Expression of E-cadherin repressors SNAIL, ZEB1 and ZEB2 by tumour and stromal cells influences tumour-budding phenotype and suggests heterogeneity of stromal cells in pancreatic cancer.

BACKGROUND There is evidence that tumour-stroma interactions have a major role in the neoplastic progression of pancreatic ductal adenocarcinoma (PDAC). Tumour budding is thought to reflect the process of epithelial-mesenchymal transition (EMT); however, the relationship between tumour buds and EMT remains unclear. Here we characterize the tumour-budding- and stromal cells in PDAC at protein and mRNA levels concerning factors involved in EMT. METHODS mRNA in situ hybridisation and immunostaining for E-cadherin, β-catenin, SNAIL1, ZEB1, ZEB2, N-cadherin and TWIST1 were assessed in the main tumour, tumour buds and tumour stroma on multipunch tissue microarrays from 120 well-characterised PDACs and associated with the clinicopathological features, including peritumoural (PTB) and intratumoural (ITB) budding. RESULTS Tumour-budding cells showed increased levels of ZEB1 (P<0.0001) and ZEB2 (P=0.0119) and reduced E-cadherin and β-catenin (P<0.0001, each) compared with the main tumour. Loss of membranous β-catenin in the main tumour (P=0.0009) and tumour buds (P=0.0053), without nuclear translocation, as well as increased SNAIL1 in tumour and stromal cells (P=0.0002, each) correlated with high PTB. ZEB1 overexpression in the main tumour-budding and stromal cells was associated with high ITB (P=0.0084; 0.0250 and 0.0029, respectively) and high PTB (P=0.0005; 0.0392 and 0.0007, respectively). ZEB2 overexpression in stromal cells correlated with higher pT stage (P=0.03), lymphatic invasion (P=0.0172) and lymph node metastasis (P=0.0152). CONCLUSIONS In the tumour microenvironment of phenotypically aggressive PDAC, tumour-budding cells express EMT hallmarks at protein and mRNA levels underlining their EMT-type character and are surrounded by stromal cells expressing high levels of the E-cadherin repressors ZEB1, ZEB2 and SNAIL1, this being strongly associated with the tumour-budding phenotype. Moreover, our findings suggest the existence of subtypes of stromal cells in PDAC with phenotypical and functional heterogeneity.

Molecular cloning, expression analysis and assignment of the porcine tumor necrosis factor superfamily member 10 gene (TNFSF10) to SSC13q34-->q36 by fluorescence in situ hybridization and radiation hybrid mapping

We have cloned the complete coding region of the porcine TNFSF10 gene. The porcine TNFSF10 cDNA has an ORF of 870 nucleotides and shares 85% identity with human TNFSF10, and 75% and 72% identity with rat and mouse Tnfsf10 coding sequences, respectively. The deduced porcine TNFSF10 protein consists of 289 amino acids with the calculated molecular mass of 33.5 kDa and a predicted pI of 8.15. The amino acid sequence similarities correspond to 86, 72 and 70% when compared with human, rat and mouse sequences, respectively. Northern blot analysis detected TNFSF10-specific transcripts (approximately 1.7 kb) in various organs of a 10-week-old pig, suggesting ubiquitous expression. Real-time RT-PCR studies of various organs from fetal (days 73 and 98) and postnatal stages (two weeks, eight months) demonstrated developmental and tissue-specific regulation of TNFSF10 mRNA abundance. The chromosomal location of the porcine TNFSF10 gene was determined by FISH of a specific BAC clone to metaphase chromosomes. This TNFSF10 BAC clone has been assigned to SSC13q34-->q36. Additionally, the localization of the TNFSF10 gene was verified by RH mapping on the porcine IMpRH panel.

