Primary cultured human umbilical vein endothelial cells were either treated or untreated with TNF-α (40 ng/mL) alone or in the presence of the specific PKC-β2 inhibitor CGP53353 (1 μM)), nitroglycerine (10 μM), propofol (100 μM), propofol plus nitroglycerin, or CGP53353 plus nitroglycerine, respectively, for 24 hours. TNF-α increased the levels of superoxide, Nox (nitrate and nitrite), malondialdehyde, and nitrotyrosine production, accompanied by increased protein expression of p-PKC-β2, gP91phox, and endothelial cell apoptosis, whereas all these changes were further enhanced by nitroglycerine. CGP53353 and propofol, respectively, reduced TNF-α induced oxidative stress and cell toxicity. CGP53353 completely prevented TNF-α induced oxidative stress and cell toxicity in the presence or absence of nitroglycerine, while the protective effects of propofol were neutralized by nitroglycerine. It is concluded that nitroglycerine comprises the protective effects of propofol against TNF-α stimulation in endothelial cells, primarily through PKC-β2 dependent NADPH oxidase activation.Continuous treatment with organic nitrates causes nitrate tolerance and endothelial dysfunction, which is involved with protein kinase C (PKC) signal pathway and NADPH oxidase activation. We determined whether chronic administration with nitroglycerine compromises the protective effects of propofol against tumor necrosis factor (TNF-) induced toxicity in endothelial cells by PKC- The mortality of myocardial infarction remains significant despite advancement in surgical techniques and pharmacological therapies. Organic nitrates  are still useful drugs and have been widely used for the prevention and treatment of ischemic heart disease for more than 100 years . Howeverpoptosis . A furthn of PKC . Despiteα induced apoptosis in primary cultured human umbilical vein endothelial cells (HUVECs) [α induced cellular toxicity by enhancing oxidative stress and nitrative stress, which was neutralized by propofol treatment [β2, a PKC isoform that may play a major role in TNF-α induced human endothelial cell apoptosis [α, PKC-δ, PKC-ε, and PKC-ξ in cardiomyocytes [β2 has not been reported, nor has it been investigated in endothelial cells in the condition of nitrate tolerance. In the present study, we hypothesize that nitrate tolerance induced by organic nitrates comprises the protective effects of propofol against TNF-α induced toxicity in endothelial cells. Our data suggests that nitroglycerine supplementation promoted PKC-β2 activation in HUVECs subjected to TNF-α stimulation, which subsequently increased the activation of NADPH oxidase and compromised the protective effects of propofol against TNF-α induced damage.Propofol, an anesthetic with demonstrated antioxidant properties , has sho(HUVECs) . Our furreatment . It is upoptosis . Of intemyocytes –18, whicμg/mL streptomycin in an atmosphere of 5% CO2 at 37°C. The medium was changed every 2-3 days until the ECs reached confluence. Cultured cells were identified as ECs by their morphology and the presence of the Factor VIII-related antigen was detected using an indirect immunocytochemistry method as described [Primary cultured HUVECs will be prepared using established procedures as previously described . Brieflyescribed . The purα  alone or TNF-α in the presence of 1 μM CGP53353 (CGP) , 10 μM nitroglycerine (NTG) , 100 μM propofol , or NTG plus propofol , and NTG plus CGP53353 , respectively, for 24 hours. In specific groups, cultured cells were pretreated with propofol for 30 min before other treatments. The concentration of TNF-α used to induce apoptosis in the present study was chosen on the basis of our previous studies [α at the dose of 40 ng/mL could significantly induce ECs apoptosis. The concentration of NTG adopted is according to the studies [μM could induce nitrate tolerance. The choice of concentration of PKC-β2 inhibitor was based on that 1 μM CGP53353 could selectively inhibit PKC-β2 activation in our previous study [μM reversed TNF-α (40 ng/mL) induced cell injury but propofol at the dose of 100 μM per se did not cause apparent apoptosis under the present experimental condition in the absence of TNF-α stimulation. Therefore, we chose the concentration of 100 μM as the treatment dose of propofol for the further mechanistic study.When the cells were at 70% confluence, the cultured cells were then randomly divided into the following groups: cells were either not treated  or treated with 40 ng/mL TNF- studies , which d studies , 21, whius study . In our Cytotoxicity will be assessed by measuring lactate dehydrogenase (LDH)  release in the medium in addition to the measurement of cell viability using the 3--2,5-diphenyltetrazolium bromide (MTT)  assay according to the manufacturer's instructions.The content of malondialdehyde (MDA), which is a marker of lipid peroxidation, was measured to evaluate the oxidative injury of ECs. After homogenizing on ice in normal saline, the MDA levels of the supernatants of cell samples was determined by the thiobarbituric acid colorimetric method using MDA assay kit  as described , 24. The2−) and nitrates (NO3−), the stable end products of nitric oxide (NO), were determined by the Griess reaction as previously described [μmol/L protein. Myocardial O2− production was determined via lucigenin chemiluminescence method [μM) and read in 96-well microplates by luminometer . Light emission, expressed as mean light units (MLU)/min/100 μg protein, was recorded for 5 minutes. Myocardial nitrotyrosine levels (μg/mg protein) in the collected supernatant were determined by chemiluminescence detection via the Nitrotyrosine Assay Kit per manufacturer's protocol .Cultured cells were homogenized in ice-cold PBS and centrifuged at 3,000 g for 15 minutes at 4°C for supernatant collection. The supernatant protein concentration was determined via a Lowry assay kit . Concentrations of nitrites  were harvested and processed as described [DNA fragments which are lost from apoptotic nuclei and nuclear DNA content can be easily measured by flow cytometry after nucleic acid staining with specific fluorochromes. Briefly, cells (1 × 10escribed . Then thβ-glycerolphosphate (1 mM), sodium pyrophosphate (2.5 mM), Triton X-100 (1%), PMSF (1 mM), DTT (l mM), leupeptin (1 μg/mL), aprotinin (1 μg/mL), and pepstatin (1 μg/mL). The homogenate was centrifuged at 1,000 g for 10 min at 4°C to collect the supernatant as total protein preparations. Equal amounts of protein were combined with 5×SDS loading buffer, boiled for 5 min, then separated via 10% SDS-PAGE, and subsequently transferred to PVDF membrane for immunoblot analysis. The membranes were blocked in 5% no fat milk for 2 hours at room temperature and then incubated overnight at 4°C with primary antibodies against p-PKC-β2 (ser660) , PKC-β2 , and gP91phox . After being washed with TBST, the membranes were incubated with proper secondary horseradish peroxidase (HRP-)conjugated antibodies  and developed with enhanced chemiluminescence reagent . The membranes were subsequently reblotted for GAPDH , and the results were normalized to GAPDH to correct for loading.Cultured cells were homogenized in cell lysis buffer containing Tris-HCl , NaCl (150 mM), EDTA (l mM), EGTA (l mM), P < 0.05 was considered statistically significant.All the data are expressed as mean ± S.E.M. Significance was evaluated by analysis of one-way variance (ANOVA) followed by Tukey's test. GraphPad Prism software program  was used for statistical analysis. α stimulation as compared with control, which was reversed by propofol treatment. The supplementation of nitroglycerine further exacerbated TNF-α induced reduction in cell viability. The treatment of propofol improved but not restored the viability of the cells subjected to TNF-α stimulation in the presence of nitroglycerine. By contrast, CGP53353, a selective inhibitor of PKC-β2, reversed the reduced cell viability induced by TNF-α with or without the presence of nitroglycerine.The cytotoxicity of the cultured endothelial cells was assessed by MTT assay and LDH release. As shown in α resulted in a significant increase in LDH release in the medium of cultured HUVECs .Stimulation of HUVECs with TNF-ic index . Nitroglα stimulation as compared to control group, which were prevented by the treatment of propofol or CGP53353. Addition of nitroglycerine further promoted the production of superoxide and MDA, which was neutralized by propofol treatment but reversed by CGP53353 treatment.As the production of ROS plays an important role in the development and progress of nitrate tolerance and endothelial dysfunction , we measα increased the levels of NOx and nitrotyrosine production, and nitroglycerine further increased their levels . A relafunction . This isoperties . Howeverβ activation has been shown to play important or critical roles in NADPH oxidase activation [β2 is preferably upregulated in failing human hearts [α production [β2 and NADPH oxidase interplay may play critical roles in mediating cellular damage in situations associated with increased TNF-α production, such as AMI, heart failure, and diabetes, as well as during cardiac surgery using cardiopulmonary bypass. In the present study, propofol prevented TNF-α induced overexpression of p-PKC-β2 and gP91phox in endothelial cells. Of interest, the selective inhibitor of PKC-β2 CGP53353 has the similar effects as propofol. Therefore, we assumed that propofol preserves endothelial cells though inhibits PKC-β2 activation signal pathway, including inhibition of NADPH oxidase.Endothelial NADPH oxidase is a major source of superoxide in blood vessels and is implicated in the oxidative stress accompanying various vascular diseases , 33. NADtivation , 37. Intn hearts , which ioduction  and NADPoduction . Therefoα stimulation in cultured HUVECs [β2 but not PKC-δ isoform pathway activation played dominant role in ROS production in this context [β2 activation was involved in NADPH oxidase activation in the condition of nitrate tolerance, a well known phenomenon that the clinical or hemodynamic response to organic nitrates  is attenuated or abolished after prolonged, continuous, or high dose nitrate treatment (see for review [β2 induced by TNF-α stimulation, accompanied with enhanced levels of gP91phox and ROS production, which were reversed by the selective inhibition of PKC-β2 with CGP53353. This suggests that excessive activation of PKC-β2 and subsequent activation of NADPH oxidase paly a critical role in nitrate tolerance induced adverse effects. Of interest, propofol treatment reversed the increased levels of superoxide, MDA, nitrotyrosine, and the elevated protein expression of PKC-β2 and gP91phox, as well as LDH release and cell apoptosis in the endothelial cells after TNF-α stimulation. In the presence of nitroglycerine administration, however, propofol attenuated but not completely prevented these changes induced by TNF-α stimulation. This means that chronic treatment with nitroglycerin neutralized the protective effects of propofol.Although there are reports on PKC involvement upon NADPH oxidase activation after TNF-d HUVECs , especia context . Howeverα induced human vascular endothelial cell injury, as well as increased ROS production by PKC-β2 dependent activation of endothelial NADPH oxidase, and that the protective effects of propofol were compromised by nitroglycerine administration in experimental settings that are associated with persistent TNF-α stimulation. Further studies need to be performed in endothelial cell with deficit of the targeted kinase enzyme derived from gene knockout animals or gene silenced with specific antisenses to confirm the findings of the current study.In summary, the results from the present study indicate that nitrate tolerance further exacerbated TNF-


Tumor necrosis factor (TNF) and TNF receptors (TNFR) superfamily members are engaged in diverse cellular phenomena such as cellular proliferation, morphogenesis, apoptosis, inflammation, and immune regulation. Their role in regulating viral infections has been well documented. Viruses have evolved with numerous strategies to interfere with TNF-mediated signaling indicating the importance of TNF and TNFR superfamily in viral pathogenesis. Recent research reports suggest that TNF and TNFRs play an important role in the pathogenesis of HIV. TNFR signaling modulates HIV replication and HIV proteins interfere with TNF/TNFR pathways. Since immune activation and inflammation are the hallmark of HIV infection, the use of TNF inhibitors can have significant impact on HIV disease progression. In this review, we will describe how HIV infection is modulated by signaling mediated through members of TNF and TNFR superfamily and in turn how these latter could be targeted by HIV proteins. Finally, we will discuss the emerging therapeutics options based on modulation of TNF activity that could ultimately lead to the cure of HIV-infected patients. The term tumor necrosis factor (TNF) came into existence in 1975 with the work of Carswell and colleagues while studying hemorrhagic necrosis by endotoxin . Role ofTill date TNF superfamily comprises of 19 ligands and 29 receptors . All memNumber of TNF ligand versus receptor suggests that some of the ligands interact with more than one receptor to achieve their goal . TNF ligMost extensively studied member of TNF superfamily is TNF-alpha. TNF-alpha is produced in response to pathological conditions like inflammation and infection mainly by activated macrophages and T lymphocytes , 7, but κB kinase (IKK) complex . T. Tin vitividuals . This meividuals . Like OXividuals , 120. Fuividuals . In addiividuals . Furtherividuals , 124. Adividuals . To be aividuals .κBalpha cytoplasmic reappearance [The persistence of HIV-1 reservoirs is a major challenge for complete viral eradication in HIV-1 infected patients –128. To pearance . Thus, tpearance . Furtherpearance . Since spearance , the usepearance .TNF ligands and TNF receptors superfamily are the integral part of our immune system. TNF signaling exerts significant impact on HIV life cycle. In turn, HIV encoded proteins also modulate TNF signaling pathways resulting in the survival of HIV-infected cells and killing of bystander cells. Anti-TNF therapy has been successfully used in several inflammatory diseases. Combinatorial therapy involving HAART, anti-TNF therapy, along with use of HDACIs/HMTI might be a viable option for the treatment of HIV infection to reach the ultimate goal, the clearance of HIV-1 from cellular reservoirs.


