Mycobacterium bovis is considered to be a possible risk factor for onward transmission of bovine tuberculosis (BTB) in infected cattle and is known to compromise diagnostic tests. A comparison is made here of M. bovis shedding (i.e. release) characteristics from 12 calves, six experimentally co-infected with BVDV and six infected with M. bovis alone, using simple models of bacterial replication. These statistical and mathematical models account for the intermittent or episodic nature of shedding, the dynamics of within-host bacterial proliferation and the sampling distribution from a given shedding episode. We show that while there are distinct differences among the shedding patterns of calves given the same infecting dose, there is no statistically significant difference between the two groups of calves. Such differences as there are, can be explained solely in terms of the shedding frequency, but with all calves potentially excreting the same amount of bacteria in a given shedding episode post-infection. The model can be thought of as a process of the bacteria becoming established in a number of discrete foci of colonization, rather than as a more generalized infection of the respiratory tract. In this case, the variability in the shedding patterns of the infected calves can be explained solely by differences in the number of foci established and shedding being from individual foci over time. Should maximum exposure on a particular occasion be a critical consideration for cattle-to-cattle transmission of BTB, cattle that shed only intermittently may still make an important contribution to the spread and persistence of the disease.Concurrent infection of cattle with bovine viral diarrhoea virus (BVDV) and  Mycobacterium bovis or bovine tuberculosis (BTB) is a global problem, important both owing to its economic cost and because it is a zoonosis. While the introduction of standardized testing regimes has led to the eradication or dramatic decline of BTB incidence in many countries, BTB remains a problem in a number of them. Notably, despite a comprehensive testing regime and enormous resources spent on its control, BTB in the UK has not only persisted but has also been on the increase for at least the past 30 years . A general concept is that the eradication of disease is not possible for those infectious agents that persist in a wild animal reservoir. The strong correlation between areas with high prevalence of BTB in the badger (Meles meles) and a high incidence of BTB in the cattle supports the argument that the two are related and that this reservoir of infection is a major reason for the continuing infection with M. bovis in the UK herd  for 7 days and aliquots of log-phase organisms frozen at −70°C. The number of colony-forming units (CFUs) was determined by titration on modified 7H11 agar (containing 10% OADC supplement and 4.16 g pyruvate per litre) and incubated aerobically at 37°C for four weeks. This strain is a fully virulent Great Britain strain isolated in 1997 from a diseased cow suffering from caseous lesions in the lung and bronchomediastinal lymph nodes, the genome sequence for which is available  is the frequency of shedding at time t, p(n) is the probability that n events will occur and thus P(x) is the cumulative probability that there are x or fewer shedding events producing measurable quantities of bacteria when a sample is taken at time t. Thus, if x=1, for example, equation λ(t) is assumed constant (one-phase model), and in the second λ(t) is assumed to start at zero and then is a constant positive value thereafter (two-phase model), i.e.λ0 is the constant frequency after time t0.First, we assume that shedding of bacteria is associated with the bursting of individual lesions. This is modelled as a random event described by a Poisson distribution, defined byB(t). Bacterial infection and replication are initially controlled by the cell-mediated immune response, with bacteria phagocytosed by macrophages (M(t)), which proliferate at a rate that depends on the density of bacteria  is the amount of bacteria recorded in a sample at time t; ϕ (log(S(t))/log(B(t))) (logs are base 10) is the cumulative probability that a sample containing S(t) CFU ml−1 will be found when the average shedding level is B(t) CFU ml−1. Technically, this allows for bacterial shedding of less than zero, but in practice the variance of the distribution is narrow enough for this to be unimportant. Data at some time points are too sparse (i.e. none) to generate typical profiles or to make any meaningful comparisons between time points. Therefore, we normalize the data at each time t using the expected mean shedding quantity at time t from the solution of the system of equations t, the ratio log(B (t))/σ(t) is constant, where σ(t) is the standard deviation of log(S(t)) at time t. In other words, if the maximum shedding level in one event is 100 CFUs per ml, for example, and the maximum in another is 1000, then the probability of collecting a sample when the current shedding level is 10 CFUs per ml in the first case is the same as the probability of collecting when the shedding level is 100 CFUs per ml in the second.If excretion is the sum of individual episodes of shedding, and each episode experiences a distinct rise and fall with finite slope, then the time past the start of the shedding episode at which a sample is taken is important for determining the quantity of bacteria recovered. Therefore, it is unlikely that the amount of bacteria recovered directly represents either the maximum shedding or the total amount released. Accordingly, we reconstruct a typical episode profile by fitting the distribution of sampling at a given time with a lognormal distribution, described byp=0.42) which is reflected in the wide 95% CIs . The random effects due to calf are not of primary interest here, but do show a similar within-calf variance in comparison to between-calf variance.The simplest analysis of the repeated measures data was to include a fixed effect for BVDV and a single random effect (intercept) for each calf . The estp=0.72).To explicitly model the time element of shedding profiles, we included a ‘day’ covariate. A good model for the data was found to be a quadratic shedding profile with random effects terms for both the linear and the quadratic terms for each calf. This accounts for between-calf variation in both the timing of peak shedding and the curvature at the peak. The model provided a significantly better fit than all nested models with simpler fixed or random effects  at the p=0.10 level (λ: since shed bacteria are detected for every sample past t0 (even though resampling might detect a gap), λ=∞ will give the best fit .As the models are nested, the likelihood ratio test is used to determine the appropriate model (one-phase or two-phase) for each shedding pattern. In 4 of the 12 calves , the two-phase model was significantly better at the 10 level . The nor10 level  is shown10 level a and  is the amount of bacteria sampled, R1 and R2 are random numbers from 0 to 1, B(t) is the solution to equations ϕ−1(R1) and p−1(R1) are the inversion functions of equations The frequency with 95% CIs is shown in Using the shedding profile parameters for equations M. bovis recovered from nasal secretions. Consideration needs to be given for whether this was due to a genuine biological phenomenon or whether it was simply a technical consequence of some unknown variability in the performance of the sampling and assays. There was a highly significant linear correlation between the extent of the lesions  and between the extent of lesions and the number of days on which bacteria were shed . Thus, it is likely that the shedding of bacteria in nasal secretions reflects the level of disease in individual calves due to variation in resistance, which is either genetic, i.e. innate or adaptive and the result of experience of other infections.Considerable variation was evident for calves with respect to quantity of It also appears that the overall level of shedding increases for the first four weeks after exposure and then begins to decline. This is consistent with the appearance of the immune response after infection and high levels of IFNγ . Taking into account the practical constraints on group size which are often faced in this field, it is important that the meta-analysis across similar experiments and the pooling of data from different investigations are first undertaken. Quantitative descriptions, such as are provided here, will aid such analyses, as well as further experimental design.M. bovis are currently removed from the herd as soon as possible, and there is no recommendation that this should change. Differences in shedding could be due to chance, or phenotypic or genotypic differences in the calves. Prior exposure to environmental organisms has been reported to impart a degree of resistance to tuberculosis in humans and in animal models designed to mimic this (The data are consistent with a model in which exposure results in the establishment of individual foci of infection and calves with more extensive disease have more foci. These individual foci then release bacteria into the respiratory tract and they are shed in the nasal secretions. These foci should not be regarded as being granulomas present later in the progression of BTB, which undergo the process of caseation and liquefaction that releases infectious material into the airways (mic this . Further


Also, HgCl2- injected rats develop glomerular cell infiltrates consisting of ED1+ cells (monocyte/macrophage), starting on day 4 and reaching a maximum on day 8. Treatment with anti-TNF-α antiserum had preventative effects as it reduced the urinary protein levels to close to the normal range and also blocked the influx of inflammatory cells in the renal glomeruli and interstitium, but circulating anti-GBM and lineal glomerular IgG deposits were unmodified. In addition, whole isolated glomeruli from HgCl2-induced nephritis secreted TNF-α commencing on day 8, being maximally detected on day 11 and preceding, between 2 to 3 days, the development of proteinuria. The administration of anti-TNF-α antiserum or anti-α4 integrin mAb completely abrogated the synthesis of TNF-α in glomeruli isolated from the respective treated groups of animals, in addition to the proteinuria. Taken together our results confirm that TNF-α plays an important role in the induction and development of HgCl2-induced nephritis and highlights the pathogenic importance of the local release of TNF in those renal diseases in which prominent glomerular macrophage accumulation is a constant feature.HgCl


The concentrations of TNF α were significantly elevated in patients with malignant tumors of the adrenal cortex and in patients with Conn’s syndrome compared to control. In patients with non-functioning adenomas and pheochromocytomas, TNF α levels were similar to those detected in the control. In subjects with myelolipomas, the serum concentration of TNF α was lower compared to the control. After adrenalectomy, the levels of TNF α were decreased in patients with malignant tumors and in patients with Conn’s syndrome, nonfunctioniong adenomas and pheochromocytomas compared to the concentration before surgery. The serum concentrations of soluble receptors of TNF α did not differ among different patient groups and compared to the control. After adrenalectomy, the blood concentrations of TNF α R1 and TNF α R2 were decreased in patients with Conn’s syndrome. However, to confirm practicality of the evaluation of TNF α and its soluble receptors in differential diagnosis in patients with adrenal tumors, a larger study group is needed.The peripheral blood levels of TNF  Most of them are incidental findings. Although the majority of adrenocortical and adrenomedullary tumors are benign, there are no clinical and laboratory markers to distinguish most of them from malignant tumors ,2. The mα is a pleiotropic cytokine that plays a central role in inflammation and apoptosis. It modifies the inflammatory reactions and immune reactions in response to injury and infection . Seven patients with malignant cortical tumors, seven patients with Conn’s syndrome, 10 patients with non-functioning adrenal adenomas, 12 patients with pheochromocytomas and three patients with myelolipomas (according to pathology data) were studied. The clinical diagnosis of the malignant tumors was suspected in patients with weight loss, high serum concentration of dehydroepiandrosterone sulfate and irregular, large  mass found by computer tomography imaging. The diagnosis of the Conn’s syndrome was based on elevated level of plasma aldosterone, low level of plasma renin and an aldosterone/renin ratio of greater than 5.4. The diagnosis of pheochromocytoma was suspected in patients with poorly controlled hypertension associated with bouts of sweating, headache, palpitations, a high serum concentration of chromogranin A and a high level of metanephrins in urine. In patients with nonfunctioning adenomas and myelolipomas, the tumors were incidentally found during computer tomography imaging.The study group was composed of 39 subjects [28 females aged 25–82  aged 36–60 (49.5 ± 8.38). The presence of adrenal tumor was excluded on the basis of physical examination, laboratory tests and ultrasound examination of the abdomen. All patients and control group were included in the study only after giving informed consent. The study was approved by the Local Ethical Committee.