inflammasome activity through NF-κB signaling in NP cells.

Pharmacological inhibition of TAK1, with the selective inhibitor takinib, alleviates clinical manifestation of arthritis in CIA mice

ObjectivesTo examine the ability of takinib, a selective transforming growth factor beta-activated kinase 1 (TAK1) inhibitor, to reduce the severity of murine type II collagen-induced arthritis (CIA), and to affect function of synovial cells.MethodsFollowing the induction of CIA, mice were treated daily with takinib (50 mg/kg) and clinical scores assessed. Thirty-six days post-CIA induction, histology was performed on various joints of treated and vehicle-treated animals. Inflammation, pannus, cartilage damage, bone resorption, and periosteal bone formation were quantified. Furthermore, pharmacokinetics of takinib were evaluated by LC-MS in various tissues. Rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) cells were cultured with 10 μM takinib and cytokine secretion analyzed by cytokine/chemokine proteome array. Cytotoxicity of takinib for RA-FLS was measured with 24 to 48 h cultures in the presence or absence of tumor necrosis factor (TNF).ResultsHere, we show takinib’s ability to reduce the clinical score in the CIA mouse model of rheumatoid arthritis (RA) (p < 0.001). TAK1 inhibition reduced inflammation (p < 0.01), cartilage damage (p < 0.01), pannus, bone resorption, and periosteal bone formation and periosteal bone width in all joints of treated mice compared to vehicle treated. Significant reduction of inflammation (p < 0.004) and cartilage damage (p < 0.004) were observed in the knees of diseased treated animals, with moderate reduction seen in the forepaws and hind paws. Furthermore, the pharmacokinetics of takinib show rapid plasma clearance (t½ = 21 min). In stimulated RA-FLS cells, takinib reduced GROα, G-CSF, and ICAM-1 pro-inflammatory cytokine signaling.ConclusionOur findings support the hypothesis that TAK1 targeted therapy represents a novel therapeutic axis to treat RA and other inflammatory diseases.

Transmembrane TNF and Partially TNFR1 Regulate TNFR2 Expression and Control Inflammation in Mycobacterial-Induced Pleurisy.

Pleural tuberculosis is one of the most frequent forms of extra-pulmonary tuberculosis observed in patients infected with Mycobacterium tuberculosis. Tumor Necrosis Factor (TNF) is a crucial cytokine needed to control tuberculosis infection that remains a leading cause of morbidity and mortality worldwide. TNF blockade compromises host immunity and may increase the risk of reactivation of latent infection resulting in overt pulmonary, pleural and extra-pulmonary tuberculosis. While TNF signaling is mainly considered pro-inflammatory, its requirement for the anti-inflammation process involved in the resolution of infection and tissue repair is less explored. Our study analyzes the role of TNF and TNF receptors in the control of the inflammatory process associated with Bacillus Calmette-Guérin (BCG)-induced pleurisy. This study shows that the absence of TNF causes exacerbated inflammation in the pleural cavity of BCG-infected mice which is controlled by the transmembrane TNF (tmTNF) expression. The lack of TNF is associated with an impaired cellular expression and shedding of TNFR2 in the pleural cavity. The presence of tmTNF restores the normal expression of TNFR2 on myeloid cells during BCG-induced pleurisy. We also show that absence of TNFR1 affects the expression of TNFR2 on pleural cells and inflammation in the pleural cavity of BCG-infected mice. In conclusion, tmTNF but not soluble TNF prevents pleural cavity inflammation leading to attenuation and the resolution of the inflammatory process caused by mycobacterial pleurisy in association with the expression of TNFR2 on myeloid cells.

Absence of Tumor Necrosis Factor Supports Alternative Activation of Macrophages in the Liver after Infection with Leishmania major.