The evolutionary implications of recombination in HIV remain not fully understood. A plausible effect could be an enhancement of immune escape from cytotoxic T lymphocytes (CTLs). In order to test this hypothesis, we constructed a population dynamic model of immune escape in HIV and examined the viral-immune dynamics with and without recombination. Our model shows that recombination (i) increases the genetic diversity of the viral population, (ii) accelerates the emergence of escape mutations with and without compensatory mutations, and (iii) accelerates the acquisition of immune escape mutations in the early stage of viral infection. We see a particularly strong impact of recombination in systems with broad, non-immunodominant CTL responses. Overall, our study argues for the importance of recombination in HIV in allowing the virus to adapt to changing selective pressures as imposed by the immune system and shows that the effect of recombination depends on the immunodominance pattern of effector T cell responses. Human immunodeficiency virus (HIV) frequently undergoes recombination HIV is known to evade the immune control, for example by escaping from the recognition by cytotoxic T lymphocytes (CTLs). These CTLs recognize and kill cells that display fragments of viral peptides on their surface. More specifically, proteins produced by the virus inside an infected cell are processed by the host cell machinery into antigenic peptides, and loaded onto MHC class I molecules. The resulting MHC:peptide complexes are then presented on the surface of the cell such that the host's CTLs, which scan the MHC:peptide complexes, can recognize and destroy the infected cells. Viruses can evade the immune response, and studies of HIV-1 and SIV infections have shown selection of CTL escape variants during both primary and chronic infections The immune escape dynamics in HIV can generate an environment which is beneficial for recombination. When the immune system generates immune responses against frequent virus strains, a strong selective pressure arises which leads to the escape of the virus. The immune system in turn produces novel responses that can recognize the escape variants and lead to further escape. The resulting coevolutionary arms race between the virus and the immune system resembles the so-called Red Queen dynamics between hosts and parasites, where recombination has been shown to play an important role In this study, we examine the role of recombination for immune escape of HIV by using a population dynamic model of HIV. We find that recombination accelerates the evolution of escape in the virus in the early stage of infection. We also find that the CTL hierarchy is of vital importance to viral/immune coevolution as well as viral recombination. In particular, we find that the immunodominance pattern of the CTL response strongly affects the mean number of escapes in the virus as well as the impact of recombination. Furthermore, we determine which parameters increase or decrease the impact of viral recombination on the immune escape, and discuss in which situations genetic shuffling could contribute to the viral immune escape as well as to adaptation of HIV to the host in general.in vivoMathematical models have proved an excellent tool for studying complex viral dynamics Three populations of cells are considered: susceptible naive CD4+ T cells  is restructured accordingly. Importantly, both mutation and recombination do not alter the number of infected cells but only the genetic composition of the provirus population.In order to perform mutation, we approximate the number of CD4+ cells infected with a single virus of type In order to perform recombination, we assume at every time-point that (i) a fraction We distinguish between two types of escape, E and EC. The escape E is the escape which only carries a mutation at the escape locus and regardless of the type of allele at the compensatory locus; the escape EC is the escape which carries a mutation at both escape locus and the compensatory locus see . Unless The average time of escape of a mutant with The mean number of escape variants in the population at a given time is calculated by the mean Hamming distance of the escape types from the wild type. For example, in order to calculate the mean number of E escape variants, we calculate the abundance of all strains with the number of E escapes equal to zero, one, two, etc., then multiply each by its Hamming distance from the wild type, and finally weight by the total virus load.Each CTL clonal sub-population has its assigned sensitivity (avidity) value The diversity of the CTL population is a measure of their distribution. At each time point in one simulation run we calculated the diversity of the CTL response by the use of the Shannon diversity index The aim of this study was to examine the impact of recombination on the immune escape of HIV. To that end, we considered a deterministic, population dynamic model of HIV infection with a stochastic implementation of mutation and recombination A key question is whether recombination can accelerate the emergence of escape mutations in HIV, and if so, whether this is beneficial for the virus. In order to assess a general impact of recombination on the evolution of escape in HIV, we measured the mean time of appearance of escape mutations during infection. In particular, we measured an average speed of accumulation of successive mutations from the time point of infection, i.e. the average time of appearance of a single mutant, the average time of appearance of a double mutant, etc. The results are presented in The figure shows that in runs with recombination successive escape variants appear faster than in the runs without recombination, and that the accumulating effect of recombination increases with the number of escape mutations. The impact of recombination is even stronger for EC than for E escape types. Since EC escapes need two point mutations, the results suggest that genetic shuffling not only helps bringing together escape mutations from different epitopes but also bringing together escape and compensatory mutations at the same epitope see . Thus, oOn the other hand, the acceleration of the emergence of escape mutations is likely to have other consequences on the virus dynamics, and thus we next examined which of the determinants of the viral population are significantly affected by genetic shuffling. We found that recombination has a considerable impact on viral diversity as well as the average number of escape mutations in the population. The results are shown in In line with this interpretation, we observed little or no reversion to the wild-type in simulations where the cost of escape was low or absent (results not shown). The higher mean number of escapes with Several measures of the T cell response are used extensively in immunological literature . However, using them to characterize the CTL dynamics during a viral infection has the disadvantage that these measures not only result from the properties of the immune cells but are are strongly affected by the interactions of the immune response with the virus and the stochastic effects. In order to characterize the response itself, one could measure the avidity distribution of the T cell population for a given antigen, but such quantification in experimental assays remains not feasible. A mathematical model, on the other hand, has the advantage that the sensitivity of each CD8+ T cell clone to the wild-type virus is defined before the simulation run  responses? The reason is that when all effector responses are similarly strong, the expected time of escape at any epitope will be comparable, meaning that all epitopes will escape on average at a similar time because they will all become advantageous at a similar time. A multiple number of escape variants increasing in frequency will in turn allow for almost any combination of those mutations to be advantageous too, possibly outgrowing variants with a smaller number of escape epitopes. This creates an environment in which genetic shuffling will have a significant effect. By contrast, immunodominant responses will favour different escape variants at different times, which will decrease the diversity in the viral population, and hence the variance which recombination can act on. This is why we see a stronger impact of recombination on the number of escapes in models with little or no immunodominance.We also examined the impact of recombination for a decreased cost of escape, and, in accordance with the intuition, we observed an increased effect of recombination on the emergence of escapes (results not shown). With decreasing cost of escape, the impact of recombination on the emergence of escape variants increased markedly. This is because fitter escape variants competed more efficiently with the wild-type variants which in turn, by increasing viral diversity, enhanced the impact of recombination. Finally, as we observed that a decreased level of immunodominance is generally associated with an increased breadth of the response and also with an increased number of CD8+ cells, we conclude that broader and stronger (higher activation and killing) immune responses are likely to increase the impact of viral recombination on the escape dynamics.In this study we show that recombination in HIV accelerates the initial phase of viral coevolution with the immune system. We demonstrate that random shuffling of the genetic material (a) accelerates the emergence of escape mutations (both with and without compensatory mutations), (b) increases viral diversity during infection . This may seem paradoxical as under positive epistasis, recombination may be expected to decelerate adaptation Depending on the total fitness cost of escape, and thus also on the number of escape loci, a mutant can arise that escapes at all loci. Because such full escape is no longer controlled by the immune response, the appearance of this mutant is accompanied by a substantial increase in viral load. The time of appearance of the full escape mutant depends strongly on the recombination rate. However, whether such total escape ever occurs in late stage patients is currently unclear.in vivo do not necessarily induce substantial changes in virus load Generally, we find that viral recombination increases the set-point viral load. Given the moderate magnitude of this increase  and the large variance seen between clinical measurements of the virus load, it is not an effect that could easily be detected in clinical data even if there were considerable variation in recombination rates between different viral isolates. The fact that in our model recombination has only a weak effect on virus load, however, is compatible with strong selective pressures on escape mutants, as observed experimentally As common strains are likely to be targeted more strongly by the immune response, a negative frequency dependent selection may ensue, leading to potentially cyclical frequency dynamics between the virus and the immune system. This behaviour shows a resemblance with the so-called Red Queen dynamics in evolutionary biology, where fluctuating selection in host-parasite systems leads to allele frequency cycles and the maintenance of genetic variants over long periods of time. One of the leading hypothesis for the evolution of recombination (Red Queen Hypothesis) states that such circumstances can explain the evolutionary maintenance of genetic shuffling Although the primary aim of this study has been the impact of recombination on the coevolution of HIV with the immune system, the results presented here might shed light on the evolutionary history of recombination in HIV (or other retroviruses like SIV). On the one hand, we have found that recombination can have a strong impact