Dual role of tumour-infiltrating T helper 17 cells in human colorectal cancer

BACKGROUND The immune contexture predicts prognosis in human colorectal cancer (CRC). Whereas tumour-infiltrating CD8+ T cells and myeloid CD16+ myeloperoxidase (MPO)+ cells are associated with favourable clinical outcome, interleukin (IL)-17-producing cells have been reported to correlate with severe prognosis. However, their phenotypes and functions continue to be debated. OBJECTIVE To investigate clinical relevance, phenotypes and functional features of CRC-infiltrating, IL-17-producing cells. METHODS IL-17 staining was performed by immunohistochemistry on a tissue microarray including 1148 CRCs. Phenotypes of IL-17-producing cells were evaluated by flow cytometry on cell suspensions obtained by enzymatic digestion of clinical specimens. Functions of CRC-isolated, IL-17-producing cells were assessed by in vitro and in vivo experiments. RESULTS IL-17+ infiltrates were not themselves predictive of an unfavourable clinical outcome, but correlated with infiltration by CD8+ T cells and CD16+ MPO+ neutrophils. Ex vivo analysis showed that tumour-infiltrating IL-17+ cells mostly consist of CD4+ T helper 17 (Th17) cells with multifaceted properties. Indeed, owing to IL-17 secretion, CRC-derived Th17 triggered the release of protumorigenic factors by tumour and tumour-associated stroma. However, on the other hand, they favoured recruitment of beneficial neutrophils through IL-8 secretion and, most importantly, they drove highly cytotoxic CCR5+CCR6+CD8+ T cells into tumour tissue, through CCL5 and CCL20 release. Consistent with these findings, the presence of intraepithelial, but not of stromal Th17 cells, positively correlated with improved survival. CONCLUSIONS Our study shows the dual role played by tumour-infiltrating Th17 in CRC, thus advising caution when developing new IL-17/Th17 targeted treatments.

Development of a Tumour Growth Inhibition Model to Elucidate the Effects of Ritonavir on Intratumoural Metabolism and Anti-tumour Effect of Docetaxel in a Mouse Model for Hereditary Breast Cancer

In a mouse tumour model for hereditary breast cancer, we previously explored the anti-cancer effects of docetaxel, ritonavir and the combination of both and studied the effect of ritonavir on the intratumoural concentration of docetaxel. The objective of the current study was to apply pharmacokinetic (PK)-pharmacodynamic (PD) modelling on this previous study to further elucidate and quantify the effects of docetaxel when co-administered with ritonavir. PK models of docetaxel and ritonavir in plasma and in tumour were developed. The effect of ritonavir on docetaxel concentration in the systemic circulation of Cyp3a knock-out mice and in the implanted tumour (with inherent Cyp3a expression) was studied, respectively. Subsequently, we designed a tumour growth inhibition model that included the inhibitory effects of both docetaxel and ritonavir. Ritonavir decreased docetaxel systemic clearance with 8% (relative standard error 0.4%) in the co-treated group compared to that in the docetaxel only-treated group. The docetaxel concentration in tumour tissues was significantly increased by ritonavir with mean area under the concentration-time curve 2.5-fold higher when combined with ritonavir. Observed tumour volume profiles in mice could be properly described by the PK/PD model. In the co-treated group, the enhanced anti-tumour effect was mainly due to increased docetaxel tumour concentration; however, we demonstrated a small but significant anti-tumour effect of ritonavir addition (p value <0.001). In conclusion, we showed that the increased anti-tumour effect observed when docetaxel is combined with ritonavir is mainly caused by enhanced docetaxel tumour concentration and to a minor extent by a direct anti-tumour effect of ritonavir.

Improving 4D plan quality for PBS-based liver tumour treatments by combining online image guided beam gating with rescanning.