α and its soluble receptors were measured by enzyme immunoassay (ELISA) . The intra-assay precision for TNF α was 4.67%, for TNF α R1 – 4.43%, and for TNF α R2 was 3.53%. The inter - assay precision for TNF α was 5.8%, for TNF α R1 – 6.1%, and for TNF α R2 was 4.07%. The statistical analysis was performed using the non - parametric Kruskal-Wallis test and Wilxocon test. A p-value of less than 0.05 was considered statistically significant.The concentration of TNF 3.α, TNF α R1 and TNF α R2 before surgery and after surgery are presented in x ± SD) concentration of TNF α in the control group was 12.09 ± 4.31 pg/mL, the mean concentration of TNF α R1 was 1,479.2 ± 388.23 pg/mL, and the mean concentration of TNF α R2 was 2,456.7 ± 521.12 pg/mL. The concentration of TNF α was significantly elevated in patients with malignant tumors of the adrenal cortex, compared to the control , and in patients with Conn’s syndrome, compared to the control . The highest levels of TNF α in patients with malignant tumors of the cortex were noted. In patients with non-functioning adenomas and pheochromocytomas, TNF α levels were not significantly different from those of the control, (p > 0.05) and were respectively 11.0 ± 2.03 pg/mL; 9.14 ± 4.58 pg/mL. In patients with myelolipomas, the serum concentration of TNF α was lower, compared to control  (The concentrations of TNF  < 0.05) .α were lower in patients with malignant tumors , and in patients with Conn’s syndrome , non-functioning adenomas  and pheochromocytomas  than before surgery    and 6.α and its receptors were found.No significant correlations between the size of the tumors and the serum level of TNF 4.α is a transmembrane protein described originally as a protein which causes the haemorrhagic necrosis of experimental tumors [α possesses proinflammatory activity and plays a key role in apoptosis [α and its receptors may increase susceptibility to infections [α is also probably involved in development of lymphoid tissue [TNF l tumors . Furtherpoptosis –12. A lofections . TNF α id tissue .α in pathogenesis of malignant tumors is not clearly understood. Mocellin and Nitti [α may represent one of the molecular links between chronic inflammation and further development of malignant tumors. Leibovich et al. [α may promote angiogenesis but others [α. Schweigerer et al. [α on the proliferation of endothelial cells derived from blood vessels of the brain and adrenal cortex. In both types of these cells, TNF α decreased the proliferative activity of endothelial cells.The role of TNF nd Nitti  suggest h et al.  revealedt others  did not r et al.  assessedα acts also as a paracrinal and autocrinal regulator of the growth and development of the adrenocortical cells. Call et al. [α and IL-6. They also proved, using immunoassay techniques, that the glomerular zone of the adrenal cortex is the site of the greatest synthesis of TNF α. The increased level of TNF α in our study in patients with Conn’s syndrome may confirm that the glomerular zone of the adrenal cortex mainly secretes TNF α. The secretion of TNF α by human adrenal glands confirmed the studies of González-Hernández et al. [et al. [α has an influence on synthesis and secretion of steroid hormones in adrenal cortex [α occur in the blood of patients with malignant tumors. Cimino et al. [α  in the blood of patients with acute leukemia. Elevated levels of TNF α were also confirmed in the peripheral blood of patients with melanoma [TNF l et al.  revealedz et al. ,20 and P [et al. . It is al cortex . Moreoveo et al.  observedmelanoma , renal cmelanoma  and pancmelanoma .α in patients with malignant tumors . It may be due to activation of the immunological system and cytokine net in patients with malignant tumors. The elevated levels of TNF α in patients with malignant tumors may be also due to activation of angiogenesis and overexpression of TNF α in the transformed cells. Interferon γ is known as the most important factor to increase the expression of TNF α. The regulation of the expression is at the transcriptional level [α may lead to overexpression of integrins and adhesive molecules [et al. [α on the expression of ZC3H10 (zinc finger protein) and GRHL-3 (grainyhead-like 3) in breast cancer cell lines (MCF-7). Expression of these proteins was increased after addition of TNF α to the incubation medium of the cells in vitro. However, ZC3H10 acts as an antagonist of cell growth in vitro, but GRHL-3 stimulates the migration of the endothelial cells.The results of our study showed the highest level of TNF al level . It has olecules . Guardio [et al.  assessedα was decreased after adrenalectomy. These results may be due to elimination of the tumor, which was the main local source of TNF α.In our patients with malignant tumors of the adrenal cortex, in patients with Conn’s syndrome and in patients with non-functioning adenomas and pheochromocytomas the concentration of TNF α in the pathogenesis of adrenal tumors is unknown. Elevated levels of these receptors were found in the amniotic fluid of pregnant women [α receptors may bind TNF α and inactivate this molecule. On the other hand, these forms of TNF α receptors can stabilize the TNF α molecule at low concentrations of TNF α [The role of the soluble receptors of TNF nt women ,31, in tnt women  and patint women . Some auof TNF α .α receptors did not differ, either among study groups or compared to the control. After adrenalectomy, the levels of the soluble forms of TNF α decreased in patients with Conn’s syndrome. It could be argued that the soluble forms of TNF α receptors are also involved in the process of tumorigenesis. The low level of TNF α and high level of soluble forms of TNF α receptors in patients with myelolipoma may confirm the hypothesis that soluble forms of these receptors inactivate this molecule. However, such a small group (3 patients) is not sufficient to draw any significant conclusions.The results of our study revealed that the concentration of soluble forms of TNF α and its soluble receptors in differential diagnosis in patients with adrenal tumors, a large study group is needed. The evaluation of the concentration of numerous cytokines in the blood has been used only in the clinical studies so far. To take advantage of these molecules in clinical practice, further studies are needed and clinical examinations, laboratory tests and imaging techniques will still be the most important means of diagnosing patients with adrenal tumors.To confirm practicality of the evaluation of TNF 5.α and its soluble receptors appear to be involved in adrenal gland oncogenesis. The peripheral blood concentrations of this cytokines before surgery may be useful for the discrimination between malignant and benign adrenal tumors.TNF


P<0.01) and size of metastases (P<0.01) of B16F10 melanoma cells. This effect is also observed when an anti-TNF neutralising monoclonal antibody is administered, confirming the essential role TNF plays in metastasis in this model. This study suggests that TNF autovaccination is a cheaper and highly efficient alternative that can block TNF and reduce metastasis in vivo and trials with TNF autovaccination are already underway in patients with metastatic cancer.TNF is a proinflammatory cytokine involved in the pathogenesis of chronic inflammatory diseases, but also in metastasis in certain types of cancer. In terms of therapy, TNF is targeted by anti-TNF neutralising monoclonal antibodies or soluble TNF receptors. Recently, a novel strategy based on the generation of self anti-TNF antibodies (TNF autovaccination) has been developed. We have previously shown that TNF autovaccination successfully generates high anti-TNF antibody titres, blocks TNF and ameliorates collagen-induced arthritis in DBA/1 mice. In this study, we examined the ability of TNF autovaccination to generate anti-TNF antibody titres and block metastasis in the murine B16F10 melanoma model. We found that immunisation of C57BL/6 mice with TNF autovaccine produces a 100-fold antibody response to TNF compared to immunisation with phosphate-buffered saline vehicle control and significantly reduces both the number ( TNF is an important proinflammatory cytokine involved in normal physiological immune and inflammatory processes. However, when inappropriately expressed, TNF also plays a role in the development of chronic inflammation and diseases associated with it .α/KC, as well as matrix metalloproteinases (MMP) and urokinase-plasminogen activator, molecules involved in ECM degradation and cellular migration (Previous studies have suggested that one of the main mechanisms by which TNF promotes tumour growth is by upregulating metastasis. TNF activates key molecules involved in metastasis such as IL-8 (an angiogenic chemokine) and GroMonoclonal antibodies and IgG fusion proteins are now an established approach to blocking TNF. We have previously used monoclonal antibodies against TNF to ameliorate disease in animal models of arthritis  emulsified with complete Freund's adjuvant (CFA), in a volume of 100 μl  three times per week or PBS. Again to reduce biased selection, the treatments were blinded coding them A and B prior to insertion into the mice and the code was broken after the data were collected and analysed. The monoclonal antibodies were a rat mouse fusion protein . The day after the initial injection, 105 B16F10 murine melanoma cells in 200 μl of PBS were injected into the tail vein of each mouse. After 3 weeks, the mice were killed and the lung tumours counted and the diameter measured as described above.C57BL/6 mice (6-week old) were injected intraperitoneally (i.p.) with 500 μg ml−1 of TNF in PBS at 100 μl well−1 and incubated overnight at 4°C. The plates were blocked with 2% bovine serum albumin (BSA) in PBS (200 μl well−1) for 1 h at 25°C and washed with 0.5% Tween in PBS after this and all subsequent steps. A measure of 100 μl of one in three serial dilutions of serum samples and the positive and negative control sera were incubated for 1 h at 25°C. The detection antibody (100 μl well−1) was diluted in 0.5% BSA in PBS and incubated for 1 h at 25°C. A sheep anti-mouse polyclonal detection antibody conjugated to horseradish peroxidase was used. These samples were developed with a peroxidase substrate system TMB, the reaction was stopped with 1 M H2SO4 and absorbance read at 450 nm. Since standard known amounts of antibodies to these antigens were unavailable, eight serial dilutions of sample sera were made and their titres taken as the dilution that gave an OD corresponding to that of the negative control sera. The negative control sera were from unimmunised mice and the positive control sera were pooled sera from previously immunised mice that had been tested by ELISA and found to have antibodies that bound TNF.Microtitre plates were coated with 1 2 in RPMI supplemented with 10% foetal bovine serum (BioWhittaker), penicillin (100 U ml−1) and streptomycin (100 μg ml−1). B16F10 cells were plated in 96-well plates at 2 × 106 cells ml−1 and 200 μl well−1. The cells were stimulated with LPS at 10 μg ml−1; some cells were further stimulated with murine TNF (mTNF) at 10 or 100 ng ml−1 and other cells with media as a control. The plates were incubated 37°C. Supernatants were removed at 24 and 72 h and stored at −20°C for immunological detection assays. KC was measured by ELISA as described previously , and then B16F10 melanoma cells were injected into the tail vein. We found that mice treated with anti-TNF monoclonal antibodies had significantly less and smaller metastases compared to the mice treated with PBS , previously described to play a role in metastasis  in keratinocytes . As we were unable to examine any cytokines in the mice sera, we examined the effects of blocking TNF inocytes , fibrobl tumours .Overall, our study shows that although metastasis is a complex multisystem process involving a large number of different molecules, in the B16F10 murine melanoma model TNF plays an important and rate-limiting role. This is likely to be partly due to its ability to increase the expression of prometastatic molecules such as KC. In addition, our study demonstrates that the TNF autovaccination technology is a very efficient strategy in producing anti-TNF antibodies in mice and preventing in that way the number and size of metastases. This strategy is also safe in this experimental system as no adverse effects or increase in tumour growth or metastasis was seen. We are currently conducting a phase I human clinical trial to evaluate the effect of blocking TNF by using TNF autovaccination in patients with a variety of cancers. Results from these trials are eagerly awaited. If this strategy is found to be effective in human trials, it would be a cost-effective alternative to infusing monoclonal antibodies and more convenient for patients who would possibly only require three injections for a 3-month benefit.


