Tumor cell lysate-pulsed human dendritic cells induce a T-cell response against pancreatic carcinoma cells: an in vitro model for the assessment of tumor vaccines.

Dendritic cells (DCs) are potent antigen-presenting cells and play a pivotal role in T cell-mediated immunity. DCs have been shown to induce strong antitumor immune responses in vitro and in vivo, and their efficacy is being investigated in clinical trials. Compared with vaccination strategies directed against a single tumor antigen, tumor-cell lysate as the source of antigen offers the potential advantage of inducing a broad T-cell response against multiple known, as well as unknown, tumor-associated antigens expressed by the individual tumor. We used pancreatic carcinoma cell lines to develop an in vitro model for monitoring T-cell responses induced by lysate-pulsed DCs. Monocyte-derived DCs of HLA-A2(+) donors were pulsed with lysate generated from the HLA-A2(+) pancreatic carcinoma cell line Panc-1. In some experiments, the immunogenic protein keyhole limpet hemocyanin (KLH) was added to the lysate. Subsequently, the antigen-loaded DCs were activated with tumor necrosis factor-alpha and prostaglandin E(2). Autologous mononuclear cells were cocultured with DCs in the presence of low-dose interleukin (IL)-2 and IL-7 and were restimulated weekly with new DCs. High levels of IL-12 and IFN-gamma could be detected in the supernatants, indicating a T-helper type 1-type immune response. This cytokine profile was associated with the expression of the activation marker CD69 on both T helper and CTLs and with an antigen-induced proliferative T-cell response. After 4 weeks, CTL-mediated cytotoxicity was assessed. Tumor cell lysis was specific for Panc-1 tumor cells and was MHC class I-restricted. Cytokine secretion, CD69 expression of T cells, and antigen-induced T-cell proliferation correlated with the cytotoxic activity and were more pronounced when KLH was added to the lysate. This is the first study to show that T cells specific for pancreatic carcinoma cells can be generated in vitro by lysate-pulsed DCs and that the T-cell response can be enhanced by KLH. This in vitro model can be applied to compare different strategies in the development of DC-based tumor vaccines.

Phosphodiesterase type 4 inhibitor suppresses expression of anti-apoptotic members of the Bcl-2 family in B-CLL cells and induces caspase-dependent apoptosis.

B cell chronic lymphocytic leukemia (B-CLL) is an incurable clonal disease which shows initial responsiveness to a number of chemotherapeutic drugs. However, in most patients the disease becomes resistant to treatment. Rolipram, a specific inhibitor of phosphodiesterase (PDE) type 4, the PDE predominantly expressed in B-CLL cells, has been shown to induce cAMP-dependent apoptosis in these cells. In the present study, we demonstrate that the extent of rolipram-induced apoptosis is similar to fludarabine-induced apoptosis in vitro. The combination of rolipram and fludarabine results in an enhancement in the number of apoptotic cells compared to apoptosis induced by either agent alone. Second, rolipram suppresses the expression of anti-apoptotic members of the Bcl-2 family and induces the pro-apoptotic protein Bax, thereby shifting the balance between pro- and anti-apoptotic members of the Bcl-2 family towards a pro-apoptotic direction. Finally rolipram-induced apoptosis is caspase-dependent. PDE 4 inhibitors are currently under investigation for chronic obstructive pulmonary disease and asthma in phase III clinical trials showing promising results with tolerable side-effects. In conclusion, by inducing apoptosis, by enhancing apoptosis induced by fludarabine, by suppressing Bcl-2, Bcl-X and by inducing Bax expression, PDE 4 inhibitors may add a new therapeutic option for patients with B-CLL.

Shock waves: a novel method for cytoplasmic delivery of antisense oligonucleotides.