on the speed of acquisition of escape mutations, and thus arguably, on the adaptation of the virus to the new hosts. This suggests that the within-host selection acting on the virus might select for non-zero recombination rates. On the other hand, the total viral load has been correlated with the transmission probability of HIV, and since we have seen that recombination moderately increases the total virus load, the between-host selection acting on the viral recombination is likely to be weak or zero. This also emphasizes the importance of the within-host stage of evolution of HIV. However, what evolutionary forces act on recombination and how, as well as whether HIV had enough time to evolve its recombination rate, remains a matter of discussion.in vivo and the number of escape mutations in the chronic phase of the infection might yield further insight into understanding the optimal recombination rate in HIV. Third, our results show that of particular importance is the estimation of the fitness of escape mutants in the context of the immune system (in vivo). The first component of this fitness is the cost that these mutations induce in the absence of the immune response, which could be measured by quantifying their effect on the replicative capacity of HIV in vitro. The other component of the fitness in vivo reflects the interaction of the virus and the immune response. This is encapsulated by the parameter avidity, which describes both the activation of the immune response by the virus as well as its subsequent recognition and the killing by the immune system. Obtaining more quantitative data on the specificity and diversity of the T cell repertoire recognizing the virus would allow better understanding of the effect of recombination on the population dynamics of the virus, and more generally would enable more quantitative approach to the causes underlying immunodominance.HIV and the immune system remain one of the best qualitatively and quantitatively characterized coevolutionary systems. However, a better insight into the coevolutionary dynamics underlying the interaction of the virus with the immune system, as well as into the population dynamical consequences of recombination, requires an improved understanding of the parameter space defining these interactions. First, the recently underlined importance of macrophages in the development of HIV infection


We previously showed that the probiotic mixture, VSL#3, prevents the onset of ileitis in SAMP/YitFc (SAMP) mice, and this effect was associated with stimulation of epithelial-derived TNF. The aim of this study was to determine the mechanism(s) of VSL#3-mediated protection on epithelial barrier function and to further investigate the “paradoxical” effects of TNF in preventing SAMP ileitis.ex vivo-cultured ilea following exposure to VSL#3 conditioned media (CM), TNF or VSL#3-CM + anti-TNF. Tight junction (TJ) proteins were assessed by qRT-PCR, Western blot, and confocal microscopy, and TNFRI/TNFRII expression measured in freshly isolated intestinal epithelial cells (IEC) from SAMP and control AKR mice.Permeability was evaluated in SAMP mice prior to the onset of inflammation and during established disease by measuring transepithelial electrical resistance (TEER) on Culture with either VSL#3-CM or TNF resulted in decreased ileal paracellular permeability in pre-inflamed SAMP, but not SAMP with established disease, while addition of anti-TNF abrogated these effects. Modulation of the TJ proteins, claudin-2 and occludin, occurred with a significant decrease in claudin-2 and increase in occludin following stimulation with VSL#3-CM or TNF. TNF protein levels increased in supernatants of SAMP ilea incubated with VSL#3-CM compared to vehicle, while IEC-derived TNFR mRNA expression decreased in young, and was elevated in inflamed, SAMP versus AKR mice.Our data demonstrate that the previously established efficacy of VSL#3 in preventing SAMP ileitis is due to direct innate and homeostatic effects of TNF on the gut epithelium, modulation of the TJ proteins, claudin-2 and occludin, and overall improvement of intestinal permeability. Although the precise etiology is currently unknown, it is generally accepted that IBD results from dysregulated immune responses to environmental factors in genetically susceptible individuals. As early as 1972, Shorter i.e., terminal ileum), histologic features, and response to standard therapies used to treat Crohn’s patients Similarly, SAMP1/YitFc (SAMP) mice display an inherent increase in small intestinal epithelial paracellular permeability that precedes the histologic onset of ileitis and is independent of commensal flora colonization Streptococcus thermophilus, three strains of Bifidobacterium and four strains of Lactobacillus, and has been proven to be efficacious in treating established colitis in IL-10 knockout (KO) mice Recently, our group reported that administration of the probiotic formulation, VSL#3, prevents the onset of ileitis in SAMP mice We provide evidence, herein that recombinant TNF or VSL#3-stimulated TNF, specifically improves transepithelial permeability during the early phases of gut inflammation in young, pre-inflamed SAMP mice, by directly regulating the TJ proteins, claudin-2 and occludin and inducing innate, proinflammatory cytokine production. In addition, VSL#3-mediated changes in epithelial permeability appear to be dependent on decreased expression of TNFRs prior to the onset of ileitis. Our findings provide evidence that the beneficial effect of VSL#3 on intestinal permeability are directly mediated by a mechanism involving modulation of TJ proteins and TNF expression in the gut epithelium. Furthermore, our studies support the concept that TNF, in addition to its known proinflammatory activities, may also possess anti-inflammatory and homeostatic properties, particularly during the earlier phases of disease.ex vivo epithelial paracellular permeability in ilea from young SAMP mice, prior to the onset of inflammation (4-wk-old), SAMP with established disease (>20 weeks of age), and age-matched AKR controls (p<0.05), but not old, SAMP mice (p<0.05). Taken together, these data indicate that both VSL#3-CM and TNF are efficacious in improving barrier function by decreasing epithelial paracellular permeability specifically in young SAMP that do not yet show overt histologic signs of ileal inflammation. In contrast, these effects are not observed in older SAMP mice with established disease or AKR controls, regardless of age. These observations are consistent with the in vivo setting wherein orally-administered VSL#3 to young non-inflamed SAMP, but not to older SAMP, effectively decreases epithelial permeability and prevents the onset of ileitis The effect of VSL#3-CM and TNF in regulating small intestinal barrier function was initially investigated by measuring AMP mice A-B. No AMP mice C. In fain vivo studies p<0.05). Moreover, other early, innate cytokines also showed a trend towards increased secretion following treatment with VSL#3-CM, including IL-1β and IL-6, with baseline levels of these cytokines greater than age-matched AKRs. Conversely, VSL#3-CM did not alter production of TNF or other innate-type cytokines from control AKR ilea. Interestingly, baseline IL-10 levels were markedly increased in AKR ilea compared to SAMP (p<0.01), but while VSL#3-CM had the ability to further induce IL-10 production from SAMP ilea (p<0.05), it did not modulate IL-10 in ilea from AKR mice. The effect of VSL#3-CM on inducing TNF secretion confirms TNF’s central role in VSL#3-mediated events, while the induction of other innate-type cytokines suggests that VSL#3 may play a role in augmenting global innate mucosal immune responses, including improving epithelial barrier function.Emerging evidence suggests that defects in innate immunity and the inability of the gut mucosa to mount appropriate innate immune responses may result in the initiation and perpetuation of chronic intestinal inflammation, such as that observed in IBD ex vivo organ culture with either VSL#3-CM or TNF (p<0.05). Interestingly, treatment with either VSL#3-CM or TNF, compared to vehicle, decreased claudin-2 and increased occludin mRNA levels (both p<0.05) in ilea from pre-inflamed SAMP mice. VSL#3- and TNF-specific modulation of claudin-2 and occludin in young, ileitis-prone SAMP mice was further confirmed by in vivo experiments on isolated ileal loops in which either VSL#3-CM or TNF was injected, followed by epithelial immunolocalization of the aforementioned TJ proteins . Interestingly, IEC derived from young, pre-inflamed SAMP displayed decreased mRNA transcripts for TNFRI and TNFRII compared to age-matched AKR (both p<0.05), while during established disease, mRNA expression for both TNFRs was markedly increased in SAMP IEC versus control AKR (both p<0.05)  or >20-wk-old (inflamed) SAMP mice A. Altho p<0.05) A.p<0.05). Together, this data suggest that TNFRs are essential in mediating VSL#3- and TNF-dependent changes in intestinal epithelial paracellular permeability, and that TNFR expression on IEC may contribute to the overall effects of VSL#3 on mucosal barrier function.Finally, we explored the effects of VSL#3-CM and TNF on epithelial paracellular permeability in mice deficient in both TNFRI and TNFRII (TNFR KO mice). ΔTEER was measured in ilea after 1 h exposure to VSL#3-CM or TNF (10 ng/ml) versus vehicle control. No differences were observed in ΔTEER from TNFR KO ilea when exposed to either VSL#3-CM or TNF. In contrast, stimulation with either VSL#3-CM or TNF markedly improved (increased TEER) epithelial barrier function of WT control mice  and WT controls were purchased from The Jackson Laboratory . All experimental mice were maintained under SPF conditions, fed standard laboratory chow , and kept on 12 h light/dark cycles. All procedures were approved by the CWRU’s Institutional Animal Care and Use Committee (IACUC)  and were in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines.SAMP mice were originally propagated at the University of Virginia and then at Case Western Reserve University (CWRU), with founders provided by S. Matsumoto  ex vivo permeability and organ culture assays. Transepithelial electrical resistance (TEER) assays were performed according to a previously described method VSL#3  conditioned media (VSL#3-CM) was prepared as previously described 9 CFU/day) mixed in their normal chow, as previously described SAMP mice were administered high-dose VSL#3 , claudin-1 (5′–ACGGTCTTTGCACTTTGGT-3′ and 5′-AGTTTGCAGGATCTGGGAT-3′), claudin-2 (5′-TATCTCTGTGGTGGGCATGA-3′ and 5′CGAAGGATGCCATGAAGATT-3′), claudin-3 (5′-CCACTACCAGCAGTCGATGA-3′ and 5′-CAGCCTGTCTGTCCTCTTCC-3′), claudin-4 (5′-AGCAAACGTCCACTGTCCTT-3′ and 5′-CCCTCATCAGTCACTCAGCA), zonula occludens-1 (ZO-1) (5′-CCTAAGACCTGTAACCATCT-3′ and 5′-CTGATAGATATCTGGCTCCT-3′), and the RT2 Primer Assay used for TNFRI and TNFRII evaluation . Target mRNA was normalized to β-actin for each sample, and all samples were run in duplicate.Total RNA was isolated from homogenized tissues or from primary isolated IEC , and subsequently converted into cDNA . cDNA was quantified using the Applied Biosystem RT-PCR detection system and software . qRT-PCR was performed using the following primers: occludin  as previously described Paracecal regions of the intestinal ileum were explanted and fixed with 4% paraformaldehyde/PBS for 12 h followed by dehydration in 30% sucrose/PBS prior to embedding in OCT freezing media . 10 µm tissue sections were cut on a CM3050s cryostat  and mounted on Superfrost Plus slides . Tissue sections were permeabilized and blocked in PBS containing 0.3% Triton X-100  and 10% goat serum , followed by staining with primary antibodies for claudin-2 (1∶300) or occludin (1∶100), and an Alexa Fluor-conjugated secondary antibody (1∶500). TJ proteins were then detected and immunolocalized using Prolong Gold or Prolong Gold plus DAPI . 