Esculentoside A (EsA) is a saponin isolated from the roots of Phytolacca esculenta. Previous experiments showed that it had strong anti-inflammatory effects. Tumour necrosis factor (TNF) is an important inflammatory mediator. In order to study the mechanism of the anti-inflammatory effect of EsA, it was determined whether TNF production from macrophages was altered by EsA under lipopolysaccharide (LPS) stimulated conditions. EsA was found to decrease both extracellular and cell associated TNF production in a dose dependent manner at concentrations higher than 1 μmol/l EsA. Previous studies have showed that EsA reduced the releasing of platelet activating factor (PAF) from rat macrophages. The reducing effects of EsA on the release of TNF and PAF may explain its anti-inflammatory effect.


Mycobacterium bovis bacille Calmette–Guérin (BCG), was tested in knock-in mice in which the endogenous TNF was replaced by a non-cleavable and regulated allele . While 100% of mice with complete TNF deficiency (TNF−/−) succumbed to infection, 50% of TNFtm/tm mice were able to control M. bovis BCG infection and survived the experimental period. Membrane expressed TNF allowed a substantial recruitment of activated T cells and macrophages with granuloma formation and expression of bactericidal inducible nitric oxide synthase (iNOS). Using virulent Mycobacterium tuberculosis infection we confirm that membrane TNF conferred partial protection. Infection in TNFtm/tm double transgenic mice with TNF-R1 or TNF-R2 suggest protection is mediated through TNF-R2 signalling. Therefore, the data suggest that membrane-expressed TNF plays a critical role in host defence to mycobacterial infection and may partially substitute for soluble TNF.Tumour necrosis factor-α (TNF-α) plays a critical role in the recruitment and activation of mononuclear cells in mycobacterial infection. The role of membrane TNF, in host resistance against  Mycobacterium tuberculosis infection, both in humans and experimental animals, is regulated by T cells, macrophages and cytokines, which include interferon-γ (IFN-γ), interleukin-12 (IL-12) and tumour necrosis factor (TNF).M. tuberculosis and succumb to the infection.6Protective immunity to A critical role for TNF in mycobacterial defence is inferred from neutralization and deletion experiments in mice.13TNF is produced by macrophages, but is expressed by a variety of cells and is a major regulator of inflammation and leucocyte trafficking.in vivo role including the control of BCG and M. tuberculosis infectionM. tuberculosisListeria monocytogenes infection.L. monocytogenes infection, but no data are available on mycobacterial infections.25Several biological functions of membrane TNF have been described, such as cytotoxicity, polyclonal activation of B cells, and induction of IL-10 by monocytes and intracellular adhesion molecule-1 expression on endothelial cells.Mycobacterium bovis BCG, while 100% of TNF-deficient mice succumb to infection. TNFtm/tm mice were able to recruit and activate macrophages and T cells, and generate mycobactericidal granuloma in response to M. bovis BCG infection. However, resistance to the virulent M. tuberculosis strain was less pronounced, suggesting substantial differences between this and the Δ1-9,K11E mouse mutant.24Here, we demonstrated that the Δ1-12 TNF mutanttm/tm), TNFR1−/−, TNFR2−/− and TNF−/−tm/tm mice were crossed on TNFR1−/− mice−/− miceAdult, 8- to 10-week-old female mice of the wild type (WT) strain C57BL/6, homozygous Δ1-12 TNF mutantM. tuberculosis H37Rv  and M. bovis Bacille Calmette–Guérin (BCG)  were grown in Middlebrook 7H9 medium , supplemented with 10% oleic acid albumin dextrose catalase (OADC)  and 0·5% glycerol  in 5% CO2 at 37° and frozen in aliquots. Prior to use, an aliquot was thawed, briefly vortexed, diluted in sterile saline and clumping disrupted by aspirating through a 29-gauge needle  20 times.tm/tm and TNF-deficient mice were injected intravenously (i.v.) at an infective dose of 2 × 106 BCG. Groups consisted of 10 mice and the experiments were repeated twice. Mice were killed at 2, 4 and 8 weeks after infection. For the aerosol infection mice were infected with 30 viable CFU of H37Rv via the aerosol route using an inhalation exposure system .WT, TNFEscherichia coli, serotype O111:B4; Sigma, St Louis, MO) with d-Gal (Sigma) was injected intraperitoneally (i.p.) to induce systemic TNF and endotoxic shock as described previously.d-Gal. Mice were monitored for 24 hr and the number of moribund mice was recorded.LPS . Plates were incubated at 37° for 19–21 days and colonies counted. Data are expressed as mean CFU per organ.g for 5 min, the supernatant was removed, aliquoted and stored at −80° for cytokine and chemokine analysis. Lungs were subsequently lavaged with four volumes of 800 μl PBS. To remove traces of red blood cells, pooled samples were incubated in 1 ml red cell lysis buffer for 5 min, washed twice with 5 ml PBS. Samples were centrifuged at 405 g for 5 min and the cells were used for flow cytometric analysis. Procedures were performed under sterile conditions and samples kept on ice for the duration of the experiment.Under anaesthesia a 20-gauge catheter  was inserted into the exposed trachea. The lungs were lavaged with two volumes of 300 μl PBS. Lavage fluid was centrifuged at 405 5cells/ml cell suspension was pipetted into each well and the liquid was drained from the well by attached Whatman filter paper. The slide was left to air dry overnight at room temperature. The cells were stained with RapiDiff staining kit, after removal of the filter paper, for differential cell counting. Unstained slides were stored at −20°, wrapped in aluminium foil. Cells were stained by immersing the slides three times (±5 s) in the following solutions: RapiDiff fixative, RapiDiff solution 1 (eosin in phosphate buffer) RapiDiff solution 2 (haematoxylin) and a final rinse in distilled H2O. Slides were left to dry for 1–2 hr at room temperature and mounted with entellan (Merck). Routinely, 100 cells per cytospin, in duplicate, per mouse were differentiated in a random fashion.Teflon coated, eight-well (6 mm) multi-well slides  were used for cytospins. A 100 μl of a 2·5 × 106) were incubated with 25 μl fluorescence-activated cell sorting (FACS) blocking solution for 30 min after which they were washed with 1 ml FACS buffer and centrifuged at 405 g for 5 min. Cells were incubated with 2 μg/ml anti-CD3 phycoerythrin  and either 2 μg/ml anti-CD4 fluoroscein isothiocyanate  or 2 μg/ml anti-CD8 FITC (clone 53-6-7) in a total volume of 50 μl for 30 min. Cells were washed with 1 ml FACS buffer, centrifuged at 405 g for 5 min and resuspended in FACS fixation buffer. Samples were kept on ice for the duration of the labelling procedure. Fluorescently labelled antibodies were purchased from BD Pharmingen . Samples were analysed on a FACSCalibur  flowcytometer using Cell Quest software.Cells  using a syringe fitted with a 29-gauge needle. The cells eluted were resuspended in DMEM supplemented with 30% L929 conditioned medium and 20% horse serum and plated at a concentration of 2 × 106cells/10 ml in Sterilin 90-mm bacteriological plates. On day 10, cells were pooled, pelleted by centrifugation and plated at 3 × 105 cells/well in a 48-well plate . Cells were stimulated with 10 μg/ml LPS  and 100 U/ml rIFN-γ (BD Pharmingen) for 72 hr. In the infection study, the cells were prestimulated with 100 U/ml rIFN-γ for 16 hr and infected with M. bovis BCG (multiplicity of infection = 1:2) and the supernatants collected after 54 hr.Mice 6–8 weeks old were killed and the femurs and tibia removed aseptically as described before by Müller TNF, IFN-γ and IL-12, IL-4, IL-5, monocyte chemoattractant protein-1 (MCP-1) and RANTES  in supernatant of bronchoalveolar lavage fluid (BALF) were measured by ELISA with a sensitivity of <15 pg/ml .t-test and anova with a level of significance of P < 0·05.Statistical evaluation of differences between the experimental groups was determined by the use of the Student’s tm/tm knock-in mice do not secrete TNF, mice were injected i.p. with 100 μg LPS and blood was collected after 90 min and serum analysed by ELISA. In contrast to WT mice that displayed significant serum TNF levels, TNF was undetectable in the sera of both TNF−/− and TNFtm/tm mice  suggesting partial protection from pulmonary infection.We and others have shown previously that the elimination of mycobacteria from the lung is absolutely dependent on TNF, while evidence for bacterial clearance from the liver and spleen in a TNF-dependent and -independent manner after systemic infection was reported. lesions . As a sunchanged . HoweverWe then determined the mycobacterial growth and elimination in the lungs over 8 weeks. All three mouse strains had similar bacilli levels (CFU) in the lungs 2 weeks after infection . At 4 wetm/tm mice and TNF−/− mice (P < 0·01) than in WT mice. TNF−/− mice displayed uncontrolled mycobacterial growth with a significant increment in CFU in the lung at 8 weeks as compared to the 4 week values (P < 0·05). In contrast to TNF−/− mice, TNFtm/tm mice were able to control infection at 8 weeks. CFU in lungs of TNFtm/tm at 8 weeks were lower than at 4 weeks (P < 0·05). In addition, CFU in the lungs of TNFtm/tm mice were significantly lower than that of TNF−/− mice (P < 0·05). Therefore, the data demonstrate that the sole presence of membrane-bound TNF is able to control M. bovis BCG infection in 50% of mice.By contrast, bacilli burden was higher at 4 weeks in both TNFM. bovis BCG infection.The establishment of granulomas is the manifestation of a vigorous cell-mediated immune response, which is crucial for inhibiting mycobacterial growth. A critical role of TNF for initiating and maintaining the structural integrity of granulomas has been demonstrated.M. bovis BCG infection, showed evidence of resolving granulomatous lesions.Pulmonary pathology in all three mouse strains at 2 weeks post-infection was characterized by areas that had normal alveolar and focal thickened septa, but the alveolar spaces were free from infiltrations  and ther−/− mice showed further thickening of septae containing inflammatory cells at 4 weeks, but did not develop granulomatous lesions and only sparse perivascular lymphocytic infiltration. At 8 weeks, a prominent perivascular and peribronchial lymphocyte infiltration was found in TNF−/− mice. There was, however, no structural