Pencil beam scanned (PBS) proton therapy has many advantages over conventional radiotherapy, but its effectiveness for treating mobile tumours remains questionable. Gating dose delivery to the breathing pattern is a well-developed method in conventional radiotherapy for mitigating tumour-motion, but its clinical efficiency for PBS proton therapy is not yet well documented. In this study, the dosimetric benefits and the treatment efficiency of beam gating for PBS proton therapy has been comprehensively evaluated. A series of dedicated 4D dose calculations (4DDC) have been performed on 9 different 4DCT(MRI) liver data sets, which give realistic 4DCT extracting motion information from 4DMRI. The value of 4DCT(MRI) is its capability of providing not only patient geometries and deformable breathing characteristics, but also includes variations in the breathing patterns between breathing cycles. In order to monitor target motion and derive a gating signal, we simulate time-resolved beams' eye view (BEV) x-ray images as an online motion surrogate. 4DDCs have been performed using three amplitude-based gating window sizes (10/5/3 mm) with motion surrogates derived from either pre-implanted fiducial markers or the diaphragm. In addition, gating has also been simulated in combination with up to 19 times rescanning using either volumetric or layered approaches. The quality of the resulting 4DDC plans has been quantified in terms of the plan homogeneity index (HI), total treatment time and duty cycle. Results show that neither beam gating nor rescanning alone can fully retrieve the plan homogeneity of the static reference plan. Especially for variable breathing patterns, reductions of the effective duty cycle to as low as 10% have been observed with the smallest gating rescanning window (3 mm), implying that gating on its own for such cases would result in much longer treatment times. In addition, when rescanning is applied on its own, large differences between volumetric and layered rescanning have been observed as a function of increasing number of re-scans. However, once gating and rescanning is combined, HI to within 2% of the static plan could be achieved in the clinical target volume, with only moderately prolonged treatment times, irrespective of the rescanning strategy used. Moreover, these results are independent of the motion surrogate used. In conclusion, our results suggest image guided beam gating, combined with rescanning, is a feasible, effective and efficient motion mitigation approach for PBS-based liver tumour treatments.

Interferon-gamma release assays for latent tuberculosis infection in child and young adult candidates or recipients of tumor necrosis factor inhibitor therapy

Background. Inhibition of tumor necrosis factor (TNF) is associated with progression of latent tuberculosis infection (LTBI) to active disease. LTBI screening prior to starting TNF inhibitor therapy is recommended. Blood tests, collectively known as interferon-gamma release assays (IGRAs), offer a means other than the tuberculin skin test (TST) of screening for LTBI. However, in the setting of immune compromise, anergy may limit the clinical utility of IGRAs. ^ Methods. A cross-sectional study was conducted in children and young adults ≤ 21 years of age who were cared for at Texas Children's Hospital in Houston, TX, during 2011 and who were candidates for, or were receiving, tumor necrosis factor (TNF)-inhibitor therapy. All subjects answered a risk factor questionnaire and were tested for LTBI by two commercially available IGRAs (QuantiFERON-Gold In-Tube assay and the T-SPOT.TB assay), along with the TST. T-cell phenotypes were evaluated through flow cytometry, both at baseline and after antigen stimulation. ^ Results. Twenty-eight subjects were enrolled. All were TST negative and none were IGRA positive. Results were negative for the 27 subjects who were tested with QuantiFERON-Gold In-Tube. However, 26% of subjects demonstrated anergy in the T-SPOT.T. Patients with T-SPOT. TB anergy had lower quantitative IFN-γ responses to mitogen in the QFT assay—the mean IFN-γ level to mitogen in patients without T-SPOT.TB anergy was 9.84 IU/ml compared to 6.91 IU/ml in patients with T-SPOT.TB anergy (P = 0.046). Age and use of TNF inhibitors, corticosteroids, or methotrexate use were not significantly associated with T-SPOT.TB anergy. Antigen stimulation revealed depressed expression of intracellular IFN-γ in subjects with T-SPOT. TB anergy. ^ Conclusions. The frequency of anergy in this population is higher than would be expected from studies in adults. There appears to be inappropriate IFN-γ responses to antigen in subjects with T-SPOT. TB anergy. This immune defect was detected by the T-SPOT. TB assay but not by the QuantiFERON-Gold In-Tube assay. Further data are needed to clarify the utility of IGRAs in this population.^

Dissection of antitumor activity by lymphokine -activated killer cells: Role of FcR+ precursors and obligatory role of tumor necrosis factor-$\alpha$ in antibody -dependent cellular cytotoxicity