The absence of tumor necrosis factor (TNF) causes lethal infection by Leishmania major in normally resistant C57BL/6J (B6.WT) mice. The underlying pathogenic mechanism of this fatal disease has so far remained elusive. We found that B6.WT mice deficient for the tnf gene (B6.TNF−/−) displayed not only a non-healing cutaneous lesion but also a serious infection of the liver upon L. major inoculation. Infected B6.TNF−/− mice developed an enlarged liver that showed increased inflammation. Furthermore, we detected an accumulating monocyte-derived macrophage population (CD45+F4/80+CD11bhiLy6Clow) that displayed a M2 macrophage phenotype with high expression of CD206, arginase-1, and IL-6, supporting the notion that IL-6 could be involved in M2 differentiation. In in vitro experiments, we demonstrated that IL-6 upregulated M-CSF receptor expression and skewed monocyte differentiation from dendritic cells to macrophages. This was countered by the addition of TNF. Furthermore, TNF interfered with the activation of IL-6-induced gp130-signal transducer and activator of transcription (STAT) 3 and IL-4-STAT6 signaling, thereby abrogating IL-6-facilitated M2 macrophage polarization. Therefore, our results support the notion of a general role of TNF in the inflammatory activation of macrophages and define a new role of IL-6 signaling in macrophage polarization downstream of TNF.

Tumor Necrosis Factor-Mediated Survival of CD169+ Cells Promotes Immune Activation during Vesicular Stomatitis Virus Infection.

ABSTRACTInnate immune activation is essential to mount an effective antiviral response and to prime adaptive immunity. Although a crucial role of CD169+ cells during vesicular stomatitis virus (VSV) infections is increasingly recognized, factors regulating CD169+ cells during viral infections remain unclear. Here, we show that tumor necrosis factor is produced by CD11b+ Ly6C+ Ly6G+ cells following infection with VSV. The absence of TNF or TNF receptor 1 (TNFR1) resulted in reduced numbers of CD169+ cells and in reduced type I interferon (IFN-I) production during VSV infection, with a severe disease outcome. Specifically, TNF triggered RelA translocation into the nuclei of CD169+ cells; this translocation was inhibited when the paracaspase MALT-1 was absent. Consequently, MALT1 deficiency resulted in reduced VSV replication, defective innate immune activation, and development of severe disease. These findings indicate that TNF mediates the maintenance of CD169+ cells and innate and adaptive immune activation during VSV infection.IMPORTANCE Over the last decade, strategically placed CD169+ metallophilic macrophages in the marginal zone of the murine spleen and lymph nodes (LN) have been shown to play a very important role in host defense against viral pathogens. CD169+ macrophages have been shown to activate innate and adaptive immunity via “enforced virus replication,” a controlled amplification of virus particles. However, the factors regulating the CD169+ macrophages remain to be studied. In this paper, we show that after vesicular stomatitis virus infection, phagocytes produce tumor necrosis factor (TNF), which signals via TNFR1, and promote enforced virus replication in CD169+ macrophages. Consequently, lack of TNF or TNFR1 resulted in defective immune activation and VSV clearance.

Transmembrane Tumor Necrosis Factor Controls Myeloid-Derived Suppressor Cell Activity via TNF Receptor 2 and Protects from Excessive Inflammation during BCG-Induced Pleurisy.

Pleural tuberculosis (TB) is a form of extra-pulmonary TB observed in patients infected with Mycobacterium tuberculosis. Accumulation of myeloid-derived suppressor cells (MDSC) has been observed in animal models of TB and in human patients but their role remains to be fully elucidated. In this study, we analyzed the role of transmembrane TNF (tmTNF) in the accumulation and function of MDSC in the pleural cavity during an acute mycobacterial infection. Mycobacterium bovis BCG-induced pleurisy was resolved in mice expressing tmTNF, but lethal in the absence of tumor necrosis factor. Pleural infection induced MDSC accumulation in the pleural cavity and functional MDSC required tmTNF to suppress T cells as did pleural wild-type MDSC. Interaction of MDSC expressing tmTNF with CD4 T cells bearing TNF receptor 2 (TNFR2), but not TNFR1, was required for MDSC suppressive activity on CD4 T cells. Expression of tmTNF attenuated Th1 cell-mediated inflammatory responses generated by the acute pleural mycobacterial infection in association with effective MDSC expressing tmTNF and interacting with CD4 T cells expressing TNFR2. In conclusion, this study provides new insights into the crucial role played by the tmTNF/TNFR2 pathway in MDSC suppressive activity required during acute pleural infection to attenuate excessive inflammation generated by the infection.