organization of the infiltrating lymphocytes and macrophages into a well-structured granuloma, and an increased recruitment of neutrophils was apparent.As expected, TNFtm/tm mice displayed typical, but small granulomas at 4 weeks post-infection, which were less well demarcated than those found in WT mice. Epitheloid macrophages and lymphocytes were distinguishable and perivascular lymphocyte recruitment was evident. These early granulomas developed into enlarged pulmonary lesions at 8 weeks post-infection.In contrast, TNFtm/tm mice are less abundant and predominantly confined to granulomas at 4 weeks and 8 weeks post-infection, whereas in TNF−/− mice with the absence of properly defined granulomas the bacilli were dispersed in the tissue and predominantly extracellular  at 4 weeks (tm/tm and TNF KO mice (P < 0·05) (In view of the granulomatous response elicited in TNF 4 weeks . The mac < 0·05) , while t < 0·05)  showed o < 0·05)  were ele+ and CD8+ T cells are critical for the generation of cell-mediated immunity. We therefore investigated whether membrane-bound TNF is sufficient to recruit specific T-cell subsets. Cells from BAL at 4 weeks post-infection were analysed by flow cytometry. In the presence of both soluble and secreted TNF (WT mice), effective recruitment of CD3+ CD4+ T cells and CD3+ CD8+ T cells occurred (+ CD4+ T cells: 21% versus 3% and CD3+ CD8+ T cells: 6% versus 0·8%). By contrast, the sole expression of membrane-bound TNF was sufficient to allow a significant recruitment of CD4 and CD8 T cells in BALF of TNFtm/tm mice, which was only 2–3 × lower than in WT mice (CD3+ CD4+ T cells: 21% versus 8% and CD3+ CD8+ T cells: 6% versus 2%).CD4occurred . In the In addition, we investigated the activation status of the recruited T cells. We found that >95% of the cells recruited were activated, as defined by the high expression of CD11a and CD44, and there was no difference among the three groups .tm/tm, but not TNF−/− mice were able to recruit abundant lymphocytes and macrophages into the lung tissue allowing the formation of granulomas, which were, however, less well developed than in WT mice. We asked whether the partial recruitment of lymphocytes as shown in BALF was sufficient to support macrophage activation. We measured iNOS as an indicator of macrophage activation, semiquantitavely and observed expression in TNFtm/tm mice, but at a lower level compared with WT mice. However, iNOS expression in TNFtm/tm mice was substantially higher than in TNF −/− mice . RANTES levels peaked at 4 weeks post-infection in WT mice and were significantly (P ≤ 0·01) reduced at 8 weeks post-infection in WT mice (tm/tm and TNF−/− mice displayed elevated levels at 8 weeks post-infection (P≤ 0·01). We were unable to detect the secretion of IL-4 or IL-5 during any stage of the infection.Levels of IL-12 at 4 weeks post-infection were significantly higher in TNF−/− mice . IFN-γ s/tm mice . As expe WT mice . TNFtm/ttm/tm mice suggest a strong Th1 response, which was confirmed by an enhanced cutaneous delayed-type reaction in infection in both TNFtm/tm and TNF−/− mice (data not shown). The higher levels of RANTES in TNF−/− mice might be related to the high bacterial burden in the lung.The high level of IFN-γ and IL-12 at 4 weeks, and RANTES at 8 weeks post-infection in TNFM. bovis BCG is applicable during a virulent infection, mice were infected with a low dose (10–30 CFU/lung) of M. tuberculosis H37Rv by aerosol inhalation. Body weight ratios were determined for 7 weeks of infection and the groups were scored for mortality. Progressive infection was paralleled by continual lowering of body weight ratios and mortality in TNF−/− mice , the TNFtm/tm mice showed at least a partial control of mycobacterial growth, with CFU values significantly less (P < 0·05) than TNF−/− mice at 45 days  than that of TNF−/− mice.We further assessed the mycobacterial burden in the lung of  45 days . CFU couan in WT . Howevertm/tm mice with TNFR1−/− or TNFR2−/− mice. We further assessed the mycobacterial burden in the lung of M. tuberculosis infected mice. In this experiment, TNF−/− mice lost weight rapidly and died within 5 weeks, while TNFtm/tm mice were less sensitive, half surviving up to 45 days. TNFtm/tm × TNFR2 KO mice seemed essentially as sensitive as TNF−/− mice, while TNFtm/tm × TNFR1 KO mice behaved more like TNFtm/tm mice (tm/tm × TNFR2 KO at 6 weeks post-infection, a time point when all TNF−/− had succumbed, essentially doubled as compared to TNFtm/tm or TNFtm/tm × TNFR1 KO mice (Lastly, we asked whether the protective effect of membrane TNF is signalled through TNFR1 or TNFR2. Therefore, we generated double transgenic mice by crossing TNF/tm mice . Therefo KO mice .Therefore, the sole expression of membrane TNF provided partial protection against H37Rv infection and the data using the double transgenic mice indicate that membrane TNF signalling through TNF-R2 confers the protective effect of membrane-bound TNF.M. bovis BCG with 50% of mice surviving the experimental period, while all TNF-deficient mice succumb to infection. TNFtm/tm mice were able to recruit and activate macrophages and T cells and generate mycobactericidal granuloma in response to BCG infection. However, resistance to the virulent M. tuberculosis strain was less pronounced, suggesting substantial differences between this and the Δ1-9,K11E mouse mutant.M. tuberculosis infectionListeria infection.28Here we demonstrated that the Δ1-12 TNF mutant,−/−, but not in TNFR1−/− mice, demonstrating the critical role of TNF and TNFR1 signalling.37A critical role of TNF for the effective control and resolution of mycobacterial infection has been demonstrated previously.tm/tm mice could indicate local cell-to-cell TNF signalling by membrane expressed TNF on T cells or macrophages at the site of infection leading to a partial activation of the immune cells. Several biological functions of membrane TNF have been reported previously and a preferential TNFR2 signalling has been suggested in vitroin vivoThe partial protection that is generated in TNFtm/tm mice. Macrophage and lymphocyte recruitment was significantly reduced in TNF−/− mice, with a significant reduction of the CD3+ CD4+ and CD3+ CD8+ T-cell subsets in the absence of TNF, which might be related to reduced chemokine production in the absence of TNF.tm/tm mice, membrane TNF on T cells or other cells might provide a partial signal for chemokine induction and hence increased cell trafficking. The contribution of CD4+ T-cells in cell-mediated immunity against mycobacteria has been investigated by the use of neutralizing antibodies and gene-deficient mice.+ typical granuloma formation in T cells was largely absent.44Although the exact mechanism of how protection is acquired through membrane TNF is unclear, membrane TNF on activated T cells might be sufficient for partial activation of macrophages; this results in the upregulation of iNOS expression, which is crucial for bacterial killing.However, in view of the only partial activation and protection, which is likely caused by direct cell–cell contact, secreted TNF and hence distal signalling, appears to be required for a full protective host response. This finding concurs with the recent data demonstrating reduced severity of autoimmune encephalomyelitis, but little effect on inflammation in the spinal cord in a similar genetic mouse model.24tm/tm× TNFR1 or R2−/− transgenic mice rescued protection in TNFtm/tm × TNFR2−/− mice and comparable protection in TNFtm/tm × TNFR1−/− mice as in TNFtm/tm mice suggest that membrane signalling though TNFR2 likely provides host protection. Our data in this infection model using the Δ1-12 TNF mutant mouse shows a reduced protective immune response during mycobacterial infection when compared to previous findings in which a membrane TNF transgenic mutant or the Δ1-9,K11E mutant were used. Our findings indicate that membrane TNF, in the Δ1-12 TNF mutant may preferentially induce protection through TNFR2. Receptor signalling in membrane TNF transgenic mutant or the Δ1-9,K11E mutant may be mediated through both TNFR1 and TNFR2 different, accounting for the differences observed in the degree of protection afforded by membrane TNF in the different mutant strains.Lastly, the infection studies in double TNFThe data suggest that sparing membrane expressed TNF in therapeutic interventions neutralizing TNF such as in rheumatoid arthritis might reduce infectious complications. In summary, we show that membrane TNF participates in cell-mediated immunity against mycobacterial infection. The protective effect of membrane-bound TNF is likely mediated through TNFR2 signalling.


Isolated limb perfusion with tumour necrosis factor alpha  and melphalan is well tolerated and highly effective in irresectable sarcoma and melanoma. No data are available on isolated hepatic perfusion (IHP) with these drugs for irresectable hepatic malignancies. This study was undertaken to assess the feasibility of such an approach by analysing hepatic and systemic toxicity of IHP with TNF-alpha with and without melphalan in pigs. Ten healthy pigs underwent IHP. After vascular isolation of the liver, inflow catheters were placed in the hepatic artery and portal vein, and an outflow catheter was placed in the inferior vena cava (IVC). An extracorporeal veno-venous bypass was used to shunt blood from the lower body and intestines to the heart. The liver was perfused for 60 min with (1) 50 microg kg(-1) TNF-alpha (n = 5), (2) 50 microg kg(-1) TNF-alpha plus 1 mg kg(-1) melphalan (n = 3) or (3) no drugs (n = 2). The liver was washed with macrodex before restoring vascular continuity. All but one pigs tolerated the procedure well. Stable perfusion was achieved in all animals with median perfusate TNF-alpha levels of 5.1 +/- 0.78 x 10(6) pg ml(-1) (+/- s.e.m). Systemic leakage of TNF-alpha from the perfusate was consistently < 0.02%. Following IHP, a transient elevation of systemic TNF-alpha levels was observed in groups 1 and 2 with a median peak level of 23 +/- 3 x 10(3) pg ml(-1) at 10 min after washout, which normalized within 6 h. No significant systemic toxicity was observed. Mild transient hepatotoxicity was seen to a similar extent in all animals, including controls. IHP with TNF-alpha with(out) melphalan in pigs is technically feasible, results in minimal systemic drug exposure and causes minor transient disturbances of liver biochemistry and histology.