3D image stacks were acquired by confocal microscopy , and images displayed as 2D maximum intensity projections.2ATP, 3.5 MgCl2, 1 mM PMSF, Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail . Cell lysis was achieved using cell douncers , and lysates were cleared of nuclear material by 1000 x g centrifugation for 10 min at 4°C. Supernatants were then centrifuged  for 1 h at 4°C to separate membrane from cytosolic fractions. Resulting supernatants were collected as cytosolic fractions and pellets (membrane fractions) were resuspended in RIPA buffer  with 1% OG (n-Octyl-β-D-Glucopyranoside) and then sonicated 3x (70% duty cycle) on ice.Freshly isolated IECs were harvested from ileal segments of experimental mice in 10 mM HEPES, pH 7.4, 100 mM KCl, 3mM NaCl, 1 mM NaWestern blot analysis was performed on total protein extracts or cellular fractions of freshly isolated IECs from experimental mice. Equal amounts of protein were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Membranes were blocked for 1 h with 5% wt/vol dry milk in Tris-buffered saline containing 0.1% Tween-20, and incubated with primary antibodies  in blocking buffer overnight at 4°C, followed by incubation with HRP-conjugated secondary antibody . Proteins were visualized using ECL detection system .p value of <0.05 was considered to be significant; p value of <0.01 was indicated as **.Statistical analyses were performed using GraphPad Prism Version 5 . Differences were evaluated by one-way ANOVA Kruskal-Wallis test with Dunns posthoc test and a ex vivo cultured pre-inflamed ileal tissues from SAMP mice. These effects were associated with decreased paracellular permeability and modulation of the epithelial TJ proteins, claudin-2 and occludin, which are inherently dysregulated in SAMP mice in vivo barrier function, whereas SAMP with established disease did not benefit therapeutically from VSL#3 treatment In the present study, we addressed the potential mechanism of action of the probiotic mixture, VSL#3, for its ability to “boost” innate mucosal immune responses by inducing TNF production, particularly during early phases of gut inflammation, in the SAMP mouse model of CD-like ileitis e.g., infliximab) IEC−/−), does not prevent dextran sodium sulfate (DSS)-induced colitis IEC−/−), spontaneously develop colitis The results of our studies show that VSL#3-CM stimulates TNF production and generates permeability changes in SAMP and C57BL/6, but not in AKR ilea, suggesting that AKR mice inherently do not respond to VSL#3. Since SAMP mice are on a mixed background The protective effects of TNF during acute phases of gut inflammation are further supported by a study reporting that TNF deletion in mice leads to increased severity and exacerbation of disease in acute DSS-induced colitis ex vivo ileal paracellular permeability and to regulate the expression of epithelial TJ proteins. Specifically, upregulation of occludin in the insoluble (membrane) fraction of IEC isolated from SAMP treated with VSL#3 suggests that occludin may function as the prominent TJ protein regulating barrier function in response to probiotic treatment. In fact, one of the beneficial roles of probiotics is to promote intestinal barrier integrity by influencing the structure of TJ proteins in vivo studies have demonstrated the global protective effects of probiotics on the gut epithelium in different animal models of intestinal inflammation in vitro treatment of epithelial cells with probiotics, or metabolites secreted by probiotics, leads to an increase in ZO-1 and occludin expression, while decreasing claudin-2 Abnormalities in SAMP small intestinal barrier function are accompanied, as previously reported Epithelial TJ proteins represent the principal determinants of intestinal paracellular permeability and, although mechanism(s) leading to changes in epithelial TJ protein expression have been extensively investigated, it remains to be proven what specific conditions lead to alterations in particular TJ proteins and overall TJ disruption. In fact, much work has focused on cytokine-dependent regulation of the TJ complex. In this context, a direct role of TNF has been implicated in causing loss of barrier function in cultured intestinal epithelial cell monolayers Our studies have significant and translational implications since it is well known that up to 50% of CD patients do not respond to anti-TNF treatment modalities Taken together, our study further support the hypothesis that CD may be initiated by a deficit in intestinal innate immunity, rather than an overly aggressive adaptive immune response to luminal antigens in which innate-type cytokines, such as TNF, play a critical role Figure S1Working hypothesis of TNF-dependent probiotic modulation of epithelial barrier function during the early phases inflammation. One of the earliest features characteristic of ileitis-prone SAMP mice are epithelial alterations, including global changes in epithelial architecture, expansion of villi, as well as Paneth and goblet cell hyperplasia. Interestingly, an inherent increase in small intestinal epithelial paracellular permeability is also present in young SAMP, prior to the onset of inflammation (left). Upon treatment of SAMP mice with VSL#3 during the early phases of inflammation, “boosting” of innate-type responses occurs, which include production of IEC-derived TNF and other early innate cytokines, including IL-1 and IL-6, as well as the anti-inflammatory cytokine, IL-10. In addition, VSL#3-induced TNF decreases ileal epithelial paracellular permeability by modulating the TJ proteins, claudin-2 (decrease) and occludin (increase), with the net effect of improving overall epithelial barrier function (right).(TIFF)Click here for additional data file.


To study the in vivo relevance, mouse lungs were irradiated with a single dose (15 Gy) and assessed at varying times for TTP alterations. Irradiation of MH-S cells caused TTP to undergo an inhibitory phosphorylation at Ser-178 and proteasome-mediated degradation, which resulted in increased TNF-α mRNA stabilization and secretion. Similarly, MH-S cells treated with TTP siRNA or macrophages isolated from ttp (−/−) mice had higher basal levels of TNF-α, which was increased minimally after irradiation. Conversely, cells overexpressing TTP mutants defective in undergoing phosphorylation released significantly lower levels of TNF-α. Inhibition of p38, a known kinase for TTP, by either siRNA or a small molecule inhibitor abrogated radiation-induced TNF-α release by MH-S cells. Lung irradiation induced TTPSer178 phosphorylation and protein degradation and a simultaneous increase in TNF-α production in C57BL/6 mice starting 24 h post-radiation. In conclusion, irradiation of lung macrophages causes TTP inactivation via p38-mediated phosphorylation and proteasome-mediated degradation, leading to TNF-α production. These findings suggest that agents capable of blocking TTP phosphorylation or stabilizing TTP after irradiation could decrease RILT.The efficacy of radiation therapy for lung cancer is limited by radiation-induced lung toxicity (RILT). Although tumor necrosis factor-alpha (TNF-α) signaling plays a critical role in RILT, the molecular regulators of radiation-induced TNF-α production remain unknown. We investigated the role of a major TNF-α regulator, Tristetraprolin (TTP), in radiation-induced TNF-α production by macrophages. For  Radiation-induced lung toxicity (RILT) limits the use of potentially curative high-dose (chemo)-radiation for patients with thoracic malignancies Although little is known regarding the mechanism by which radiation increases TNF-α production, there has been substantial investigation into TNF-α regulation in other systems. Stimulation of lung macrophages, the major TNF-α producing cells in mice 52 and Ser178), and mutation of these serine residues to alanine enhances the effectiveness of TTP in inhibiting LPS-mediated TNF-α production The most important regulator of TTP phosphorylation in LPS-treated lung macrophages appears to be the p38-MK2 pathway ttp knockout mice and further analyzed TTP expression and phosphorylation in mouse lung, in an attempt to evaluate the importance of post-translational modification-dependent TTP inactivation as a mechanism of radiation-induced TNF-α production.Because of the importance of TTP in the regulation of LPS-induced TNF-α production in macrophages, we thought it was possible that radiation might stimulate TNF-α production through similar mechanisms. Therefore, we assessed the changes in expression and p38-mediated phosphorylation of TTP in response to radiation in a mouse macrophage cell line MH-S. We also assessed the effects of radiation on macrophages isolated from Here we confirm that all the animal studies conducted here were approved by the University Committee on Use and Care of Animals (UCUCA) of the University of Michigan (protocol # PRO00001915).Ser178 antibody was a kind gift from Dr. Georg Stoecklin (Germany). Total, phospho-specific p38 antibodies and antibodies against GAPDH were purchased from Cell Signaling Technology . Rat monoclonal (Cl: A3-1) F4/80 antibody was purchased from Abcam Inc. (ab6640). siRNA against mouse TTP (sc-36761), mouse p38 (sc-29434) and control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology. Kinase inhibitors were purchased from the following sources: SB203580 , Wortmannin , GSK3 inhibitors, SB216763 and SB415286 . 35S-methionine (7 mCi/0.680 ml) was purchased from MP Biomedicals.The mouse alveolar macrophage cell line MH-S was obtained from ATCC and cultured in regular DMEM supplemented with 10% FBS (Sigma). Chinese hamster ovary (CHO) cells were cultured in RPMI medium with 10% FBS. Rabbit polyclonal TTP antibodies were purchased from Santa Cruz Biotechnology (sc-14030), Abcam Inc. (ab36558 and ab83579) and Sigma (T5452). Rabbit polyclonal Phospho-TTPConstructs containing Myc/His-tagged wild type (pcDNA3-TTP-wt-Myc/His) and serine-to-alanine mutants of TTP  have been described To determine the rate of TNF-α mRNA synthesis and simultaneously determine the stability of transcribed RNA, a BrU pulse-chase labeling assay was followed as described previously  GAG AAA CCT GCC AA GTA 3′ and GAPDH R: 5′ TGT TGC TGT AGC CGT ATT CA 3′), TTP (TTP F: 5′AAA TTC AGT GTT TGG GTG GA 3′ and TTP R: TGT AAC CCC AGA ACT TGG AA 3′) and TNF-α  by quantitative reverse-transcription PCR (qRT-PCR) using SYBR green chemistry (Applied Biosystems). The PCR products were resolved by electrophoresis on 1.5% agarose gels, and melting curve analysis was carried out to confirm the specificity of the product. The δδCt method was used to analyze the data as described elsewhere Total RNA was isolated using the RNAeasy Mini kit  according to the manufacturer’s instructions. RNA (1 µg) was reverse transcribed to cDNA using the High Capacity cDNA Archive kit  and purified . Diluted cDNA was used to amplify GAPDH (GAPDH F: 5′ CTGimax (Invitrogen) with 20 nM of either a control siRNA duplex (sc-37007) or an siRNA duplex targeting mouse TTP (sc-36761) or mouse p38 (sc-29434) using protocol as described previously. 