Role of Gut Inflammation in Altering the Monocyte Compartment and Its Osteoclastogenic Potential in HLA-B27-Transgenic Rats.

To investigate the relationship between intestinal inflammation and the central and peripheral innate immune system in the pathogenesis of HLA-B27-associated spondyloarthritis using an HLA-B27-transgenic (B27-Tg) rat model. The myeloid compartment of the blood and bone marrow (BM) of B27-Tg rats, as well as HLA-B7-Tg and non-Tg rats as controls, was evaluated by flow cytometry. Plasma from rats was assessed by enzyme-linked immunosorbent assay for levels of CCL2 and interleukin-1α (IL-1α). Rats were treated with antibiotics for 4 weeks, and the myeloid compartment of the blood and BM was evaluated by flow cytometry. The osteoclastogenic potential of BM-derived cells from antibiotic-treated rats, in the presence or absence of tumor necrosis factor (TNF), was evaluated in vitro. B27-Tg rats had substantially higher numbers of circulating Lin-CD172a+CD43low monocytes as compared to control animals, and this was significantly correlated with higher levels of plasma CCL2. Antibiotic treatment of B27-Tg rats markedly reduced the severity of ileitis, plasma levels of CCL2 and IL-1α, and number of BM and blood Lin-CD172a+CD43low monocytes, a cell subset shown in the present study to have the greatest in vitro osteoclastogenic potential. Antibiotic treatment also prevented the TNF-dependent enhancement of osteoclastogenesis in B27-Tg rats. Microbiota-dependent intestinal inflammation in B27-Tg rats directly drives the systemic inflammatory and bone-erosive potential of the monocyte compartment.

Tumor Necrosis Factor and Its Receptors Are Crucial to Control Mycobacterium bovis Bacillus Calmette-Guerin Pleural Infection in a Murine Model

Tumor necrosis factor (TNF) is crucial to control Mycobacterium tuberculosis infection, which remains a leading cause of morbidity and mortality worldwide. TNF blockade compromises host immunity and may cause reactivation of latent infection, resulting in overt pulmonary, pleural, and extrapulmonary tuberculosis. Herein, we investigate the roles of TNF and TNF receptors in the control of Mycobacterium bovis bacillus Calmette-Guerin (BCG) pleural infection in a murine model. As controls, wild-type mice and those with a defective CCR5, a receptor that is crucial for control of viral infection but not for tuberculosis, were used. BCG-induced pleural infection was uncontrolled and progressive in absence of TNF or TNF receptor 1 (TNFR1)/TNFR2 (TNFR1R2) with increased inflammatory cell recruitment and bacterial load in the pleural cavity, and heightened levels of pleural and serum proinflammatory cytokines and chemokines, compared to wild-type control mice. The visceral pleura was thickened with chronic inflammation, which was prominent in TNF(-/-) and TNFR1R2(-/-) mice. The parietal pleural of TNF(-/-) and TNFR1R2(-/-) mice exhibited abundant inflammatory nodules containing mycobacteria, and these mice developed nonresolving inflammation and succumbed from disseminated BCG infection. By contrast, CCR5(-/-) mice survived and controlled pleural BCG infection as wild-type control mice. In conclusion, BCG-induced pleurisy was uncontrolled in the absence of TNF or TNF receptors with exacerbated inflammatory response, impaired bacterial clearance, and defective mesothelium repair, suggesting a critical role of TNF to control mycobacterial pleurisy.