Sensitisation to TNF-induced apoptosis was prevented by the broad caspase inhibitor zVAD-fmk, whereas overexpression of Bcl-2 had no effect. Mithramycin also potentiated cell death induced by Fas agonistic antibodies. In contrast, mithramycin reduced the percentage of cells undergoing apoptosis due to factor withdrawal. TNF-induced activation of NF-kappaB (NF-κB)-dependent gene expression was not modulated by mithramycin treatment. Concomitantly with the increased sensitivity, the protein level of the short-spliced cFLIP variant was downregulated. These results indicate that mithramycin enhances TNF-induced cell death in an NF-κB-independent manner, and suggest that the Fas-associated death domain protein plays a crucial role in the TNF-sensitising effect of mithramycin.In this report we show that mithramycin considerably increases the direct cytotoxic effect of tumour necrosis factor (TNF) on tumour cells  Mithramycin A (trade name Plicamycin) is an anticancer drug used to treat several types of cancer including chronic myeloid leukaemia and acute myeloid leukaemia (κB). Nuclear factor-kappaB is believed to be activated via TRADD and TNF receptor-associated factor-2(TRAF2), and/or receptor interacting protein (RIP) . Differential responses to this cytokine are thought to be due to different intracellular signalling pathways . One majin vivo, have limited the use of TNF as an antitumour agent  and 10% hGM-CSF-conditioned medium (complete medium). hGM-CSF-conditioned medium was obtained from stably transduced human embryonic kidney 293 cells with a pLHCX plasmid  encoding the human GM-CSF protein. The HL60 promyelocytic leukaemia and U937 human histiocytic lymphoma cell lines were maintained in RPMI 1640 medium containing 10% FCS. The GM-CSF producing HEK-293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS. The caspase inhibitor zVAD-fmk, the transcription inhibitor actinomycin D and the antimitotic agent aphidicolin were supplied by Sigma, Dublin, Irl. Recombinant human TNF was purchased from R&D systems, Abington, UK. Anti-Fas antibodies  and anti-FLIP γ/δ antibodies were obtained from MBL, MA, USA and Calbiochem, Nottingham, UK respectively. Mithramycin A was purchased from Tocris, Bristol, UK.All human cell lines were obtained from the ATCC. TF-1, a human GM-CSF-dependent erythroleukaemic cell line  staining according to Darzynkiewicz  expressing a hrGFP gene controlled by a synthetic promoter that contains five binding sites for NF-κB. A measure of 5 μg of reporter plasmid was transfected with lipofectin  with complete protease inhibitor mix from Roche, East Sussex, UK. In total, 30 μg of protein were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (12% SDS–PAGE), transferred to a nitrocellulose membrane and immunoblotted with the indicated antibody. Immunoreactive proteins were detected by enhanced chemiluminescence .Proteins were extracted in RIPA buffer . We therefore investigated the expression levels of the short-spliced form of cFLIP in TF-1 cells. As shown in Figure 8Since TNF-induced apoptosis, but not TNF-mediated NF-This current report demonstrates that a combination of TNF and mithramycin enhanced apoptosis of TF-1 cells within 24 h, relative to single treatments of TNF or mithramycin alone. Apoptosis induced by TNF and mithramycin treatment was effectively blocked by treatment with z-VAD-fmk peptide, an inhibitor of caspases, indicating that caspases played a critical role in the execution phase of apoptosis induced by TNF in the presence of mithramycin. Studies on the involvement of the mitochondria in the regulation of apoptosis revealed that TF-1 cells overexpressing the antiapoptotic protein Bcl-2 were not protected against apoptosis induced by TNF and mithramycin. These results further indicate that in the presence of mithramycin, TNF induced apoptosis via a caspase-signalling cascade that executed apoptosis independently of the proapoptotic machinery of the mitochondria. These results support the hypothesis that mithramycin targets an inhibitor of apoptosis involved in TNF-induced apoptosis, at the level of death receptor signalling.Mithramycin also significantly increased apoptosis mediated by another member of the death receptor family, Fas (CD95/APO-1), and could not be suppressed by overexpression of Bcl-2, suggesting the existence of a common mithramycin-sensitive inhibitor of apoptosis. TNF- and Fas-induced signalling pathways leading to caspase activation and apoptosis converge at the level of FADD  FADD is a common mediator of Fas and TNF receptor-induced apoptosis, (ii) in the TNF-receptor signalling complex, FADD is immediately downstream TRADD, a molecule that allows TNF-mediated NF-The ability of mithramycin to enhance the direct cytotoxic effects of TNF (and TNF family members) has wide ranging therapeutic implications. Since the mithramycin/TNF combination is capable of inducing apoptosis in TF-1 cells independently of the mitochondrial pathway, combining TNF treatment with mithramycin could have potential for elimination of tumour cells that express large amounts of Bcl-2. In addition, apoptosis induced by TNF in the presence of mithramycin was enhanced in three cell lines with different p53 status. As loss of p53 function contributes to resistance of some tumour cells to TNF-induced cytotoxicity -induced cytotoxicity, thus representing a novel alternative in cancer therapy. Indeed, TRAIL or Apo-2L, a member of the TNF family, induces apoptosis preferentially in tumour cells also through binding to its cognate death receptors and recruitment of FADD (Recent data indicate that induction of tumour cell death by stimulation of death receptors such as Fas constitutes a more prominent mechanism in the defence against tumours than has been thought previously . As bloc of FADD . Unlike Since mithramycin has been used for many years in the clinic to treat several types of cancer, the sensitising effect of mithramycin is not only intriguing but merits further study to realize the full potential of mithramycin as an adjunctive therapy in death receptor-mediated apoptosis.


Secondary bone marrow (BM) myelodysplastic syndromes (MDS) are increasingly common, as a result of radio or chemotherapy administered to a majority of cancer patients. Patients with secondary MDS have increased BM cell apoptosis, which results in BM dysfunction (cytopenias), and an increased risk of developing fatal acute leukemias. In the present study we asked whether TNF-α, known to regulate cell apoptosis, could modulate the onset of secondary MDS.in vitro and in vivo. In contrast to irradiated wild type (WT) mice, TNF-α deficient (TNF-α KO) mice or WT mice treated with a TNF-α-neutralizing antibody were partially protected from the apoptotic effects of irradiation. Next we established a 3-cycle irradiation protocol, in which mice were sub-lethally irradiated once monthly over a 3 month period. In this model, irradiated WT mice presented loss of microsatellite markers on BM cells, low white blood cell (WBC) counts, reduced megakaryocyte (MK) and platelet levels (thrombocytopenia) and macrocytic anemia, phenoypes that suggest the irradiation protocol resulted in BM dysfunction with clinical features of MDS. In contrast, TNF-α KO mice were protected from the irradiation effects: BM cell apoptosis following irradiation was significantly reduced, concomitant with sustained BM MK numbers and absence of other cytopenias. Moreover, irradiated WT mice with long term (≥5 months) BM dysfunction had increased BM angiogenesis, MMPs and VEGF and NFkB p65, suggestive of disease progression.We show that TNF-α is induced by irradiation and regulates BM cells apoptosis Taken together, our data shows that TNF-α induction following irradiation modulates BM cell apoptosis and is a crucial event in BM dysfunction, secondary MDS onset and progression. Tumour Necrosis Factor-α (TNF-α) is a pro-inflammatory cytokine secreted by activated macrophages and T lymphocytes, but also by keratinocytes and fibroblasts [reviewed in 1]. Its role in inflammatory processes is based both on tissue destruction and subsequent recovery of tissue homeostasis de novo, or primary, MDS, has been relatively modest TNF-α has also been implicated in BM diseases such as Fanconi anemia Despite some evidence pointing for a putative role of TNF-α in regulating BM disease onset, studies exploiting its involvement in secondary (irradiation-induced) BM failure/MDS are lacking, and were the subject of the present study. Patients with secondary MDS (which develop following chemotherapy or radiotherapy for other cancers) have worse prognosis than primary MDS We reasoned an increase in BM TNF-α might correlate with BM cell apoptosis induced by irradiation. To test this hypothesis, we analysed the effects of sub-lethal irradiation in BM cell turnover over a 3 day (72 h) period (“short-term irradiation effect”). As shown in in vitro, and measured the levels of TNF-α released into the culture supernatants by ELISA. As shown in Next we tried to find a causal relationship between the increase in TNF-α levels and the incidence in BM cell apoptosis following irradiation. For this purpose, we irradiated whole BM mononuclear cells and BM stromal cells in vitro. Nevertheless, we cannot exclude other undisclosed factor(s) may also promote BM cell apoptosis in response to the irradiation stimulus.Next, we hypothesized that TNF-α might be responsible for the incidence in BM cell apoptosis, and thus neutralizing its activity might exert a protective effect. Therefore, we exposed subsets of BM cells to the supernatants described earlier, and tested the protective effects of adding a TNF-α neutralizing antibody. As shown in in vivo, we compared the “short term irradiation effects” in wild type (WT) and TNF-α deficient (KO) mice BM content. As shown in in vitro results, irradiated TNF-α KO mice were partially resistant to the apoptotic effects of irradiation .To test the importance of TNF-α in BM apoptosis following irradiation To overcome possible differences between the BM microenvironment of TNF-α KO and WT mice, we also tested the effects of irradiation in the BM content of WT mice treated with anti-TNF-α Ab. In Taken together, these experiments suggest irradiation induces TNF-α production in the BM, and that the released TNF-α is partially responsible for the increase in BM cell apoptosis following irradiation.Next, we developed a 3-cycle irradiation protocol (to test the “long term effects of irradiation”), and characterized its effects in inducing BM dysfunction. First, we showed the irradiation protocol induces loss of microsatellite markers by BM cells, suggesting it had carcinogenic/transforming capacity . Taken together, these data suggest that in 3xirradiated mice sustained BM TNF-α levels induce MMP activity, VEGF production/release and NFkB expression, thereby promoting disease progression.MDS patients are at a higher risk of developing secondary acute leukemias In the present report we exploited the possibility that irradiation, known to induce BM malignant transformation, namely secondary acute myeloid leukemias in vitro exposure to supernatants from irradiated BM stroma, treated or untreated with a neutralizing antibody to TNF-α, demonstrated the TNF-α released in response to irradiation is partially responsible for BM cell apoptosis induction. Other studies have highlighted the role of TNF-α signalling in hematopoietic progenitor turnover in vitroin vivo thatWT mice treated with anti-TNF-α Ab as well TNF-α deficient mice were more resistant to irradiation-induced BM cell turnover than non-treated WT mice. Irradiation-induced production of TNF-α have also been reported in other systems like lung First, we observed that