24 h following a second transfection, cells were either sham radiated or irradiated with 4 Gy, and the culture supernatant, RNA or cell lysates were harvested as described previously MH-S cells were transfected using Lipofectamine RNAttp wild type (+/+) and null (−/−) mice. The bone marrow was flushed out and the cells were cultured in RPMI supplemented with 30% L929 cell conditioned medium, 20% heat inactivated FBS in 100 mm culture dishes for 7–8 days. A day before radiation, equal number of cells was replated and was either sham-irradiated or irradiated with 4 Gy. To confirm macrophage differentiation, few cells were immunostained with F4/80 marker. RNA samples were collected at different time points and quantitated for TNF-α transcripts as described above.Murine bone marrow derived macrophages were prepared from bone marrow of 8–10 weeks old mice following the procedure described previously C57BL/6 male mice 6 to 8 weeks of age were purchased from Charles River laboratories and were either sham irradiated or irradiated  with a single dose of 15 Gy to the whole lung as described previously TNF-α secreted into the culture supernatant was quantified using an ELISA kit (R&D Systems) according to manufacturer’s instructions.For immunoblotting, cell lysates were prepared as described previously 35S-met (0.2 mCi) was added into each plate, and cells were incubated for 3 h. Labeling medium was then removed and the cells were washed with ice cold PBS, and cell lysates were prepared as described above. Cell lysates were then subjected to immunoprecipitation using TNF-α antibody. The immunocomplexes were resolved in 12% bis-tris gel, and the gel was fixed and incubated with Amplify . The gel was then vacuum dried at 80°C and developed by autoradiography.For metabolic labeling, 48 h after 4 Gy radiation, MH-S cells were washed twice with PBS and twice with DMEM [methionine (met)/cysteine (cys) free] supplemented with 10% dialyzed serum. Cells were incubated with 3 ml of such medium for 1 h to deplete the intracellular pools of sulfur-containing amino acids. 6 cells were plated in DMEM medium containing 10% FBS and incubated overnight. Cells were then washed twice with PBS and twice with DMEM (-met/−cys) with 10% dialyzed serum. Cells were incubated for 1 h with 3 ml of the Met/Cys-free DMEM containing dialyzed serum and pulse labeled with 0.2 mCi of 35S-met for 1 h. A set of plates were then sham-irradiated and another set were received 4 Gy of radiation. Labeling medium was then removed and cells were washed twice with PBS and twice with DMEM medium with 10% FBS and 100 mM cold methionine. Cells were then incubated with excess Met-containing DMEM medium with 10% FBS for different time periods as indicated. For the 0 h time point, cell lysates were prepared immediately after radiation both from sham-irradiated and irradiated plates. Immunoprecipitation was performed using anti-TTP antibody, followed by SDS-PAGE analysis and autoradiography was performed as described above.For pulse-chase experiment to calculate the TTP protein half life in sham-irradiated or 4 Gy irradiated MH-S cells, 1×10Immunostaining was performed as described previously P value of <0.05 was considered as significant.Data are expressed as means ± S.D. The unpaired Student’s t-test was used to compare the differences between two groups, and a 35S-methionine followed by immunoprecipitation of TNF-α using specific antibody showed that radiation did not change the rate of TNF-α translation up to 48 h post-irradiation  of radiation and quantified TNF-α secretion into the culture supernatant using ELISA between 30 min till 48 h post-radiation. Two Gy had no major effects on TNF-α release until 48 h after radiation (data not shown); however 4 Gy consistently induced the secretion of the cytokine at 48 h post-irradiation . To undeadiation . From thP<0.001; ttp (+/+) and ttp (−/−) mice and differentiated them to macrophages as evidenced by the F4/80 positive staining , Ser178A (S178A), and SS52/178AA] and inactivated caused a significant inhibition of radiation-induced TNF-α secretion 48 h post-radiation compared to vector transfected cells (52/178AA showed only a modest increase of about 1.3±0.1 fold (P<0.0017). These experiments demonstrate TTP involvement in radiation-induced TNF-α secretion.Tristetraprolin (TTP) regulates mRNA stability of various inflammatory cytokines, most notably TNF-α see . As the staining . As showadiation . Conversed cells . Vector 178 phosphorylation of TTP, which gradually declined with time  to study the effect of radiation on post-translational modifications of TTP. To determine if the effect of radiation was mediated by TTP phosphorylation, cell lysates were prepared from both non-irradiated and irradiated MH-S cells at various times starting at 10 min after radiation. Within 10 minutes after radiation, there was a significant increase in Serith time . A similith time . Interesith time . There with time . These o35S-methionine pulse-chase assay. As shown in To better understand the radiation-induced TTP down-regulation, endogenous TTP protein’s half-life in MH-S cells was determined using 178-phosphorylation of TTP, whereas GSK3 inhibitors had no effect  to determine which, if any, inhibit radiation-induced TTP phosphorylation. Among these kinase inhibitors, SB203580 and Wortmannin significantly blocked the radiation-induced Sero effect . Similaro effect . As we eo effect . To furto effect  inset cao effect  inset. Tin vivo, mice were irradiated, and the lungs were assessed at different time points post-radiation. As shown in 178) phosphorylation and a down-regulation of steady state TTP levels between 72 h-2 weeks post-radiation. Furthermore, reprobing the membrane with Thr180 and Tyr182 phospho-specific p38 antibody showed increased phosphorylation as well. Finally, immunofluorescence staining of cryo-preserved lung sections confirmed that radiation caused an increase in TNF-α , and the kinetics of TTP inactivation coincided well with the previously published literature on an enhanced second burst of a biphasic TNF-α release by mouse lung following a single dose of radiation in vitro and in vivo systems reflect differences in the cellular cross-talk remain to be elucidated. Alternatively, it is also plausible to hypothesize that radiation-induced TTP phosphorylation may not be sufficient enough to completely inactivate TTP function to cause early increase in TNF-α production; instead at later time points TTP is completely inactive via proteasome-mediated degradation thus allowing TNF-α release at later time points.Radiation has long been known to activate various stress-activated signaling via phosphorylation of mitogen-activated protein (MAP) kinases including p38, ERK and JNK ttp-null animals). Such observations point to the fact that although TTP may be a major negative regulator of TNF-α levels, there may be additional regulator(s) involved in radiation-induced TNF-α production. Previous studies have identified that ADAM17/TACE as a metalloprotease involved in TNF-α shedding, thus helps in cytokine secretion Although our studies have identified TTP as a major regulator of radiation-induced TNF-α production, it also raised the existence of alternative signaling in the process. We have not only observed increased basal level of TNF-α secretion by TTP-deficient macrophages as reported previously, we have also noted that radiation can further promote modest but consistent increase in TNF-α secretion in TTP-deficient cells , a free radical scavenger Figure S14 Gy irradiation does not alter TNF-α rate of translation in MH-S cells. MH-S cells were either sham-irradiated or irradiated with 4 Gy and left for 48 h. Cells were then metabolically labeled with 35S-Met for 3 h. Cell lysates were then subjected to immunoprecipitation using TNF-α antibody, separated in SDS-PAGE and autoradiographed.(TIF)Click here for additional data file.


Ubiquitination now ranks with phosphorylation as one of the best-studied post-translational modifications of proteins with broad regulatory roles across all of biology. Ubiquitination usually involves the addition of ubiquitin chains to target protein molecules, and these may be of eight different types, seven of which involve the linkage of one of the seven internal lysine (K) residues in one ubiquitin molecule to the carboxy-terminal diglycine of the next. In the eighth, the so-called linear ubiquitin chains, the linkage is between the amino-terminal amino group of methionine on a ubiquitin that is conjugated with a target protein and the carboxy-terminal carboxy group of the incoming ubiquitin. Physiological roles are well established for K48-linked chains, which are essential for signaling proteasomal degradation of proteins, and for K63-linked chains, which play a part in recruitment of DNA repair enzymes, cell signaling and endocytosis. We focus here on linear ubiquitin chains, how they are assembled, and how three different avenues of research have indicated physiological roles for linear ubiquitination in innate and adaptive immunity and suppression of inflammation. Ubiquitination was first recognized for its function in tagging proteins for destruction by the proteasome -5, but iet al. [The assembly of linear ubiquitin chains is unusual in three ways. First, as we have already mentioned, the linkage does not involve any of the lysine residues in the ubiquitin molecule, but occurs between the amino-terminal methionine of one ubiquitin and the carboxy-terminal glycine of the next in the chain. For this reason, linear ubiquitin chains are also known as M1-linked chains. The second unusual feature of linear ubiquitin chain assembly is that it is the E3 that determines the nature of the linkage in these chains  - a deciet al. , who alsSubsequent research, however, revealed that LUBAC also contains a third component, SHARPIN (SHANK-associated RH domain interacting protein), whose carboxy-terminal region has high sequence similarity with the amino-terminal part of HOIL-1 -21. The in vitro [in vitro by LUBAC reveals linear ubiquitin linkages [Several lines of evidence indicate that LUBAC generates exclusively linear ubiquitin chains: (i) LUBAC can generate ubiquitin chains with lysine-less (K0) ubiquitin in vitro ,18,21;  can; moreover, overexpression of these combinations is also capable of activating NF-κB, one of the key transcription factors activated by TNF (see below) [There are two classes of E3s: RING  or U-box-type E3s catalyze the E2-mediated transfer of ubiquitin to target proteins ,24, where below) -21.in vitro. It is possible, however, that interactions with partners other than HOIP and SHARPIN, or perhaps post-translational modification, may induce its activation.This is in line with experiments showing that, despite the fact that HOIL-1 and HOIP both contain an RBR domain , may hint at the mechanism whereby LUBAC promotes the formation of ubiquitin chains. They showed that HHARI, and possibly also Parkin, functions as an HECT-like E3 ligase, through a conserved cysteine residue in the second RING domain, RING2, that accepts a charged ubiquitin in a thioester intermediate before transferring the bound ubiquitin to a substrate . This inClearly we are only just beginning to explore the biochemistry of linear ubiquitin chain formation by LUBAC, and much remains to be discovered about the specificity of this complex in the exclusive generation of linear ubiquitin chains, and the exact actions of the different components within the protein complex.Ubiquitination by K63- and K48-linked chains was already known, before the discovery of linear ubiquitin chains, to play an important part in the activation of NF-κB, arguably the most crucial output of TNFR1 signaling. Activation of the TNFR1 pathway occurs when trimeric TNF crosslinks three TNFR1 monomers to initiate formation of the TNFR1 signaling complex (TNF-RSC). As schematically illustrated in Figure NF-κB is a central transcriptional regulator in the induction of immune response genes that, in the absence of activating signals, is located in the cytoplasm. Activation of NF-κB occurs through the action of a kinase complex, referred to as the IkB kinase (IKK) complex, which consists of two catalytic subunits, IKKα (IKK1) and IKKβ (IKK2), and a critical regulatory subunit called NEMO (IKKγ). This complex is required to phosphorylate the inhibitor of NF-κB (IκB), thereby inducing its degradation