Fatal Leishmaniasis in the Absence of TNF Despite a Strong Th1 Response.

Induction of inducible nitric oxide synthase in mononuclear phagocytes by IFN-γ and innate tumor necrosis factor (TNF) provide the basis for an effective immune response to the intracellular parasite Leishmania (L.) major. In previous experiments, we observed a fatal visceral form of leishmaniasis in L. major-infected C57BL/6 TNF-/- mice. To further delineate the protective function of TNF and its receptor requirements, we comparatively assessed L. major-infected C57BL/6 mice that were either deficient for membrane and soluble TNF (Tnf-/-), for soluble TNF alone (memTnfΔ/Δ), or the TNF receptors type 1 (Tnfr1-/-) or type 2 (Tnfr2-/-). We detected locally and systemically increased levels of the cytokine IFN-γ in the absence of the TNF-TNFR1-signaling pathway. An analysis of transcription factors and cytokines revealed that activated Tnf-/- CD4+ T cells displayed a highly active Th1 phenotype with a strong usage of the T cell receptor Vβ5.1/2. From these data we conclude that the fatal outcome of L. major infection in Tnf-/- mice does not result from a skewed or deficient Th1 differentiation.

TNF Modulates The Gut Microbiota And Drives Inflammation

Inflammatory Bowel Disease (IBD) consists of Ulcerative Colitis and Crohn's Disease (CD). The etiology remains unclear; however tumor necrosis factor (TNF) is known to promote the inflammation seen in these diseases. Many patients with IBD have elevated systemic and local levels of TNF and are treated with anti‐TNF drugs with variable success. It is also clear that the gut microbiota play a role in the development and perpetuation of IBD. We hypothesize that TNF modulates the gut microflora and that the severity of colitis correlates with specific microbiota shifts. To address our hypothesis, we examined the gut microbiota in mouse models of acute and chronic Trinitrobenzenesulfonic acid (TNBS) induced colitis in the presence (Wildtype, WT) and absence of TNF (TNF KO). Mice were given 1intra‐rectal (IR) injection (acute) TNBS. Feces were collected for DNA extraction and microbiome analyses. TNBS treated WT mice had more inflammation than TNBS treated TNF KOmice, proving that TNF drives inflammation in the acute model. Analyses of the fecal microbial communities at both days 0 and 10 revealed significant,Tnf‐dependent, differences in alpha and beta diversity, as well as notable differences in many species that were also primarily Tnf‐dependent. The results of the microbial analyses coupled with the results of the pathology support our hypothesis that TNF modulates the gut microbiota, which contributes to the severity of acute colitis. Analyses of the chronic studies are ongoing. We expect to see similar inflammatory results WT and TNF KOmice, with the potential for additional shifts in the microbiota as disease progresses from acute to chronic.

Possible chondroprotective effect of canakinumab: An in vitro study on human osteoarthritic chondrocytes

Canakinumab is a human IgGκ monoclonal antibody that neutralizes the activity of interleukin (IL)-1β blocking interaction with IL-1β receptors. Our study aimed to evaluate the in vitro effect of canakinumab on human osteoarthritic (OA) chondrocytes cultivated in the presence or absence of tumor necrosis factor (TNF)-α. Articular cartilage was obtained from the femoral heads of patients with osteoarthritis (OA). Chondrocytes were incubated with two concentrations (1μg/ml and 10μg/ml) of canakinumab alone or with TNF-α (10ng/ml) for 48h. We evaluated cell viability, release of proteoglycans (PG) and nitric oxide (NO) in culture medium, inducible nitric oxide synthase (iNOS) and metalloproteinanes (MMP)-1,3,13 gene expression, apoptosis, necrosis and morphological feature by transmission electron microscopy (TEM). Canakinumab alone did not have cytotoxic effect. Cell viability was reduced significantly (p<0.001) by TNF-α and restored by canakinumab at both concentrations used. TNF-α determined a significant decrease of PG (p<0.001) and an increase of NO (p<0.001) and MMP-1,3,13 gene expression. Canakinumab significantly increased the PG levels and decreased (1μg/ml, p<0.01; 10μg/ml, p<0.01) NO levels in cells cultured with TNF-α. The NO data were confirmed by the immunocytochemistry assay for iNOS. A significant reduction of MMP-1,3,13 gene expression was induced by canakinumab. Our experiments confirmed the pro-apoptotic effect of TNF-α and demonstrated a protective role of canakinumab. The results concerning biochemical data were further confirmed by the morphological findings obtained by TEM. We showed that canakinumab counteracts the negative effects of TNF-α on OA chondrocyte cultures and may have a potential chondroprotective role in OA.