Human peripheral blood lymphocytes (PBL) cultured for varying lengths of time in IL-2 are able to mediate antibody independent cellular cytotoxicity (AICC) as well as antibody dependent cellular cytotoxicity (ADCC) against a wide range of tumor targets. The objective of our study is to determine the cytotoxic potential of the subset of LAK cells involved in ADCC, the tumor recognition mechanism in ADCC, the kinetics of ADCC mediated by PBL cultured under various conditions and the role of TNF-$\alpha$ in the development and maturation of ADCC effectors in the LAK population.^ The model system in this study for ADCC used a monoclonal antibody 14G2a (IgG2a), that recognizes the GD2 epitope on human melanoma cell line, SK-Mel-1. The target recognition mechanism operative in AICC (traditionally known as lymphokine activated killing or LAK) is an acquired property of these IL-2 activated cells which confers on them the unique ability to distinguish between tumor and normal cells. This recognition probably involves the presence of a trypsin sensitive N-linked glycoprotein epitope on tumor cells. Proteolytic treatment of the tumor cells with trypsin renders them resistant to AICC by PBL cultured in IL-2. However, ADCC is unaffected. This ADCC, mediated by the relatively small population of cells that are positive for the Fc receptor for IgG (FcR), is an indication that this subset of "LAK" cells does not require the trypsin sensitive epitope on tumor cells to mediate killing. Enriching PBL for FcR+ cells markedly enhanced both AICC and ADCC and also reduced the IL-2 requirement of these cells.^ The stoichiometry of Fc receptor (FcR) expression on the cytotoxic effectors does not correlate with ADCC lytic activity. Although FcRs are necessary to mediate ADCC, other factors, appear to regulate the magnitude of cytolytic activity. In order to investigate these putative factors, the kinetics of ADCC development was studied under various conditions (in IL-2 (10u/ml) and 100u/ml), in IL-2(10u/ml) + TNF$\alpha$ (500u/ml) and in TNF-$\alpha$ (500u/ml) alone). Addition of exogenous TNF-$\alpha$ into the four hour cytotoxicity assay did not increase ADCC, nor did anti-TNF antibodies result in inhibition. On the other hand, addition of anti-TNF antibodies to PBL and IL-2 for 24 hours, resulted in a marked inhibition of the ADCC, suggesting that endogenous TNF-$\alpha$ is obligatory for the maturation and differentiation of ADCC effectors. ^

A distinct element involved in the lipopolysaccharide activation of the tumor necrosis factor -alpha promoter in a promonocytic cell line, THP-1

TNF-α is a pleiotropic cytokine involved in normal homeostasis and plays a key role in defending the host from infection and malignancy. However when deregulated, TNF-α can lead to various disease states. Therefore, understanding the mechanisms by which TNF-α is regulated may aid in its control. In spite of the knowledge gained regarding the transcriptional regulation of TNF-α further characterization of specific TNF-α promoter elements remains to be elucidated. In particular, the T&barbelow;NF-α A&barbelow;P-1/C&barbelow;RE-like (TAC) element of the TNF-α promoter has been shown to be important in the regulation of TNF-α in lymphocytes. Activating transcription factor-2 (ATF-2) and c-Jun were shown to bind to and transactivate the TAC element However, the role of TAC and transcription factors ATF-2 and c-Jun in the regulation of TNF-α in monocytes is not as well characterized. Lipopolysaccharide (LPS), a potent activator of TNF-α in monocytes, provides a good model to study the involvement of TAC in TNF-α regulation. On the other hand, all-tram retinoic acid (ATRA), a physiological monocyte-differentiation agent, is unable to induce TNF-α protein release. ^ To delineate the functional role of TAC, we transfected the wildtype or the TAC deleted TNF-α promoter-CAT construct into THP-1 promonocytic cells before stimulating them with LPS. CAT activity was induced 17-fold with the wildtype TNF-α promoter, whereas the CAT activity was uninducible when the TAC deletion mutant was used. This daft suggests that TAC is vital for LPS to activate the TNF-α promoter. Electrophoretic mobility shift assays using the TAC element as a probe showed a unique pattern for LPS-activated cells: the disappearance of the upper band of a doublet seen in untreated and ATRA treated cells. Supershift analysis identified c-Jun and ATF-2 as components of the LPS-stimulated binding complex. Transient transfection studies using dominant negative mutants of JNK, c-Jun, or ATF-2 suggest that these proteins we important for LPS to activate the TNF-α promoter. Furthermore, an increase in phosphorylated or activated c-Jun was bound to the TAC element in LPS-stimulated cells. Increased c-Jun activation was correlated with increased activity of Jun N-terminal kinase (JNK), a known upstream stimulator of c-Jun and ATF-2, in LPS-stimulated monocytes. On the other hand, ATRA did not induce TNF-α protein release nor changes in the phosphorylation of c-Jun or JNK activity, suggesting that pathways leading to ATRA differentiation of monocytic cells are independent of TNF-α activation. Together, the induction of TNF-α gene expression seems to require JNK activation, and activated c-Jun binding to the TAC element of the TNF-α promoter in THP-1 promonocytic cells. ^