and releasing NF-κB to relocate to the nucleus and bind to the promoters of immune genes. The IKK complex is recruited to the TNF-RSC through NEMO, and this results in activation of the kinase activity of this complex. MAPKs are activated as a result of recruitment of the TAB/TAK complex into the TNF-RSC. Whilst the TAB/TAK complex is currently thought to be recruited exlusively to K63-linked chains within the TNF-RSC, the IKK complex can be recruited to this complex via linear chains and, albeit with lesser affinity, also via K63- and K11-linked chains .LUBAC activity was first implicated in signaling from TNFR1 when TNF-mediated NF-κB activation was shown to be impaired in primary hepatocytes from HOIL-1 knockout mice, and LUBAC was shown to form part of the signaling complex that forms on binding of TNF by the receptor, and moreover to be crucial both to the stability of the TNF-RSC and in determining the outcome of TNF signaling -18. How LUBAC also linearly ubiquitinates NEMO itself in the native TNF-RSC . TNF-indin vivo. In the absence of LUBAC components the TNF-RSC still forms and activation of NF-κB still occurs, albeit at significantly reduced levels [Together, these findings indicate a functional role for linear ubiquitination in full gene activation by the signaling pathways triggered by TNF d levels ,21. Exped levels  and can d levels ,36-39.cpdm mice. These mice, which are genetically deficient in SHARPIN and thus lack functional LUBAC complexes [chronic proliferative dermatitis (cpdm)) at about 4 to 6 weeks of age [cpdm mice is apparently paradoxical, because it is generally thought that aberrantly high TNF-induced gene activation is the source of inflammation induced by this cytokine. Our finding that TNF stimulation results in aberrant death of cpdm-derived cells, and that this cell death has both an apoptotic and a necroptotic (and thus inflammatory) component [cpdm mice could be due to inflammatory cell death consequent on the absence of SHARPIN-requiring LUBAC activity. To investigate this possibility, we crossed cpdm mice with TNF-deficient mice, and were able to show that even partial genetic ablation of TNF prevented the formation of inflammatory lesions in cpdm mice, indicating that TNF-induced cell death is indeed causative for the inflammatory phenotype that characterizes these mice [cpdm mice.Absence of LUBAC components also renders cells sensitive to TNF-induced cell death ,20. Intromplexes , have pls of age . The infomponent ,41,42, sese mice . It is pHence, linear ubiquitination is implicated in two different physiological processes: the development of the immune system and the prevention of chronic inflammation, where the latter effect is achieved through interference with TNF-induced cell death. Whether the aberrant cell death in the absence of LUBAC is due to reduced gene-inducing capacity of TNF, to a more direct effect of absence of linear ubiquitin chains from the signaling complexes induced by TNF, or perhaps to a combination of both these effects remains to be established. Our current suggestion for the contribution of LUBAC to these pathways is schematically illustrated in Figure in vivo have provided a platform for addressing new challenges in the field. Among them are proteomic studies of the linear ubiquitinome - the set of linearly ubiquitinated proteins in cells; analysis at atomic resolution of protein complexes implicated both in conjugation and recognition functions; and the possibility of finding novel regulatory components of LUBAC by identification of regulatory principles of LUBAC functions and of novel linear ubiquitin binding domains (LUBIDs). Interestingly, the new LUBIDs include the zinc finger (ZF) domain of HOIL-1, which has recently been shown to recognize specifically linear ubiquitin chains [The discovery of linear ubiquitin chains and their specific ligase complex (LUBAC) has sparked considerable interest in the physiological roles of these cellular signals. Rapid progress in the delineation of protein assemblies involved in conjugation and recognition of linear ubiquitination n chains . One of n chains .


With increasing use, evidence is collected for the association of sarcoid-like granulomatous disease developing after the initiation of anti-TNF- We report a case of a 37-year-old married Pakistani-born woman, resident in the UK since the age of 19, who developed pulmonary sarcoidosis on treatment with etanercept for psoriatic arthritis. She first presented in 2009 with dactylitis affecting her left foot during the post partum period. She later went onto develop psoriatic plaques over her limbs, scalp, and trunk with worsening small and large joint arthritis. Her psoriatic arthritis was treated first with sulphasalazine. Due to side effects, her treatment was changed to methotrexate, with the later addition of leflunomide. Despite full-dose combination DMARD therapy and maintenance low-dose oral prednisone (5–10 mg/day), her disease remained active, requiring intramuscular and on occasion intravenous pulsed methyl-prednisolone to achieve even indifferent control and allow her to cope with looking after her 3 young children (PsARC score tender joints (TJ) 5, swollen joints (SJ) 6, physician global (PhG) 4/5, patient global (PtG) 4/5, DAS28 = 6.12, CRP 40 mg/L). Following screening, including a full history of potential exposure to tuberculosis (TB) and a normal baseline chest X-ray, she was commenced on etanercept 50 mg weekly sub.cut. in April 2010 and leflunomide was discontinued. She made a prompt response, achieving near joint and skin remission.  and her maintenance prednisolone was phased out over the subsequent 3 months. She discontinued methotrexate on her own initiative 4 months after starting etanercept. She remained well until seven months into her etanercept treatment when she presented to clinic with a 3-week history of persistent dry cough and mild exertional breathlessness, having just returned from a 5-week stay in Pakistan.Chest X-ray showed widespread reticulonodular shadowing with prominence of the of right hilum suggestive of lymphadenopathy ; staininHigh resolution computed tomography showed multiple small nodules throughout the lung, few peribronchovascular nodules, with multiple enlarged mediastinal and hilar lymph nodes Figures . BronchoHistological analysis showed the presence of noncaseating granulomata with small number of surrounding lymphocytes. Special stains performed showed that there were no identifiable acid fast bacilli on Ziehl-Nielson stain or fungal element on PAS or Grocott stains Figures . The investigations therefore make a diagnosis for miliary TB highly unlikely and thus suggestive of sarcoidosis given her clinical picture. Etanercept was discontinued and prednisolone commenced. α (TNF-α) is produced by a number of inflammatory cells such as macrophages and its role is implicated in the pathogenesis of granulomatous inflammation; blockade of TNF-α offers a potential role for targeted therapy. However, a series of nation-wide case reports have reported the association of sarcoid-like granulomatous disease after initiation of anti-TNF-α therapy, with disease reversal after discontinuation. A possible mechanism for this association is that anti-TNF-α therapies modulate a CD4+ Th1 cytokine response, key to the immunopathogenesis of sarcoidosis. CD4+ T cells interact with antigen presenting cells which initiate the formation and maintenance of granulomas, resulting in differentiation of selective Th1 cells secreting IFN-γ and IL-2 [α, IL-12, IL-18 are the main cytokines produced. These cytokines are key in driving the Th1 commitment in the granulomatous process. Therefore blockade of TNF-α should have a therapeutic effect on sarcoidosis [α blockade. Etanercept, a soluble TNF-α receptor fusion protein, is thought to enhance T-cell production of IFN-γ which is a key cytokine in the formation of granulomas in the acute stages of sarcoidosis [Tumour necrosis factor-coidosis .α in treatment of sarcoidosis, but their role remains questionable. A study using etanercept for stage II or III pulmonary sarcoidosis in seventeen patients was terminated early due to treatment failure when compared to conventional corticosteroid therapy [A number of studies have been carried out assessing the role of anti TNF- therapy . Likewis therapy . These tα, the right thing to do? Our patient continues to improve on a reducing regimen of oral corticosteroid therapy, with disappearance of symptoms and resolution of pulmonary nodulosis. However, the question remains on what to do next when inflammatory arthritis becomes active. There is limited data available regarding treatment options in such patients; with what we know is rechallenging with another anti TNF-α, thus their role is controversial in granulomatous conditions. Consequently the use of corticosteroids is still the cornerstone of treatment in patients requiring systemic therapy.In contrast, two retrospective series reported symptomatic improvement in patients receiving infliximab when used in patients with chronic extrapulmonary disease , refractory to oral corticosteroid therapy or in patients who had not responded to etanercept . Howeverα therapy. Unfortunately our patient's psoriatic arthropathy failed treatment with leflunomide.Again, although the evidence is limited, leflunomide has been reported as an efficacious alternative therapy in treatment of sarcoidosis, thus offering the possibility of use for patients where sarcoidosis has developed following anti TNF-α, and until then corticosteroids remain the preferred option. The evidence available remains limited in order to draw firm conclusions. Further studies are required to clarify the role of a second anti TNF-


Additionally, TNF-α administration caused hemorrhagic transformation and enhanced VEGF and MMP-9 expression in MMQ pituitary adenoma cell xenografts in mice. Blockers of VEGF or MMP-9, either alone or in combination, attenuated but not abrogated TNF-α mediated hemorrhagic transformation in xenografts. This study suggests that TNF-α may play a role in the development of intratumoral hemorrhage in pituitary adenomas via up-regulation of VEGF and MMP-9.Pituitary apoplexy is a clinical syndrome with unknown pathogenesis. Therefore, identifying the underlying mechanisms is of high clinical relevance. Tumor necrosis factor alpha (TNF-α) is a critical cytokine mediating various hemorrhagic events, but little is known about its involvement in pituitary apoplexy. Here we show that TNF-α may be an important regulator of hemorrhagic transformation in pituitary adenomas. In this study, sixty surgical specimens of hemorrhagic and non-hemorrhagic human pituitary adenomas were examined. Hemorrhagic pituitary adenomas displayed higher protein and mRNA levels of TNF-α, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) compared with those of non-hemorrhagic tumors. Exposure of MMQ pituitary adenoma cells to TNF-α induced VEGF and MMP-9 expression  Pituitary apoplexy is a potentially life-threatening syndrome resulting from hemorrhage, hemorrhagic infarction or infarction of a pre-existing pituitary adenoma . The repet al. [VEGF, a growth factor which has essential roles in vasculogenesis, angiogenesis and vascular permeability , has beeet al.  found a et al. –19.MMP-9 is known to degrade extracellular matrix around blood vessels leading to increased vascular permeability ,21. MMP-in vitro and in vivo.Based on the fact that TNF-α and TNFR1 are present in normal and tumoral anterior pituitary cells ,27, we hn = 30) with hemorrhagic pituitary adenomas, histopathological assessment revealed that 18 patients had prolactinomas, five patients had non-functional adenomas, three had multihormonal adenomas, two had gonadotropinomas and two had GH-secreting adenomas. In the patients (n = 30) with non-hemorrhagic pituitary adenomas, 21 patients had prolactinomas, five patients had non-functioning adenomas, two had gonadotropinomas, and two had GH-secreting adenomas. Invasive tumors were found in four patients (hemorrhagic tumor in one patient and non-hemorrhagic tumors in three). Intratumoral hemorrhage was not observed to correlate with tumor size, hormone types, angiogenesis and tumor invasion (data not shown).In our study, there were 30 patients harboring hemorrhagic adenomas  and 30 patients with non-hemorrhagic adenomas . The numbers of clinical and subclinical pituitary apoplexy patients were 8 and 22, respectively. The eight patients with clinical pituitary apoplexy had several common clinical symptoms, including headache (8/8), nausea (7/8), vomiting (7/8), visual field defect (7/8), decreased visual acuity (6/8), ophthalmoplegia (4/8), febrile (3/8), and altered consciousness (1/8). For the patients (P < 0.05). To further elucidate the potential mechanisms by which TNF-α induces hemorrhage in pituitary adenomas, the expression levels of VEGF and MMP-9 were determined. Western blotting showed that the protein levels of TNF-α, VEGF and MMP-9 in hemorrhagic pituitary adenomas were significantly higher than those in non-hemorrhagic specimens (P < 0.05) (P < 0.05)  . The mRN < 0.05) .P = 0.01) and 0.521 (P = 0.01), respectively.As statistically significant higher expression levels of TNF-α protein were found in hemorrhagic pituitary adenomas, correlations between TNF-α and hemorrhage-associated genes (VEGF and MMP-9) expression in pituitary adenoma tissues were analyzed. The Pearson correlation coefficients for VEGF and MMP-9 versus TNF-α protein expression in pituitary adenomas were 0.674  or 0.9% saline into subcutaneous MMQ pituitary tumor cell xenografts and then analyzed the effect of blood injection on protein expression of VEGF and MMP-9 in these tumors. The results indicated that blood has no significant enhancing effect on protein expression of VEGF and MMP-9 in MMQ cell xenografts .P < 0.05) and 16 ± 4.2% (P < 0.05), respectively. Based on this result, we used TNF-α at concentration of 20 ng/mL for following stimulation. After 24 h of TNF-α (20 ng/mL) exposure, MMQ cells showed significantly higher protein and mRNA levels of VEGF and MMP-9 expression (P < 0.05), as determined by western blot analysis (P > 0.05).To define the role of TNF-α in stimulating hemorrhage-associated genes expression, the effect of TNF-α on cell proliferation and hemorrhage-associated genes (VEGF and MMP-9) expression was examined in the MMQ pituitary adenoma cell line. MMQ cells were treated with different concentrations of TNF-α  for 24 h. A dose-dependent inhibitory effect on cell proliferation was observed. After a 24 h incubation period, TNF-α stimulation at concentrations of 10 and 20 ng/mL inhibited basal cell growth by 9 ± 3.4%  in comparison to vehicle control (0 out of 6)  and a broad MMP inhibitor (GM6001), we administered either or both of them to different groups of mice one week before and during TNF-α treatment, and then the rate of TNF-α induced hemorrhage in each group was examined using HE staining. One out of five mice in the VEGF plus MMP-9 inhibitors group displayed hemorrhagic xenograft tumors, while two in the VEGF inhibitor group and three in the MMP-9 inhibitor group were found (To translate our above ut of 6) . We next control . As MMP-re found . The resThe mechanism underlying hemorrhagic transformation in pituitary adenomas remains unclear. In the present study, we provide the first evidence that TNF-α plays a key role in hemorrhagic transformation in pituitary adenomas. Our results reveal an association of TNF-α elevation with up-regulation of VEGF and MMP-9 in hemorrhagic pituitary adenomas and demonstrate that TNF-α promotes hemorrhagic transformation in MMQ pituitary adenoma cell xenografts via up-regulation of VEGF and MMP-9.TNF-α is a critical inflammatory cytokine that plays a central role in initiating and promoting inflammatory responses ,6. PreviConsidering that TNF-α may be involved in hemorrhagic transformation of pituitary adenomas, we next explored the possible mechanisms underlying its role in hemorrhagic transformation. TNF-α has been shown to stimulate VEGF expression . VEGF inIn addition to VEGF, MMP-9 is also known to be regulated by TNF-α and has been implicated in hemorrhagic transformation . MMP-9 hin vivo results may raise a question as whether the remaining hemorrhagic transformation in tumors treated with inhibitors is caused by the inhibitor drug itself. However, in this study, inhibitor treatment for one week before the TNF-α administration had not caused obvious intratumoral hemorrhage in xenograft tumors during the week. This observation excluded the possibility of side effect of the inhibitory drugs on hemorrhage transformation.In this study, elevated VEGF and MMP-9 expression in hemorrhagic pituitary adenomas was demonstrated. Furthermore, TNF-α administration induced VEGF and MMP-9 expression and caused hemorrhagic transformation in MMQ cell xenografts. These findings confirm the pivotal role of TNF-α in mediating VEGF and MMP-9 up-regulation in hemorrhagic transformation of pituitary adenomas. Contrary to our expectations, the application of both VEGF and MMP-9 inhibitors attenuated but not abrogated TNF-α mediated hemorrhagic transformation. These results suggest that, besides VEGF and MMP-9, other targets may also contribute to hemorrhagic transformation. Our There is an inherent limitation of our experimental results from patient samples, because whether they are a cause or an effect of hemorrhagic incites remains unclear. However, the effects of hemorrhage have been reported to generally abate over the first week in animal studies ,35. As oApoplectic pituitary adenomas display three different pathologic types: hemorrhage, hemorrhagic infarction and infarction, and these three pathologic types are present in nearly equal numbers . MoreoveThe study was approved by the Research Ethics Committee of the Huazhong University of Science and Technology, Wuhan, China. Prior informed consent was obtained from all patients enrolled in this study. All specimens were made anonymous according to ethical standards.From May 2009 to December 2010, a series of 60 surgically removed pituitary adenomas were obtained from patients undergoing transsphenoidal surgery at the Department of Neurosurgery, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China. Each sample was bisected. One half was immediately frozen at −80 °C until protein and RNA extraction. The other half was fixed with 10% formalin and embedded in paraffin for histology and immunofluorescence.The sections were deparaffinized and rehydrated in successive xylene and alcohol, and then stained with Hematoxylin and Eosin for the morphological diagnosis. For immunofluorescence, paraffin sections of tumors were deparaffinized and rehydrated by successive washes with xylene, graded ethanol. Antigen retrieval was accomplished in microwave oven in 100 mM sodium citrate buffer (pH 6.0) for 10 min and then cooled to room temperature for 20 min. Subsequently, slides were incubated with 10% normal serum followed by the primary anti-TNF-α antibody  and incubated overnight at 4 °C. The slides were then incubated with corresponding Alexa fluor-labeled secondary antibody (Invitrogen) for 30 min. For quantification, 3 to 5 “hotspot” areas were captured for each slide by two individuals. Digital images were analyzed and quantified using Image-Pro Plus.MMQ, a rat prolactin-secreting pituitary tumor cell line, was purchased from ATCC  and routinely cultured in F-12 medium supplemented with 2.5% fetal bovine serum and 15% equine serum at 37 °C in a humidified air atmosphere containing 5% carbon dioxide in T-75 flasks. MMQ cells were stimulated with 20 ng/mL TNF-α (Abcam) or saline for 24 h, and then were analyzed for possible effects of TNF-α on VEGF and MMP-9 expression. In order to evaluate the impact of VEGF blockade on TNF-α mediated MMP-9 expression in MMQ cells, another group of cells were pretreated with VEGF inhibitor bevacizumab (Roche) (5 mg/mL) for 72 h and then TNF-α (20 ng/mL) was added for an additional 24 h. After incubation, cells were harvested for western blot and real-time RT-PCR analysis.The effect of TNF-α on cell viability was carried out using Cell Counting Kit-8 (Boster Co.). Briefly, MMQ cells were plated in 96-well plates . The cells were treated with TNF-α at concentrations of 0, 10, 20 ng/mL. After 24 h incubation, 10 μL of the CCK-8 solution was then added to each well of the plate, and then the plate was incubated for another 1 h. The absorbance was measured at 450 nm using a 96-well plate reader.Expression of VEGF and MMP-9 protein from adenoma tissues, cells and xenograft tumors were determined by western blot analysis. Tumors or cells were thawed on ice and homogenized at 4 °C in lysis buffer for extracting total protein. The protein was quantitated using a bicinchoninic acid (BCA) reagent. Equivalent amounts of protein (30 μg) from each sample were separated, under denaturing conditions, by electrophoresis on 10% SDS-PAGE gels (polyacrylamide gels containing sodium dodecyl sulfate). The protein bands were transferred by electroblotting to nitrocellulose membranes. The blots were incubated 1 hour with PBS contain 0.1% Tween 20 (PBST) and 5% powdered milk, and then incubated overnight with anti-human/rat MMP-9 antibody , anti-human/rat VEGF antibody  and anti-human/rat GAPDH antibody . The membranes were washed six times with TBST , and then were incubated with relative second antibodies and washed six times with TBST. Bands were visualized by ECL (Thermo-Pierce). In each tumor sample, protein levels were normalized to that of GAPDH as a loading control.6) were cultured with TNF-α (20 ng/mL) or TNF-α plus VEGF inhibitor bevacizumab (5 mg/mL) in serum free DMEM medium, after 24 h incubation, the supernatant was collected, and centrifuged at 2000 rpm for 10 min. Protein concentration was determined and electrophoresed on 7.5% non-reducing SDS–PAGE gels containing 2 mg/mL gelatin as a substrate at constant voltage. After electrophoresis, the gels were rinsed in renaturation buffer (2.5% Triton X-100) on a shaker for 30 min to remove SDS and were incubated overnight at 37 °C in a water bath in activation buffer composed of 50 mmol/L Tris-HCl (pH 7.5), 5 mmol/L CaCl2. Gels were stained using 0.5% Coomassie blue R-250 (Bio-Rad) for 2 h, followed by appropriate destaining. MMP activity was detected as a band on a dark blue background and quantified densitometrically using Image-Pro Plus.MMQ cells  according to the manufacturer’s instructions. Real-time RT-PCR was done using TaqMan one-step RT-PCR master mix kit and gene specific taqman gene expression assay kits in a 7300 real-time PCR system (Applied Biosystems). PCR amplification was carried out in a 20 μL reaction volume containing 200 ng of cDNA, 1.8 μM each of forward and reverse primers, and 10 μL Fast SYBR Green Master Mix (Applied Biosystems). The PCR reactions were initiated with denaturation at 95 °C for 10 min; followed by 40 amplification cycles at 95 °C for 15 s and 60 °C for 1 min. Samples were run in triplicate and results were averaged. CT was calculated by analysis software SDS 2.2.1 (Applied Biosystems). Analysis of gene expression changes were calculated using the 2-ΔΔCT method. The normalized expression ratio (fold induction) was calculated by 2-ΔΔCT = fold induction. Statistical analyses were performed using the CT ± SD values. The primer sequences used for real-time RT-PCR for Human TNF-α F: TGGAGAAGGGTGACCGACTC, R: TGCCCAGACTCGGCAAAG; Human MMP-9 F: CCCTGGAGACCTGAGAACCA, R: CCCGAGTGTAACCATAGCGG; Human VEGF F: TGTGAATGCAGACCAAAGAAAG, R: GCTTTCTCCGCTCTGAGCAA; Human