Transient TNF regulates the self-renewing capacity of stem-like label-retaining cells in sphere and skin equivalent models of melanoma.

BackgroundIt is well established that inflammation promotes cancer, including melanoma, although the exact mechanisms involved are less known. In this study, we tested the hypothesis that inflammatory factors affect the cancer stem cell (CSC) compartment responsible for tumor development and relapse.ResultsUsing an inducible histone 2B-GFP fusion protein as a tracer of cell divisional history, we determined that tumor necrosis factor (TNF), which is a classical pro-inflammatory cytokine, enlarged the CSC pool of GFP-positive label-retaining cells (LRCs) in tumor-like melanospheres. Although these cells acquired melanoma stem cell markers, including ABCB5 and CD271, and self-renewal ability, they lost their capacity to differentiate, as evidenced by the diminished MelanA expression in melanosphere cells and the loss of pigmentation in a skin equivalent model of human melanoma. The undifferentiated cell phenotype could be reversed by LY294002, which is an inhibitor of the PI3K/AKT signaling pathway, and this reversal was accompanied by a significant reduction in CSC phenotypic markers and functional properties. Importantly, the changes induced by a transient exposure to TNF were long-lasting and observed for many generations after TNF withdrawal.ConclusionsWe conclude that pro-inflammatory TNF targets the quiescent/slow-cycling melanoma SC compartment and promotes PI3K/AKT-driven expansion of melanoma SCs most likely by preventing their asymmetrical self-renewal. This TNF effect is maintained and transferred to descendants of LRC CSCs and is manifested in the absence of TNF, suggesting that a transient exposure to inflammatory factors imprints long-lasting molecular and/or cellular changes with functional consequences long after inflammatory signal suppression. Clinically, these results may translate into an inflammation-triggered accumulation of quiescent/slow-cycling CSCs and a post-inflammatory onset of an aggressive tumor.

SAT0443 In Vitro Effects of Canakinumab on Human Osteoarthritic Chondrocytes

BackgroundCanakinumab is a human IgGκ monoclonal antibody targeting Interleukin (IL)-1β; its action involves the neutralization of the signaling of IL-1β, the most important cytokine in the pathogenesis of Osteoartrhitis (OA)....

Antibody‐mediated control of Trypanosoma vivax infection fails in the absence of tumour necrosis factor

Trypanosoma vivax causes a wasting disease affecting livestock breeding and agriculture in developing countries of sub-Sahara Africa and South America. Being an extracellular parasite, control of T.vivax has been proposed to be mediated by host antibodies. However, the use of a comparative infection model of wild-type (WT) and tumour necrosis factor (TNF) knockout (TNF(-/-) ) mice shows that the latter is unable to control first-peak parasitaemia, despite the presence of specific antitrypanosome antibodies. In contrast, WT mice parasitaemia peak control coincides with a combined early onset of TNF production and induction of specific antibodies. TNF is mainly produced by liver-associated monocytes and neutrophils. In this study, no other correlation between cellular immunomodulations and peak parasitaemia control was observed, underscoring the importance of the role of TNF in the control of T.vivax infections.