Caracterización de la necrosis sistémica inducida por la interacción sinérgica entre el Virus X de la patata y potyvirus en Nicotiana benthamiana

Los virus de plantas pueden causar enfermedades severas que conllevan serias pérdidas económicas a nivel mundial. Además, en la naturaleza son comunes las infecciones simultáneas con distintos virus que conducen a la exacerbación de los síntomas de enfermedad, fenómeno al que se conoce como sinergismo viral. Una de las sintomatologías más severas causadas por los virus en plantas susceptibles es la necrosis sistémica (NS), que incluso puede conducir a la muerte del huésped. Este fenotipo ha sido comparado en ocasiones con la respuesta de resistencia de tipo HR, permitiendo establecer una serie de paralelismos entre ambos tipos de respuesta que sugieren que la NS producida en interacciones compatibles sería el resultado de una respuesta hipersensible sistémica (SHR). Sin embargo, los mecanismos moleculares implicados en el desarrollo de la NS, su relación con procesos de defensa antiviral o su relevancia biológica aún no son bien entendidos, al igual que tampoco han sido estudiados los cambios producidos en la planta a escala genómica en infecciones múltiples que muestran sinergismo en patología. En esta tesis doctoral se han empleado distintas aproximaciones de análisis de expresión génica, junto con otras técnicas genéticas y bioquímicas, en el sistema modelo de Nicotiana benthamiana para estudiar la NS producida por la infección sinérgica entre el Virus X de la patata (PVX) y diversos potyvirus. Se han comparado los cambios producidos en el huésped a nivel genómico y fisiológico entre la infección doble con PVX y el Virus Y de la patata (PVY), y las infecciones simples con PVX o PVY. Además, los cambios transcriptómicos y hormonales asociados a la infección con la quimera viral PVX/HC‐Pro, que reproduce los síntomas del sinergismo entre PVX‐potyvirus, se han comparado con aquellos producidos por otros dos tipos de muerte celular, la PCD ligada a una interacción incompatible y la PCD producida por la disfunción del proteasoma. Por último, técnicas de genética reversa han permitido conocer la implicación de factores del huésped, como las oxilipinas, en el desarrollo de la NS asociada al sinergismo entre PVXpotyvirus. Los resultados revelan que, respecto a las infecciones con solo uno de los virus, la infección doble con PVX‐PVY produce en el huésped diferencias cualitativas además de cuantitativas en el perfil transcriptómico relacionado con el metabolismo primario. Otros cambios en la expresión génica, que reflejan la activación de mecanismos de defensa, correlacionan con un fuerte estrés oxidativo en las plantas doblemente infectadas que no se detecta en las infecciones simples. Además, medidas en la acumulación de determinados miRNAs implicados en diversos procesos celulares muestran como la infección doble altera de manera diferencial tanto la acumulación de estos miRNAs como su funcionalidad, lo cual podría estar relacionado con los cambios en el transcriptoma, así como con la sintomatología de la infección. La comparación a nivel transcriptómico y hormonal entre la NS producida por PVX/HC‐Pro y la interacción incompatible del Virus del mosaico del tabaco en plantas que expresan el gen N de resistencia (SHR), muestra que la respuesta en la interacción compatible es similar a la que se produce durante la SHR, si bien se presenta de manera retardada en el tiempo. Sin embargo, los perfiles de expresión de genes de defensa y de respuesta a hormonas, así como la acumulación relativa de ácido salicílico (SA), ácido jasmonico (JA) y ácido abscísico, en la interacción compatible son más semejantes a la respuesta PCD producida por la disfunción del proteasoma que a la interacción incompatible. Estos datos sugieren una contribución de la interferencia sobre la funcionalidad del proteasoma en el incremento de la patogenicidad, observado en el sinergismo PVX‐potyvirus. Por último, los resultados obtenidos al disminuir la expresión de 9‐LOX, α‐DOX1 y COI1, relacionados con la síntesis o con la señalización de oxilipinas, y mediante la aplicación exógena de JA y SA, muestran la implicación del metabolismo de las oxilipinas en el desarrollo de la NS producida por la infección sinérgica entre PVXpotyvirus en N. benthamiana. Además, estos resultados indican