the effects of whole body “short term irradiation”, resulting in BM cell turnover and subsequent recovery, correlated with the levels of BM TNF-α. Moreover, in vivo model involved the loss of microsatelite markers in chromosome 2) are strong indications of an MDS-like phenotype We developed a model of BM dysfunction induced by prolonged (long-term) irradiation exposure, to study the importance of BM TNF-α in this setting. Our aim was to establish a reproducible and clinically relevant model of BM dysfunction that resembled secondary  MDS. It has been known for several years, that dogs chronically exposed to low daily doses of whole-body γ-radiation are prone to develop hematologic changes consistent with a myeloproliferative disorder in vitro. Under these conditions, MMP-9 activity in culture supernatants was diminished in the presence of the TNF-α antibody (data not shown) suggesting TNF-α release from the irradiated stroma induces a rapid MMP release.WT mice with sustained MDS-like symptoms beyond 5 months after the last irradiation had increased BM angiogenesis, as determined by quantification of laminin staining in BM sections and also by quantification of collagen IV and laminin in whole BM extracts (since laminin and collagen IV are components of the vessels basement membranes), suggestive of disease progression. The BM of 3x irradiated WT mice also showed increased MMP activity, increased VEGF and NFkB p65, in contrast to the BM of 3x irradiated TNF-α KO mice, where these parameters were unchanged. TNF-α is an important modulator of MMP activity, namely gelatinaseB/MMP9 Therefore, besides inducing cell apoptosis, overproduction of TNF-α in the BM microenvironment following irradiation, may induce MMP-9 and possibly other undisclosed factors, contributing towards degradation of the endothelial basement membrane and release of pro-angiogenic factors such as VEGF, which in turn may promote angiogenesis. Moreover, our data also shows that TNF-α stimulates VEGF production and release from different BM cells, in particular MK. Concerning the involvement of NFkB, TNF-α has been shown to exert some of its effects via NF-kB Our recent unpublished data suggested that MK are major producers of ECM molecules in the BM microenvironment (manuscript in preparation) namely fibronectin, suggesting their survival, observed in TNF-α KO mice, and the resistance of TNF-α KO mice to the effects of irradiation, may involve the production (maintenance) of such ECM molecules in the BM microenvironment. Lack of integrin α4  has been shown to restrict stem-cell BM repopulating capacity and to limit BM stem cells self-renewal Two recent elegant studies demonstrated unequivocally that the interaction between hematopoietic stem cells (HSC) and the BM microenvironment is strictly regulated by Rb and retinoic acid receptor signalling Strategies to block TNF-α activity, seeking to neutralize its inflammatory role, have met some success in the treatment of epithelial cancers, and to a lesser extent also in subsets of patients with primary MDS, although with varying degrees of therapeutic benefit in vitro and in vivo), and as such, strategies designed to block the effects of TNF-α in the BM microenvironment may be an attractive option to treat patients with secondary MDS.Taken together, we suggest that BM TNF-α is a critical factor in the onset and subsequent progression of irradiation-induced BM dysfunction with clinical features of secondary MDS (shown TNF-α knock-out (KO) and wild-type (WT) mice were obtained from the Jackson Laboratory, maintained on a C57BL/6 background. Animals were kept under specific pathogen-free conditions and handled in compliance with the Instituto Gulbenkian de Ciência or the IBMC-INEB Animal House guidelines and the European Convention.To determine the effects of short term irradiation in BM turnover and cytokine induction, 10 week-old WT mice were sub-lethally irradiated (300 cGy) and BM collected (from flushing one femur cavity with PBS) at different time-points after irradiation . Cell suspension was incubated with red cell lysis buffer (RCLB) for 15 minutes, centrifuged at 1200 rpm for 5 minutes and the supernatant (mononucleated BM cells) collected for further analysis.The TNFα neutralizing antibody was delivered to 10 week-old WT males by intraperitoneal injections of 1 mg daily, beginning two days prior sub-lethal irradiation. Control animals received equivalent amounts of PBS. Following irradiation, BM cells were isolated at different time-points as described above and processed for flow cytometry.The percentage of apoptotic cells during BM recovery following irradiation was estimated by flow cytometry in TNF-α KO or WT  mice. Total BM mononuclear cells were double-stained with Annexin V-PE (BD Biosciences) and FITC-conjugated antibodies against lineage markers, such as CD11b (Biolegend) and Sca1 (BD Biosciences) for hematopoietic mature (mostly myeloid) and precursor cells, respectively. The cells where analyzed on a FACSCalibur flow cytometer, using the “Cell Quest” software.Ten week-old TNF-α KO and WT mice were irradiated sub-lethally monthly over a three month period (3x irradiation). Haematological parameters  from irradiated and control mice were determined 7 days after each irradiation and 3–6 months after the last irradiation. Peripheral blood was collected from the cheek pouch in EDTA-coated tubes  and analyzed with a Hemavet 950FS cell counter . Apoptotic cells in frozen and paraffin-embedded BM sections, obtained from mice sacrificed at the same time-points defined above, were detected by transferase-mediated dUTP nick-end labeling (TUNEL) assay. We used the in Situ Cell Death Detection Kit, POD and DAB Substrate Kits (Roche), following the manufacturer's instructions. The percentage of apoptotic cells was determined by counting stained (TUNEL positive) nuclei in a total of six high power fields (200× magnification) per condition. These quantifications were done in triplicate (3 independent counts/BM section).Finally, when exhibiting signs of disease  animals were sacrificed by CO2 asphyxiation, their BM and other internal organs removed and processed for further analysis as described below. As a control, healthy non-irradiated mice were sacrificed at the end of the experimental period, approximately 6 months after the last irradiation. Isolated BM cells or BM sections were obtained and analysed as described below.Total BM mononuclear cells from WT mice and murine stromal cell line S17 were cultured in complete RPMI medium, 10% fetal bovine serum (Gibco BRL), overnight (o.n.) at 37°C. For all experimental procedures, serum was removed from the cultures. To determine whether irradiation induced TNF-α from BM cells, and whether this induced TNF-α leads to BM cell apoptosis, these BM mononucleated cells were irradiated with 1200 cGy; some of them were previously incubated with anti-mouse TNF-α/TNFSF1A antibody (R&D Systems), at a concentration of 0.06 µg/mL. Twenty four hours after irradiation, supernatants were collected and added to new BM mononuclear cell cultures which were incubated or not with the anti-mouse TNF-α/TNFSF1A antibody and irradiated (1200 rad) or not (controls) on the following day. At the end of the experiment, the incidence of apoptosis in BM cells submitted to different conditions was determined by flow cytometry (see above).TNF-α and VEGF protein levels were determined by enzyme-linked immunosorbent assay (ELISA). Briefly, supernatants from irradiated and non-irradiated BM cells (isolated for lineage surface markers CD41 (MK), CD11 (myeloid), Flk1 (endothelium) and sca1 (hematopoietic precursors) and from cultured stromal cells were collected, and loaded onto an ELISA kit  following the manufacturer's protocol.BM from TNF-α and WT mice were fixed with 2% paraformaldayde in 0,12 M phosphate buffer pH 7.2 o.n. and frozen on dry ice embedded in 0,12 M phosphate buffer pH 7.2, 15% sacarose, 7.5% gelatine.Eight µm sections from frozen BM were stained for basement membranes with anti-laminin (Sigma), 1∶100, and anti-CD41-FITC for megakaryocytes (Chemicon) antibodies. Briefly, sections were incubated with primary antibodies diluted in PBS, 0.1% BSA, o.n., 4°C, incubated in secondary antibodies, anti-rabbit Alex Fluor 488 (Molecular Probes) diluted 1∶1000 in PBS, 0.1% BSA. At the end, slides were mounted on Vectashield mounting-medium with Dapi (Vector) and analyzed by fluorescence microscopy on an Axioplan Microscope (Zeiss).Total RNA was extracted from mononuclear BM cells and cDNA synthesized following conventional protocols. D2Mit447 probe was design for a microsatellite located in a highly instable region of chromosome 2, prone to be lost after irradiation. TNF-α and D2Mit447 microssatelite mRNA quantification was performed by real time polimerase chain reaction (RQ-PCR) using the SYBR Green Master Mix kit (Applied Biosystems). Mouse β-actin gene (endogenous housekeeping gene) was used as standard reference . The relative expression of TNF-α and D2Mit447 was calculated by using the comparative threshold cycle (CT) method.Total protein extracts from all BM were obtained by lysing the samples in cold RIPA buffer , in the presence of protease and phosphatase inhibitors. After 30 minutes on ice, lysates were centrifuged for 15 minutes at 4°C and 14000 rpm. Equal protein amounts from each sample were used for dot-blotting and western blotting assays.For dot-blotting, primary antibodies were anti-human fibronectin (Sigma), 1∶500, anti-laminin (Sigma), 1∶250, anti-collagen IV, (Chemicon), 1∶100 and anti-β-actin (Sigma), 1∶250.For Western blotting the following antibodies and respective dilutions were used: anti-VEGF (Santa Cruz), 1∶400, anti-NFkB p65 (Santa Cruz), 1∶1000, and anti-β-actin (Sigma), 1∶250.For both assays, nitrocellulose membranes were blocked in PBS, 1% BSA, 0.1% Tween-20 or PBS, 5% milk, depending on the antibody manufacturer's protocol, for 1 hour at room temperature. Secondary antibodies were anti–rabbit IgG-HRP (Santa Cruz), anti–mouse IgG-HRP (Santa Cruz) and anti–goat IgG-HRP (Santa Cruz), all at 1∶6000. The electrochemiluminescence (ECL) detection system (Amersham Biosciences) was used to visualize the presence of specific proteins on the nitrocellulose blots. The blots obtained by dot-blotting were quantified with Image J program and represent relative expression levels of each protein.Presence or absence of latent or active species of MMP-2 and −9 were monitored by gelatin zymography. Briefly, equal volumes (40 µl) of supernatant obtained from cell cultures, both from bone marrow mononucleated and stromal cells were mixed with 5x sample buffer  and loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel containing 0.12% gelatin . The gel was run under nonreducing conditions at constant voltage of 180 V for 5 hours. To remove SDS and allow the MMPs to renuture, the gel was incubated in 2.5% Triton X-100 at room temperature for 1 h in an orbital shaker, and then incubated in low salt collagenase buffer  at 37°C, overnight. To reveal the lysis zones, the gel was stained in stain/destain solution for 30 minutes, and washed in distilled water.Results are expressed as mean ± standard deviation. Data were analyzed using the unpaired two-tailed student's t test or the one-way ANOVA Turkey test. P values of <0.05 were considered significant.Figure S1Irradiation induces the loss of microsatelite markers. Total BM cells were obtained from control and 3xirradiated mice. The results show the loss of microssatelite markers (D2Mit230) in irradiated mice BM cells, suggesting the irradiation protocol induces chromosomal abnormalities in irradiated mice bone marrow cells. *: p<0.01(0.20 MB TIF)Click here for additional data file.