Transient TNF regulates the self-renewing capacity of stem-like label-retaining cells in sphere and skin equivalent models of melanoma

It is well established that inflammation promotes cancer, including melanoma, although the exact mechanisms involved are less known. In this study, we tested the hypothesis that inflammatory factors affect the cancer stem cell (CSC) compartment responsible for tumor development and relapse. Using an inducible histone 2B-GFP fusion protein as a tracer of cell divisional history, we determined that tumor necrosis factor (TNF), which is a classical pro-inflammatory cytokine, enlarged the CSC pool of GFP-positive label-retaining cells (LRCs) in tumor-like melanospheres. Although these cells acquired melanoma stem cell markers, including ABCB5 and CD271, and self-renewal ability, they lost their capacity to differentiate, as evidenced by the diminished MelanA expression in melanosphere cells and the loss of pigmentation in a skin equivalent model of human melanoma. The undifferentiated cell phenotype could be reversed by LY294002, which is an inhibitor of the PI3K/AKT signaling pathway, and this reversal was accompanied by a significant reduction in CSC phenotypic markers and functional properties. Importantly, the changes induced by a transient exposure to TNF were long-lasting and observed for many generations after TNF withdrawal. We conclude that pro-inflammatory TNF targets the quiescent/slow-cycling melanoma SC compartment and promotes PI3K/AKT-driven expansion of melanoma SCs most likely by preventing their asymmetrical self-renewal. This TNF effect is maintained and transferred to descendants of LRC CSCs and is manifested in the absence of TNF, suggesting that a transient exposure to inflammatory factors imprints long-lasting molecular and/or cellular changes with functional consequences long after inflammatory signal suppression. Clinically, these results may translate into an inflammation-triggered accumulation of quiescent/slow-cycling CSCs and a post-inflammatory onset of an aggressive tumor.

An anti-TNFR1 scFv-HSA fusion protein as selective antagonist of TNF action

IZI-06.1 is a humanized anti-TNFR1 single-chain fragment variable (scFv) that selectively inhibits binding of tumor necrosis factor (TNF) and lymphotoxin alpha to tumor necrosis factor receptor 1 (TNFR1) but not TNFR2. Recently, IZI-06.1 was converted into a fully human IgG1 antibody (ATROSAB) for the treatment of inflammatory diseases. Here, we compare the bivalent ATROSAB with a monovalent scFv-human serum albumin (HSA) fusion protein lacking any antibody-associated effector functions and possessing approximately only half the molecular mass of an IgG, which should facilitate accumulation in inflamed tissues. Furthermore, the half-life of the scFv should be strongly extended while maintaining monovalent binding, avoiding a possible signal transduction by receptor cross-linking in the absence of TNF. The scFv-HSA fusion protein was produced by stably transfected Chinese hamster ovary cells and purified by affinity chromatography. The fusion protein bound specifically to TNFR1 in enzyme-linked immunosorbent assay and TNFR1-transfected mouse embryonic fibroblasts. Affinity determined by quartz crystal microbalance was reduced compared with ATROSAB, which resulted also in a reduced inhibitory activity. Compared with the scFv fragment, the half-life of the fusion protein was significantly increased, although not reaching the long half-life of ATROSAB. In summary, the scFv-HSA may provide an alternative to the full-length IgG1 with the ability to selectively inhibit TNFR1 and exploiting the pharmacokinetic properties of albumin.

A functional genomics screen for microRNA regulators of NF-kappaB signaling.