que la PCD asociada a esta infección, al igual que ocurre en interacciones incompatibles, no contiene necesariamente la acumulación viral, lo cual indica que necrosis e inhibición de la multiplicación viral son procesos independientes. ABSTRACT Plant viruses cause severe diseases that lead to serious economic losses worldwide. Moreover, simultaneous infections with several viruses are common in nature leading to exacerbation of the disease symptoms. This phenomenon is known as viral synergism. Systemic necrosis (SN) is one of the most severe symptoms caused by plant viruses in susceptible plants, even leading to death of the host. This phenotype has been compared with the hypersensitive response (HR) displayed by resistant plants, and some parallelisms have been found between both responses, which suggest that SN induced by compatible interactions could be the result of a systemic hypersensitive response (SHR). However, the molecular mechanisms involved in the development of SN, its relationship with antiviral defence processes and its biological relevance are still unknown. Furthermore, the changes produced in plants by mixed infections that cause synergistic pathological effects have not been studied in a genome‐wide scale. In this doctoral thesis different approaches have been used to analyse gene expression, together with other genetic and biochemical techniques, in the model plant Nicotiana benthamiana, in order to study the SN produced by the synergistic infection of Potato virus X (PVX) with several potyviruses. Genomic and physiological changes produced in the host by double infection with PVX and Potato virus Y (PVY), and by single infection with PVX or PVY have been compared. In addition, transcriptional and hormonal changes associated with infection by the chimeric virus PVX/HC‐Pro, which produces synergistic symptoms similar to those caused by PVX‐potyvirus, have been compared with those produced by other types of cell death. These types of cell death are: PCD associated with an incompatible interaction, and PCD produced by proteasome disruption. Finally, reverse genetic techniques have revealed the involvement of host factors, such as oxylipins, in the development of SN associated with PVX‐potyvirus synergism. The results revealed that compared with single infections, double infection with PVX‐PVY produced qualitative and quantitative differences in the transcriptome profile, mainly related to primary metabolism. Other changes in gene expression, which reflected the activation of defence mechanisms, correlated with a severe oxidative stress in doubly infected plants that was undetected in single infections. Additionally, accumulation levels of several miRNAs involved in different cellular processes were measured, and the results showed that double infection not only produced the greatest variations in miRNA accumulation levels but also in miRNA functionality. These variations could be related with transcriptomic changes and the symptomatology of the infection. Transcriptome and hormone level comparisons between SN induced by PVX/HCPro and the incompatible interaction produced by Tobacco mosaic virus in plants expressing the N resistance gene (SHR), showed some similarities between both responses, even though the compatible interaction appeared retarded in time. Nevertheless, the expression profiles of both defence‐related genes and hormoneresponsive genes, as well as the relative accumulation of salicylic acid (SA), jasmonic acid (JA) and abscisic acid in the compatible interaction are more similar to the PCD response produced by proteasome disruption. These data suggest that interference with proteasome functionality contributes to the increase in pathogenicity associated with PVX‐potyvirus synergism. Finally, the results obtained by reducing the expression of 9‐LOX, α‐DOX1 and COI1, related with synthesis or signalling of oxylipins, and by applying exogenously JA and SA, revealed that oxylipin metabolism is involved in the development of SN induced by PVX‐potyvirus synergistic infections in N. benthamiana. Moreover, these results also indicated that PVX‐potyvirus associated PCD does not necessarily restrict viral accumulation, as is also the case in incompatible interactions. This indicates that both necrosis and inhibition of viral multiplication are independent processes.

Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for tumor necrosis factor-induced cytokine expression

The p38 mitogen-activated protein kinase is activated by treatment of cells with cytokines and by exposure to environmental stress. The effects of these stimuli on p38 MAP kinase are mediated by the MAP kinase kinases (MKKs) MKK3, MKK4, and MKK6. We have examined the function of the p38 MAP kinase signaling pathway by investigating the effect of targeted disruption of the Mkk3 gene. Here we report that Mkk3 gene disruption caused a selective defect in the response of fibroblasts to the proinflammatory cytokine tumor necrosis factor, including reduced p38 MAP kinase activation and cytokine expression. These data demonstrate that the MKK3 protein kinase is a critical component of a tumor necrosis factor-stimulated signaling pathway that causes increased expression of inflammatory cytokines.

Direct adenovirus-mediated gene transfer of interleukin 1 and tumor necrosis factor α soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects

Adenoviral vectors were used to deliver genes encoding a soluble interleukin 1 (IL-1)-type I receptor-IgG fusion protein and/or a soluble type I tumor necrosis factor α (TNFα) receptor-IgG fusion protein directly to the knees of rabbits with antigen-induced arthritis. When tested individually, knees receiving the soluble IL-1 receptor had significantly reduced cartilage matrix degradation and white blood cell infiltration into the joint space. Delivery of the soluble TNFα receptor was less effective, having only a moderate effect on white blood cell infiltration and no effect on cartilage breakdown. When both soluble receptors were used together, there was a greater inhibition of white blood cell infiltration and cartilage breakdown with a considerable reduction of synovitis. Interestingly, anti-arthritic effects were also seen in contralateral control knees receiving only a marker gene, suggesting that sustained local inhibition of disease activity in one joint may confer an anti-arthritic effect on other joints. These results suggest that local intra-articular gene transfer could be used to treat systemic polyarticular arthritides.

Hepatitis B virus HBx protein sensitizes cells to apoptotic killing by tumor necrosis factor α

Persistent infection with hepatitis B virus (HBV) is a leading cause of human liver disease and is strongly associated with hepatocellular carcinoma, one of the most prevalent forms of human cancer. Apoptosis (programmed cell death) is an important mediator of chronic liver disease caused by HBV infection. It is demonstrated that the HBV HBx protein acutely sensitizes cells to apoptotic killing when expressed during viral replication in cultured cells and in transfected cells independently of other HBV genes. Cells that were resistant to apoptotic killing by high doses of tumor necrosis factor α (TNFα), a cytokine associated with liver damage during HBV infection, were made sensitive to very low doses of TNFα by HBx. HBx induced apoptosis by prolonged stimulation of N-Myc and the stress-mediated mitogen-activated-protein kinase kinase 1 (MEKK1) pathway but not by up-regulating TNF receptors. Cell killing was blocked by inhibiting HBx stimulation of N-Myc or mitogen-activated-protein kinase kinase 1 using dominant-interfering forms or by retargeting HBx from the cytoplasm to the nucleus, which prevents HBx activation of cytoplasmic signal transduction cascades. Treatment of cells with a mitogenic growth factor produced by many virus-induced tumors impaired induction of apoptosis by HBx and TNFα. These results indicate that HBx might be involved in HBV pathogenesis (liver disease) during virus infection and that enhanced apoptotic killing by HBx and TNFα might select for neoplastic hepatocytes that survive by synthesizing mitogenic growth factors.

Tumor necrosis factor (TNF)-mediated kinase cascades: Bifurcation of Nuclear Factor-κB and c-jun N-terminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor 2

TNF-induced activation of the transcription factor NF-κB and the c-jun N-terminal kinase (JNK/SAPK) requires TNF receptor-associated factor 2 (TRAF2). The NF-κB-inducing kinase (NIK) associates with TRAF2 and mediates TNF activation of NF-κB. Herein we show that NIK interacts with additional members of the TRAF family and that this interaction requires the conserved “WKI” motif within the TRAF domain. We also investigated the role of NIK in JNK activation by TNF. Whereas overexpression of NIK potently induced NF-κB activation, it failed to stimulate JNK activation. A kinase-inactive mutant of NIK was a dominant negative inhibitor of NF-κB activation but did not suppress TNF- or TRAF2-induced JNK activation. Thus, TRAF2 is the bifurcation point of two kinase cascades leading to activation of NF-κB and JNK, respectively.