Persistent infection by high risk HPV types  is the main risk factor for development of cervical intraepithelial neoplasia and cervical cancer. Tumor necrosis factor (TNF) is a key mediator of epithelial cell inflammatory response and exerts a potent cytostatic effect on normal or HPV16, but not on HPV18 immortalized keratinocytes. Moreover, several cervical carcinoma-derived cell lines are resistant to TNF anti-proliferative effect suggesting that the acquisition of TNF-resistance may constitute an important step in HPV-mediated carcinogenesis. In the present study, we compared the gene expression profiles of normal and HPV16 or 18 immortalized human keratinocytes before and after treatment with TNF for 3 or 60 hours.In this study, we determined the transcriptional changes 3 and 60 hours after TNF treatment of normal, HPV16 and HPV18 immortalized keratinocytes by microarray analysis. The expression pattern of two genes observed by microarray was confirmed by Northern Blot. NF-κB activation was also determined by electrophoretic mobility shift assay (EMSA) using specific oligonucleotides and nuclear protein extracts.KLK7 , SOD2 (superoxide dismutase 2), 100P , PI3 , CSTA (cystatin A), RARRES1 (retinoic acid receptor responder 1), and LXN (latexin). The differential expression of the KLK7 and SOD2 transcripts was confirmed by Northern blot. Moreover, we observed that SOD2 expression correlates with the differential NF-κB activation exhibited by TNF-sensitive and TNF-resistant cells.We observed the differential expression of a common set of genes in two TNF-sensitive cell lines that differs from those modulated in TNF-resistant ones. This information was used to define genes whose differential expression could be associated with the differential response to TNF, such as: This is the first in depth analysis of the differential effect of TNF on normal and HPV16 or HPV18 immortalized keratinocytes. Our findings may be useful for the identification of genes involved in TNF resistance acquisition and candidate genes which deregulated expression may be associated with cervical disease establishment and/or progression. Human papillomaviruses (HPVs) are double-stranded DNA tumor viruses that infect keratinocytes of the anogenital tract epithelium . PersistPersistence of HPV infections and development of neoplasia is influenced by local cell-mediated immune response . Tumor nThe aim of the present study was to characterize and compare the global transcription profile of normal and HPV-immortalized keratinocytes. Furthermore, we sought to analyze their response to TNF in order to identify differences that contribute to explain their divergent response to this cytokine. For this purpose, we used microarray analysis to determine transcriptional changes upon 3 and 60 hours after TNF exposure. The 3 hours treatment would favor the identification of immediate early TNF regulated genes. On the other hand, the 60 hours treatment was used because the cytostatic effect exerted by this cytokine on normal and HPV16-immortalized keratinocytes reaches its maximum at this time-point ,23. Our Cultures of primary human keratinocytes (PHK), recovered from newborn foreskin , were maintained in keratinocyte serum-free medium – KSFM  for 3 to seven passages . HF698 aFor each sample, total RNA was extracted using TRIzol Reagent  following the procedure recommended by the manufacturer. Three micrograms of target and reference  total RNA were linearly amplified using T7-based protocol, converted to cyanine-modified cDNA, and labeled as described previously .Hybridizations were performed in duplicate, using dye-swap, on a cDNA platform of ORESTES representing 4,600 unique genes with known full-length sequence selected from the clone collection derived from the Human Cancer Genome Project . cDNA amt-test and determined the nominal p-value for each individual gene. Those nominal p-values can be conservatively adjusted for multiple testing with the Bonferroni correction by multiplying them by the number of genes in our chip. For clustering samples on the basis of their expression profile, we applied hierarchical clustering based on correlation distance and complete linkage.Data analysis was performed with R project for statistical computing  and tool32P]dCTP (3000 Ci/mmol). Nylon filters were exposed to Kodak Hyperfilm  with intensifying screen.For Northern blot analysis, 15 μg of total RNA was fractionated through a 1% denaturing agarose gel and transferred by capillarity onto Hybond N filters . Prehybridization, hybridization, and washes were performed as described by Church and Gilbert . The KLKNuclear extracts were obtained from monolayer cultures of PHK, and from cell lines HPV16 and HPV18 treated with 2 nM of human recombinant TNF for 1 h. Briefly, cell plates were washed with ice-cold PBS and cells were scraped in 5 ml of PBS. Cells were transferred to a 15 ml Falcon tube and centrifuged at 3000 rpm for 3 min. Cell pellets were ressuspended in 4 ml of lysis buffer , incubated on ice for 5 min, centrifuged and ressuspended in 4 ml of lysis buffer. Nuclei obtained were centrifuged at 2000 rpm for 2 min, ressuspended in 100 μl of extraction buffer  and incubated on ice for 30 min. Finally, the samples were centrifuged at 12000 rpm for 15 min at 4°C. The supernatants were stored at -80°C. The protein concentration was determined by the Bradford method .32]ATP  using TK polynucleotide kinase according to the manufacturer instructions  and purified using Sephadex G50 columns followed by phenol:chloroform extraction and precipitation using 10 μg of salmon sperm DNA as a carrier . DNA pellets were ressuspended in binding buffer  to a final concentration of 2,5 fmol/μl. The incorporated radioactivity was quantitated using a LS6500 scintillation counter .For gel retardation the following double-stranded oligonucleotide, corresponding to the NF-κB binding sequence, was used: forward-5'-GCCTGGGAAAGTCCCCTCAACT-3'  was used. The annealed oligonucleotide was labeled with ATP 5'-end-labeled double-stranded oligonucleotide probe was added, and the incubation was continued for an additional 15 min at 30°C. The DNA-protein complexes were resolved on 4% nondenaturing polyacrylamide gels (29:1 cross-linking ratio), dried, and exposed overnight to X-ray films .Glass arrays containing 4.800 cDNA sequences were used in order to determine the effects of HPV infection in human keratinocytes as well as the impact of TNF treatment on global gene expression, in HPV negative or positive cells.In order to identify differentially expressed genes as a function of a unique variable (cell type or TNF-treatment) our dataset was first analyzed by one way ANOVA. The comparisons performed allowed us to determine 1) genes that are differentially expressed between TNF-sensitive and TNF-resistant cells; 2) identify genes that are differentially modulated by TNF at two-time points (3 and 60 hours); 3) analyze the effect of HPV-induced immortalization on TNF-regulated genes and, 4) find genes that are differentially expressed between cells immortalized by two different high-risk HPV types  were grouped hierarchically, using correlation distance and complete linkage , epidermal development, differentiation and proliferation , proteolysis regulation , and cell adhesion . The name and function of the genes described are listed in Table -2,9) are listed in Table Among the differentially regulated genes we found some related with inflammatory response  are listed in additional file Since our experimental setting included the comparative analysis of global gene expression at two time points (3 or 60 hours), we searched for genes that best differentiate our samples as a function of time. We found 48 genes that clearly differentiate samples from the analyzed time points. The name and annotated function of the identified genes , epidermis development, differentiation and proliferation , protein ubiquitination (APPBP1) and cell adhesion , among others, were differentially expressed between TNF-sensitive and TNF-resistant cells Table  and 5.superoxide dismutase 2), S100P , PI3 , CSTA (cystatin A), RARRES1 (retinoic acid receptor responder 1), and LXN (latexin). Based on the reported function as well as the expression profile observed, KLK7 and SOD2 genes were selected for further analysis. The expression pattern of these genes observed by microarray was confirmed by Northern Blot in control and TNF-treated (60 hours) samples from all cell lines used , SOD2  using specific oligonucleotides and nuclear protein extracts. Interestingly, we observed that normal as well as HPV16-immortalized keratinocytes exhibited a clear activation of NF-κB as shown by the increase of this factor levels in nuclear protein extracts after TNF treatment  expression pattern was also validated by Northern blot Figure . This gePrevious reports have shown that activation of the transcription factor NF-κB is essential for the induction of SOD2 by TNF and IL-1β ,61. Herekallikrein 7), SOD2 (superoxide dismutase 2), S100P , PI3 , CSTA (cystatin A), RARRES1 (retinoic acid receptor responder 1), and LXN (latexin). The differential expression of the KLK7 and SOD2 transcripts was further confirmed at the RNA level. Moreover, we present evidence that differential SOD2 expression correlates with the levels of NF-κB activation exhibited by TNF-sensitive and TNF-resistant cells.Progression of HPV-associated lesions depends on the many alterations caused by this virus in the infected cells. We have identified multiple genes differentially regulated by TNF in HPV16 and HPV18 immortalized keratinocytes. Among them we found KLK7 ; HPV16-immortalized keratinocytes: controls and treated for 3 or 60 hours with TNF, respectively ; HPV18-immortalized keratinocytes: controls and treated for 3 or 60 hours with TNF, respectively .Hierarchical grouping based on differentially expressed genes as a function of time. These genes where identified by the ANOVA method and the samples where grouped considering the correlation distance and complete linkage. After sample grouping the genes were hierarchically grouped by their correlation distances. High gene expression is shown in red, low gene expression is shown in green and black indicates non-differential gene expression, p values <10Click here for fileTable of differentially expressed genes obtained by pair-wise comparison of all the samples and their respective expression ratio ordered by p-value. PHK_3H (primary human keratinocytes – control/3 hours), PHK_60H (primary human keratinocytes – control/60 hours), HPV16_3H , HPV16_60H , HPV18_3H , HPV18_60H , PHK_TNF3H (primary human keratinocytes – TNF/3 hours), PHK_TNF60H (primary human keratinocytes – TNF/60 hours), HPV16_TNF3H , HPV16_TNF60H , HPV18_TNF3H , HPV18_TNF60H .Click here for fileSupervised hierarchical grouping based on the 30 genes that best differentiate normal and HPV 16-immortalized keratinocytes from the HPV 18-immortalized ones after treatment with TNF for 60 hours. High gene expression is shown in red, low gene expression is shown in red and black indicates non-differential gene expression. PHK_3H (primary human keratinocytes – control/3 hours), PHK_3H TNF (primary human keratinocytes – TNF/3 hours), HPV16_3H , HPV16_3H TNF , HPV18_3H , HPV18_3H TNF .Click here for fileTable of differentially expressed genes that best distinguish TNF-resistant (HPV18) from TNF-sensitive cells (PHK and HPV16), in normal culture conditions or upon treatment with TNF for 60 hours.Click here for fileAnalysis of TNF-induced NF-κB activation in normal and HPV16 or 18-transformed keratinocytes. Nuclear protein extracts were obtained from sub confluent cultures of normal keratinocytes (PHK), HPV16-positive (SiHa) and HPV18-positive (SW756 and HeLa) cervical cancer-derived cell lines treated with 2 nM of TNF for 1 h. For each EMSA reaction, 5 mg of nuclear protein were incubated with 50 fmol of [γ-32P]ATP-labeled double-stranded oligonucleotide and a 50X excess of consensus unlabeled oligonucleotide . Specificity of binding was further demonstrated by incubation of nuclear extracts with the described amount of labeled consensus oligonucleotide and a 50X excess of a labeled oligonucleotide carrying a single-base mutation at the NF-κB binding site  and incubation of nuclear extract in the absence of any labeled probe (lane 15). NF-κB-DNA binding reactions were carried out as described under "Material and Methods". HeLa nuclear extracts obtained from two different experiments were included as controls of NF-κB activation levels in HPV-positive cell lines. DNA binding complexes are indicated.Click here for file


Tumour necrosis factor (TNF) has been found to be an important immunomodulator. Among other functions TNF activates natural killer (NK) cells and stimulates monocytes/macrophages in an autocrine fashion. TNF production and NK activity in peripheral blood mononuclear cells were determined in a clinical phase I study in which recombinant human (rh) TNF was administered as a continuous infusion weekly for a period of 8 weeks. Even though TNF production and NK activity were significantly reduced directly after rhTNF infusion the effect proved to be transient and most pronounced at the first rhTNF administration. One day after completion of the rhTNF infusion the peripheral cells released more TNF into the supernatant compared to TNF activity immediately before the rhTNF infusion. This effect was conspicuous in non-stimulated cultures. After repeated rhTNF infusions both stimulated and non-stimulated TNF production of the peripheral blood mononuclear cells was increased. NK cell activity was also enhanced after repeated cycles of rhTNF administration as compared to early rhTNF treatment. Thus, repeated rhTNF infusions lead to a stimulatory effect on TNF production and NK activity of peripheral blood cells.