GAPDH F: GAAGGTGAAGGTCGGAGTC R: GAAGATGGTGATGGGATTTC; Rat VEGF F: TGTGCGGGCTGCTGCAATGAT, R: TGTGCTGGCTTTGGTGAGGTTTGA; Rat MMP-9 F: CTTTGTAGGGTCGGTTCTG, R: GAGTGGATAGCTCGGTGG; Rat GAPDH F: GTCTTCACTACCATGGAGAAGG, R:TCATGGATGACCTTGGCCAG; Real-time PCR results were carried out by three replicate measurements of each sample.6 cells per mouse inoculation). rhTNF-α, bevacizumab and GM6001 were purchased from Weike Shanghai Biopharmaceutical Limited, Roche and Chemicon, respectively. Mice were randomized into five groups: control  group, rhTNF-α group, rhTNF-α plus bevacizumab (a VEGF inhibitor) group, rhTNF-α plus GM6001 (a broad MMP inhibitor) group, rhTNF-α plus bevacizumab and GM6001 group, with five mice in each group. Treatments were started two weeks after cell injection. rhTNF-α , bevacizumab (10 mg/kg i.p.) and GM6001 (10 mg/kg i.p.) were implemented twice per week. Both bevacizumab and GM6001 were administrated one week before and during rhTNF-α treatment. Tumors were harvested after one week of rhTNF-α treatment. Half of each tumor was frozen in liquid nitrogen for protein analysis and the other half was fixed in 10% formalin for histology analysis.Six week-old athymic nude mice were inoculated subcutaneously in the lower rear flank with MMQ cells suspended in 0.1 mL of PBS  or 0.9% saline into subcutaneous MMQ cell xenografts, respectively. Two days after injection, the mice were sacrificed and tumors were collected. Tumors from whole blood group (3 cases) and saline group (3 cases) were immediately frozen and then western blot analysis was performed for protein expression of MMP-9 and VEGF.in vitro and in vivo studies, values were expressed as mean ± SEM. Student t-tests were used to compare hemorrhagic adenomas versus non-hemorrhagic tumors on protein and mRNA levels. Pearson correlation test was used to determine the correlations between VEGF, MMP-9 and TNF-α protein expression in tissues. Results from animal and cell culture studies were analyzed using Student t-tests. Probability values of less than 0.05 were considered statistically significant.Statistical analyses were processed and analyzed with the aid of SPSS 17.0 software. For experiment with patient samples, results are expressed as the mean ± SD. For Taken together, our results show that TNF-α significantly correlates with intratumoral hemorrhage in pituitary adenomas and induces pituitary adenoma hemorrhage through up-regulation of VEGF and MMP-9. Based on these findings, our study demonstrates that TNF-α may play a role in the development of hemorrhage within pituitary adenomas.


A novel web-based tool PedWiz that pipelines the informatics process for pedigree data is introduced. PedWiz is designed to assist researchers in the analysis of pedigree data. It provides a convenient tool for pedigree informatics: descriptive statistics, relative pairs, genetic similarity coefficients, the variance-covariance matrix for three estimated coefficients of allele identical-by-descent sharing as well as mean allele sharing, a plot of the pedigree structures, and a visualization of the identity coefficients. With a renewed interest in linkage and other family based methods, PedWiz will be a valuable tool for the analysis of family data. When a researcher has collected or is provided with a set of nuclear family or extended pedigree data for genetic analysis, the first thing that needs to be done is to find out what information is available on the family or families before proceeding in the analysis of phenotype and/or genotype data to study the characteristics of a certain disease or trait, i.e., pedigree informatics. This can include descriptive statistics, visualization of family data, the degree of genetic relatedness among members of a family, and so on.kinship , and this is the only currently available program that outputs the nine condensed coefficients of IBD. The R package ibdreg by sim.ibd.var and exact.ibd.var, to calculate the variance-covariance of mean allele sharing, but not the variance-covariance of the individual coefficients of IBD. An essential part of score tests is the choice of the denominator variance, and some of these tests for genetic linkage require the variance-covariance of allele IBD sharing statistics under the null, i.e., of the coefficients of IBD. It would be useful to make available the variance-covariance matrix of these coefficients for a pedigree independent of the choice of test statistics, so that it can be used for different choices of test statistics. Currently, no such tools are available.A brief survey of available R packages with their relevant components of pedigree informatics is shown in Pedigree Informatics Wizard) is designed to fulfill this need as a web-based tool for pedigree informatics, to assist researchers in the analysis of pedigree data. It provides a convenient “one-stop-shop” for pedigree informatics. It provides all the genetic similarity coefficients mentioned above, including the nine condensed coefficients of IBD and the variance-covariance matrix of the one-locus three marginal coefficients of allele IBD sharing, as well as other pedigree descriptive statistics. Additionally, it provides a plot of the pedigree structure and a visualization of the identity coefficients, something that no other program provides. PedWiz is an automated pipeline for extracting pedigree informatics before conducting specialized analyses of phenotype and/or genotype data.PedWiz (The web interface of PedWiz is implemented using a combination of XHTML (eXtensible HyperText Markup Language), CSS (Cascading Style Sheets), and PHP (Hypertext Preprocessor) on an Apache web server. The interactivity is provided by JavaScript and Ajax technologies. Custom Python modules handle the overall flow of the pipeline by calling pre-existing programs written in C++ or R.PedWiz accepts a plain ASCII text file format for pedigree input. Since PedWiz extracts the information contained in a pedigree structure, it requires a pedigree file to have five essential columns: pedigree ID, individual ID, the two parents’ IDs and sex. These five columns do not need be in any specific order, nor need they be consecutive. If a pedigree file contains other columns, they are ignored. The pedigree file is required to be in either tab-delimited or comma-delimited format. It may optionally contain a header line specifying the names of the columns. The user inputs configuration information and the location of the pedigree file through a user-friendly interface, and then submits it to start the analysis pipeline.Figure 1). The complete process utilizes many internal Python scripts (which are not detailed here) to create junctions between the programs (format compatibility) and to create the necessary R scripts.Once the user submits a pedigree file and configuration information, the informatics process starts by running the first tool. Currently, the PedWiz process consists of six main tools (each_pedigree = true option). PEDINFO provides many useful descriptive statistics on pedigree data including means, standard deviations; family, sibship and pedigree sizes; and counts of each type of relative pair. The results are parsed and reported to the user by PedWiz as a table on the website. From here, the user selects a pedigree to proceed with other tools.This tool is used to calculate the descriptive statistics for each pedigree contained in the user-submitted pedigree file. PedWiz utilizes the existing C++ program PEDINFO of the S.A.G.E. package , and the other is the matrix of nine condensed coefficients of IBD. The coefficients of relationship, which are twice the kinship coefficients, can be easily calculated from the kinship/inbreeding coefficients. The resulting matrices are reported to the user on the website as a text file.This tool is used to visualize the two matrices generated by the genetic similarity tool. PedWiz uses a custom R script to represent a matrix graphically as a heat map. The resulting heat maps are reported to the user as a pdf file on the website.This tool is used to find the variance-covariance matrix of the coefficients reported by the genetic similarity tool. PedWiz uses an internal C++ program to perform this task. The variance-covariance matrix of kinship coefficients is estimated by an exact method given by . Here we present an application example using pedigree data from the Madeline 2.0 website . The resulting output from each tool for the example pedigree is shown also.After configuration information and the location of the pedigree file have been submitted by the user, PedWiz produces a table with the descriptive statistics for each pedigree on the website as shown in We developed a novel web-based tool PedWiz that pipelines the informatics process for pedigree data. PedWiz is designed to assist researchers in the analysis of pedigree data. It provides a convenient tool for pedigree informatics: descriptive statistics, relative pairs, genetic similarity coefficients, the variance-covariance matrix of three coefficients of allele IBD sharing, as well as mean allele sharing, a plot of the pedigree structure, and visualization of identity coefficients. PedWiz is an automated pipeline for extracting pedigree informatics before conducting specialized analysis of phenotype and/or genotype data.Emerging availability of whole genome sequence data has led to a renewed interest in linkage and other family based methods . Many reAdditionally, the results from each tool in Pedwiz will be useful for later analysis of phenotype and/or genotype data. As stated before, an essential part of score tests is the choice of the denominator variance, and some of these tests for genetic linkage require the variance-covariance of the coefficients of IBD. No software tools are currently available to provide this information independent of the choice of test statistics. The variance-covariance of the genetic similarity tool of PedWiz provides this need, so that it can be used for different choices of test statistics. The information from the genetic similarity tool of PedWiz can be used for weighting pedigrees of different sizes. Another potential use of this tool is for selecting families with the most information in terms of genetic relatedness that would best suit a phenotype/genotype analysis of choice. Selecting families with multiple affected subjects, or families with extreme values, is known to provide improved ability to measure, and detect, the effects of rare variants . The strFigure 1 with dotted lines. PedWiz is currently specialized to deal with the information contained within pedigree structures only. Therefore, it is very fast and safe with regard to data transfer over the web. However, it is always possible to add more pipeline modules that could process the information from phenotype and/or genotype data. Good candidates for this addition would be simulation conditional on given phenotype and/or genotype data, and imputation. Another extension that could be added on is the inclusion of a backend database to save data and results for reuse.With a modular design, each analysis tool within PedWiz is independent of the others, so it is very easy to extend and add more tools. Planned additions in the near future are simulation and pedigree split tools, shown in The genetic similarity tool of PedWiz is specifically designed to provide the information on within-pedigree relatedness. As a reviewr pointed out, a tool that addresses between-pedigree relatedness, especially for pedigrees from a relatively isolated population like the Hutterites, would be a useful addition to PedWiz. Cryptic relatedness among unrelated individuals can be estimated by incorporating a number of dense markers across different chromosomes . There aThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.