BackgroundThe nuclear factor-KappaB (NF-κB) pathway is conserved from fruit flies to humans and is a key mediator of inflammatory signaling. Aberrant regulation of NF-κB is associated with several disorders including autoimmune disease, chronic inflammation, and cancer, making the NF-κB pathway an attractive therapeutic target. Many regulatory components of the NF-κB pathway have been identified, including microRNAs (miRNAs). miRNAs are small non-coding RNAs and are common components of signal transduction pathways. Here we present a cell-based functional genomics screen to systematically identify miRNAs that regulate NF-κB signaling.ResultsWe screened a library of miRNA mimics using a NF-κB reporter cell line in the presence and absence of tumor necrosis factor (+/- TNF). There were 9 and 15 hits in the -TNF and +TNF screens, respectively. We identified putative functional targets of these hits by integrating computational predictions with NF-κB modulators identified in a previous genome-wide cDNA screen. miR-517a and miR-517c were the top hits, activating the reporter 86- and 126-fold, respectively. Consistent with these results, miR-517a/c induced the expression of endogenous NF-κB targets and promoted the nuclear localization of p65 and the degradation of IκB. We identified TNFAIP3 interacting protein1 (TNIP1) as a target and characterized a functional SNP in the miR-517a/c binding site. Lastly, miR-517a/c induced apoptosis in vitro, which was phenocopied by knockdown of TNIP1.ConclusionsOur study suggests that miRNAs are common components of NF-κB signaling and miR-517a/c may play an important role in linking NF-κB signaling with cell survival through TNIP1.

Abstract 1183: Monoallelic expression in codon 72 of TP53 in heterozygote individuals

Abstract Background &amp; Aim: Correlations between DNA variation and human phenotypic differences, such as susceptibility to certain diseases, are not well understood. Polymorphisms can contribute to the observed variation in complex human traits, but their relative contributions remain to be determined. Expression differences between alleles of the same gene have been observed, contributing to phenotypic variation between individuals. Studies showed that variations exist in the relative allelic expression levels in certain genes of heterozygote individuals. Polymorphism at codon 72 of TP53 results in either Arginine or Proline, whose functional significance in carcinogenesis is controversial. Studies showed that the Pro72 is less efficient than Arg72 allele in suppressing cell transformation and inducing apoptosis. We have investigated if the expression of these p53 polymorphs is selectively regulated, using mRNA and DNA from colorectal cancer (CRC). Methods: 89 non-related patients treated at ACCamargo Hospital/Brazil were evaluated. DNA/RNA were isolated from frozen tumor tissue using phenol/chloroform protocol and evaluated by sequencing. Associations were analyzed by Pearson Chi-Square or Fisher Exact tests and Multiple Logistic Regression. Results: We found 24 (27%) heterozygotes tumors and 10 (41.7%) of these showed monoallelic expression: 70% (7/10) of them preferentially expressed the Pro allele. Thus, the allele frequencies showed a discrete decrease, from 0.26 for Pro allele of expression analysis to 0.25 for Pro allele of tumor genomic analysis. Previous results revealed that the p53 codon 72 expression was significantly associated with gender (P= .037), recurrence (P= .005), dirty tumor necrosis (P= .025), border pattern of tumor growth (P= .05), post chemoradiotherapy use (P= .002), p53 immunohistochemistry expression (P= .041) and TP53 mutation (P= .004), suggesting that the presence of at least one Arg72 allele is associated with better prognosis. According to multiple logistic regression the variable independently associated with the presence of the Arginine allele is the absence of tumor necrosis factor (OD= 8.64/P= .047). This is the first report describing the monoallelic expression of the p53 codon 72 in CRC and revealed that heterozygotes preferentially express the Pro allele, suggesting that the Pro allele is selectively activated during CRC development. Conclusions: The expression of the different p53 polymorphs is selectively regulated in Pro72Arg heterozygotes individuals. The silent Arg allele seems to have a negative relation with cancer susceptibility, since it is better in inducing apoptosis. The differences in the expression of heterozygotes resulted in an increase in the total numbers of Pro expressers in this cancer population. Thus, the expression status of the p53 polymorphs, rather than the genotypic status, might be a useful indicator for cancer susceptibility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1183. doi:1538-7445.AM2012-1183