Real-time reverse transcription–polymerase chain reaction showed that TNF-α mRNA was expressed in large quantities in the implanted tumour. Gefitinib inhibited EGF- but not hepatocyte growth factor (HGF)-induced activation of mitogen-activated protein kinase (MAPK) cascades, suggesting selectivity of the inhibitor. However, gefitinib inhibited the TNF-α-induced activation of MAPKs and Akt. In addition, TNF-α-induced metastatic properties including adhesion to fibronectin, mRNA expression of integrin αv, production of matrix metalloproteinase-9 and invasion were inhibited by gefitinib without affecting cell proliferation. Furthermore, the TNF-α-induced responses except for NF-κB activation were blocked by metalloprotease inhibitors, suggesting that gefitinib inhibited the transactivation of EGFR induced by TNF-α. These results suggest that the TNF-α signalling pathway is a possible target of gefitinib in suppressing the intrahepatic metastasis of hepatocellular carcinoma.We have reported that the selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, gefitinib , suppressed intrahepatic metastasis of hepatocellular carcinoma CBO140C12 cells. In this study, we focused on the tumour necrosis factor- Upon lig cancer . There iα (TNF-α) was first identified as the macrophage-derived product responsible for tumouricidal activity  and matrix metalloproteinase-9 (MMP-9)  signalling pathway  cascades and Akt as well as TNF-α-induced metastatic properties in vitro possibly by inhibiting EGFR transactivation.Recently, we have shown that gefitinib is effective in inhibiting intrahepatic metastasis of murine hepatocellular carcinoma CBO140C12 cells by blocking EGFR-dependent metastatic properties (in vitro study. Recombinant murine EGF were purchased from Upstate Biotechnology and murine hepatocyte growth factor (HGF) and human TNF-α were purchased from Genzyme/Techne. Metalloprotease inhibitors, GM6001, GM6001 negative and TAPI-1, were purchased from Calbiochem, Darmstadt, Germany.Gefitinib was kindly provided by AstraZeneca . It was dissolved in DMSO for the Female 5-week-old specific pathogen-free B6C3F1 mice were purchased from Japan SLC . The mice were maintained under specific pathogen-free conditions and used according to institutional guidelines. Orthotopic implantation of CBO140C12 tumour fragments into mouse liver was performed as described previously (μg) using oligo(dT) primer and SuperScript II reverse transcriptase . The cDNA was conducted in a 25 μl final volume mixture containing primer, probe and TaqMan Universal PCR Master Mix. Probes were labelled with fluorescent reporter dye at the 5′-end : VIC; TNF-α: FAM; MMP-9: FAM) and a quencher dye at the 3′-end . Plates were analysed on a TaqMan ABI Prism 7700 Sequence Detector . Cycling parameters were: 50°C for 2 min, 95°C for 10 min, 40 cycles for GAPDH and TNF-α, 50 cycles for MMP-9 at 95°C for 15 s and 60°C for 1 min. Cycle threshold detection was converted into number of cDNA contents in the starting material and a standard curve was constructed using the known amounts of cDNA. Test gene mRNA values were extrapolated from the standard curve and expressed in arbitrary units.Total RNA from CBO140C12 cells was extracted using the RNeasy Mini Kit . First strand cDNA was prepared from the total RNA . The sequences of the primers were as follows: integrin αv, 5′-CAAGCTCACTCCCATCAC-3′ and 5′-GGGTGTCTTGATTCTCAAAGGG-3′; GAPDH, 5′-GGTGAAGGTCGGTGTGAACGGATTT-3′ and 5′-GATGCCAAA GTTGTCATGGATGACC-3′. Polymerase chain reaction was preformed in a thermocycler for specified cycles of denaturation , annealing  and extension . The PCR products were electrophoresed on 1.2% agarose gels and detected by ethidium bromide staining.Amplification of integrin −1L-glutamine. A549 cells were maintained in DMEM supplemented with 10% FCS.The CBO140C12 murine hepatocellular carcinoma cell line was kindly provided by Dr K Ogawa  and maintained in DMEM : F-12 supplemented with 10% FCS, 320 mg l4 cells well−1) were seeded in 100 μl of medium containing 0.5%. FCS in 96-well plates and allowed to adhere for 24 h. After preincubation with 90 μl of medium containing gefitinib  for 15 min, cells were stimulated with 10 μl of medium containing TNF-α  for 12 or 72 h. Cell proliferation was determined by using a cell counting kit (Dojindo).Cells (1 × 10M Tris-HCl (pH 6.8), 5% w v−1 glycerol, 1% w v−1 SDS, 0.05% w v−1 bromophenol blue) and were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to Immobilon-P membranes (Millipore). Blots were probed using primary antibodies described above and horseradish peroxidase-conjugated secondary antibodies  followed by enhanced chemiluminescence . Antibodies against EGFR and phospho-EGFR, phospho-ERK, phospho-c-Jun-N-terminal kinase (JNK), phospho-Akt, phospho-p38 and phospho-p65 were purchased from Cell Signaling Technology, Beverly, USA and anti-p38, JNK, p65 and Akt antibodies were obtained from Santa Cruz Biotechnology, California, USA.Cells were cultured in a medium containing 0.5% FBS for 24 h. After indicated treatment, cell lysates were prepared with sample buffer  were precoated with 1 μg of fibronectin on the lower surface, and then 5 μg of Matrigel was applied to the upper surface of the filter. Cells in 0.1% BSA medium were pretreated with gefitinib for 15 min, and then stimulated with TNF-α for 12 h. In all, 3 × 104 cells were added to the upper compartment of the chamber and incubated for 6 h at 37°C. The cells were stained with haematoxylin and eosin and were counted using the mean of five windows (× 400 magnification) per filter.The invasion assay was performed using Transwell culture chambers (Corning Costar). Polyvinylpyrrolidone-free polycarbonate filters with an 8.0 −1) and incubated at 37°C for 24 h. Enzyme-digested regions were quantified by the Chemi Doc XRS system (Bio-Rad).Gelatin zymography was performed as previously described  as well as the downstream signalling pathways including Akt, ERK, JNK and p38 MAPK in CBO140C12 cells . In contα signalling, therefore, we next examined the effects of gefitinib on the TNF-α-induced responses. Interestingly, TNF-α-induced activation of MAPKs was significantly inhibited by gefitinib in CBO140C12 cells (α and gefitinib (data not shown). It should be noted that the inhibition was more potent compared with the inhibitory activity against EGF-induced MAPK activation. Gefitinib completely inhibited TNF-α-induced activation of ERK and JNK at 0.1 μM  and the gelatinase activity (α-induced invasion of CBO140C12 cells (Adhesion to the extracellular matrix and invasion across the matrix and basement membrane are the critical steps in tumour metastasis. CBO140C12 cells were stimulated with TNF-efitinib . This isefitinib . In addiactivity  of MMP-912 cells .α in CBO140C12 cells are mediated by EGFR transactivaton, effects of metalloprotease inhibitors were examined. GM6001, a broad metalloprotease inhibitor, blocked TNF-α-induced phosphorylation of ERK1/2 (κB p65 at Ser-536 was not inhibited (α-induced phosphorylation of ERK1/2 as well as phosphorylation of NF-κB (α-induced phosphorylation of ERK1/2 was inhibited by TAPI-1 (α-induced signalling pathways. Moreover, TNF-α-induced metastatic properties including the cell adhesion to fibronectin (αv (α-induced cellular responces.To elucidate the possibility that responses to TNF-f ERK1/2 . Activatnhibited . In contof NF-κB . It has y TAPI-1 . Both GMronectin  and mRNActin (αv  were inhα induces EGFR transactivation via metalloproteinase-dependent release of EGFR ligands. Therefore, we have tired to examine the effects of gefitinib on the TNF-α-induced cellular responces.Epidermal growth factor receptor is a promising target for cancer therapy and a number of anti-EGFR agents have been developed . Preclinα promote malignant cell growth and metastatic potential (α induced the expression of several different adhesive molecules including several integrin subunits, ICAM-1 and VCAM-1 (α also induced the activation of MAPKs, which has been shown to be involved in MMP-9 expression and invasion. In this study, we found that gefitinib inhibited not only EGF-induced but also TNF-α-induced activation of MAPKs and metastatic properties including adhesion and invasion. In addition, we have previously demonstrated that gefitinib inhibited intrahepatic metastasis of CBO140C12 cells (α mRNA in primary tumour of orthotopically implanted CBO140C12 cells in the liver support the idea that TNF-α-meidated cellular activities are potential targets for the antimetastatic activity of gefitinib. Moreover, TNF-α is one of the angiogenic factors associated with tumour-induced neovascularisation (α signalling pathways in both tumour and endothelial cells. In our previous observation, gefitinib inhibited the incidence of metastasis as well as the growth of primary tumour (α-induced reactions may be one way gefitinib exerts its antitumour activity.There has been accumulating evidence that inflammatory mediators such as TNF-α and EGF signalling pathways have been found to play a physiological function in TNF-α signalling (α, one of the EGFR ligands, is one possible mechanism of EGFR transactivation by TNF-α (et al proved that MMPs, especially ADAM17, is responsible for the transactivation of EGFR by TNF-α. Here we confirmed that an ADAMs inhibitor TAPI-1 inhibited the activation of TNF-α-induced MAPK activation. In addition, TNF-α-induced metastatic properties in vitro such as adhesion were also inhibited by a broad MMP inhibitor GM6001. These results demonstrated that TNF-α-induced metastatic properties were mediated via MMP activities. The fact that gefitinib inhibited the TNF-α-induced cellular responses suggested the TNF-α-induced EGFR transactivation by shedding EGFR ligands. Identification of the ligands and MMPs will provide more information for the mechanism of anti-TNF-α activity of gefitinib.Recently, TNF-α triggers several intracellular signalling pathways, in which MAPK cascades and NF-κB are the main pathways. In contrast to MAPK cascades, phosphorylation of NF-κB p65 was not inhibited by both gefitinib and MMP inhibitors. This is correlated with the finding that AG1478, an EGFR inhibitor, did not inhibit TNF-α-induced phosphorylation of IκBα, another event essential for activation of NF-κB, in hepatocytes (α-induced expression of chemokine RANTES, one of the NF-κB target genes, was not blocked by the EGFR-neutralizing monoclonal antibody 225 in mammary epithelial cells (α-induced NF-κB activation is independent of EGFR (Tumour necrosis factor-α-induced EGFR signalling are the possible targets. Recent genome-wide approaches identified genes that may be associated with sensitivity to gefitinib (In summary, we have demonstrated that gefitinib shows antimetastatic activity using an intrahepatic metastasis model, in which TNF-