Intracytoplasmic delivery of oligonucleotides (ODN) can improve ODN-based strategies such as the antisense approach and the use of immunostimulatory CpG dinucleotide containing ODN. Shock waves are established for the treatment of nephrolithiasis and other diseases. Here we describe the use of shock waves as a new physical method for the direct transport of antisense ODN into the cytoplasm and the nucleus of cells. Human peripheral blood mononuclear cells together with antisense ODN were exposed to shock waves generated by an electrohydraulic lithotripter. ODN uptake was examined by flow cytometry and fluorescence microscopy. By optimization of physical parameters we achieved the transfer of high amounts of ODN which were detected within less than 5 min after shock wave exposure, with viability of cells higher than 95%. Transfection of human peripheral blood mononuclear cells with an antisense ODN directed against tumor necrosis factor (TNF) alpha resulted in a reduction in lipopolysaccharide-induced TNF production by 62% (n=5, P=0.006). Specificity of TNF suppression was confirmed with a four-mismatch oligonucleotide. Positive atmospheric pressure abolished antisense-mediated inhibition of TNF synthesis by blocking shock wave-induced cavitation and formation of oscillating air bubbles. Electroporation was less effective. The use of shock waves is thus an efficient physical tool for ODN delivery to cells. Shock waves may allow the evaluation of target proteins in cell types difficult to transfect with other methods and thus may improve the antisense technique for the analysis of unknown genes.

Suppression of TNF-alpha production in human mononuclear cells by an adenosine kinase inhibitor.

Adenosine exerts potent anti-inflammatory activities through inhibition of cytokine synthesis by activated monocytes. Adenosine is rapidly phosphorylated intracellularly by adenosine kinase. GP515, an adenosine kinase inhibitor, prevents the phosphorylation of adenosine to AMP and thereby locally enhances the adenosine concentration. GP515 has exhibited significant anti-inflammatory effects in several murine models of inflammation. In this study we investigated the effect of GP515 alone and in combination with exogenous adenosine or with rolipram, a phosphodiesterase inhibitor, on tumor necrosis factor alpha (TNF-alpha) synthesis in human peripheral blood mononuclear cells (PBMC) or whole blood. Lipopolysaccharide (LPS; 10 ng/mL)-stimulated PBMC were incubated in the absence or presence of these substances. GP515 alone showed a dose-dependent suppression of TNF-alpha production with an IC50 of 80 microM. The TNF-alpha-inhibiting effects of adenosine and GP515 were reversed in the presence of the cAMP antagonist (Rp)-cAMPS, supporting the hypothesis of a cAMP-mediated pathway. Combinations of GP515 with either adenosine or rolipram led to an additive inhibition of TNF-alpha synthesis. These experiments are the first to demonstrate efficacy of an adenosine kinase inhibitor in TNF-alpha suppression in cells of human origin. The findings form a basis to investigate these strategies in animal models of TNF-alpha-mediated chronic inflammatory diseases.

Anti-inflammatory activities of cAMP-elevating agents: enhancement of IL-10 synthesis and concurrent suppression of TNF production.

Interleukin-10 (IL-10) and tumor necrosis factor (TNF) exert key roles in some acute and chronic inflammatory diseases. In this study we investigated (1) the potency of different cAMP-elevating agents in enhancing IL-10 synthesis, (2) the involvement of protein kinase A in this enhancement, and (3) the mutual dependence of cAMP-enhanced IL-10 formation and cAMP-suppressed TNF synthesis. Rolipram, a specific phosphodiesterase inhibitor and cicaprost, a prostacyclin analogue, were applied as cAMP-elevating agents. The stable cAMP antagonist (Rp)-cAMPS was used to abrogate activation of protein kinase A. Human peripheral blood mononuclear cells were stimulated with lipopolysaccharide (LPS). TNF was quantified by radioimmunoassay, IL-10 by enzyme-linked immunosorbent assay, and mRNA by reverse transcriptase-polymerase chain reaction. After LPS stimulation alone 253+/-45 pg/mL IL-10 was synthesized, which increased to 644+/-117 pg/mL in the presence of 1 microM rolipram. (Rp)-cAMPS reversed this increase of IL-10 formation. In the same samples, the LPS-stimulated production of TNF was markedly attenuated by rolipram or cicaprost. A kinetic analysis revealed a significant increase in TNF production before IL-10 formation was detectable. These results demonstrate that (1) cAMP-elevating agents enhance IL-10 synthesis and suppress TNF production; (2) these regulative functions of cAMP-elevating agents are mediated by activation of protein kinases A; (3) suppression of TNF synthesis by cAMP in the early phase is not mediated by endogenous IL-10. Taken together, rolipram and cicaprost exert a dual regulatory function by enhancing IL-10 formation and attenuating TNF synthesis.

The hairy cell leukemia cell line Eskol spontaneously synthesizes tumor necrosis factor-alpha and nitric oxide.

Tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) exert a wide array of immunoregulatory, partly related effects. We examined the production of these two mediators by the human hairy cell leukemia cell line Eskol. Combined cell lysate and supernatant of Eskol cells (0.5 x 10(6) cells ml(-1)) incubated for 18 h, contained a mean of 1.5 ng ml(-1) TNF-alpha. This spontaneous TNF-alpha synthesis was enhanced by phorbol ester (PMA) and phytohemagglutinin (PHA) and decreased by dexamethasone. Nitrite, the stable product of NO, accumulated in the supernatant of Eskol cells after prolonged incubation. Maximal nitrite concentrations (range: 0.8-3.5 microM at 2 x 10(6) cells ml(-1)) were detected after 7 days of incubation. NO production was augmented by PHA and reduced by PMA. The inhibitors of NO synthase N(G)-monomethyl-L-arginine (L-NMMA) and aminoguanidine decreased NO synthesis. Simultaneous activation with the proinflammatory cytokines, interferon-gamma, interleukin-1beta and TNF-alpha, increased NO synthesis. These results suggest that NO production in Eskol cells results from inducible NO synthase activity. This is the first direct demonstration of NO formation in human lymphoid cells. The cell line, Eskol, may serve as a model to study regulation of TNF-alpha and NO synthesis in human B-cell leukemia.

The clinical relevance of circulating tumor necrosis factor-alpha in acute decompensated chronic heart failure without cachexia.

STUDY OBJECTIVE: To evaluate the clinical relevance of circulating tumor necrosis factor-alpha (TNF alpha) in subjects with advanced acutely decompensated congestive heart failure (CHF) and to determine the modulatory effect of clinical interventions on short-term elaboration of this cytokine. DESIGN: Prospective, case-controlled study. SETTING: Inpatient and outpatient (hospital and clinic), at regional academic medical center. PATIENT INTERVENTIONS: Plasma concentrations of TNF alpha were determined in 25 healthy, normal control subjects and in 29 noncachectic patients with advanced CHF (mean ejection fraction = 16 +/- 6%) who required hospitalization for i.v. diuretic and/or inotropic therapy despite optimization of oral medical regimens. CHF patients were divided into two groups: diuretic responsive (group A; n = 6) and diuretic resistant requiring inotropic support (group B; n = 23). Group B was randomly allocated to receive either i.v. dobutamine (n = 13) or milrinone (n = 10) for 72 h. TNF alpha levels in CHF patients were measured serially at baseline, at 6 h, at 48 h, at 72 h, and at 1-week follow-up after hospital discharge. RESULTS: Plasma TNF alpha levels at baseline in CHF patients were 4.0 +/- 1.1 pg/mL (range, 0.5 to 6.5 pg/ mL) and 2.5 +/- 0.6 pg/mL (range, 0.5 to 6.8 pg/mL) in groups A and B, respectively, which were significantly different (p < 0.002) from normal subjects (0.89 +/- 0.40 pg/mL; range, 0.5 to 9.7 pg/mL). Despite clinically successful therapy with i.v. diuretics, dobutamine, or milrinone, plasma levels of this cytokine remained unchanged. Plasma TNF alpha in CHF patients measured in recovery (1 week after hospital discharge) was 5.1 +/- 1.2 pg/mL (range, 1.0 to 9.9 pg/mL) and 3.9 +/- 0.8 pg/mL (range, 0.5 to 8.7 pg/mL) in groups A and B, respectively. CONCLUSION: These findings suggest that although noncachectic patients with chronic heart failure who suffer acute decompensation elaborate significantly higher circulating levels of TNF alpha compared with healthy control subjects, no significant reduction or alteration in circulating TNF alpha is noted in the short-term follow-up despite clinical improvement.

Cicaprost and the type IV phosphodiesterase inhibitor, rolipram, synergize in suppression of tumor necrosis factor-alpha synthesis.

Suppression of tumor necrosis factor-alpha (TNF) synthesis is one major target in pharmacological immunomodulation. We now showed the synergistic suppressive effect of the specific type IV phosphodiesterase inhibitor, rolipram, and of the stable prostacyclin analogue, cicaprost, on TNF synthesis. This effect was seen with lipopolysaccharide and Staphylococcus epidermidis as stimuli in human peripheral blood mononuclear cells and in whole blood. Lipopolysaccharide-induced TNF synthesis by mononuclear cells decreased from 3.4 ng/ml to 1.5 ng/ml in the presence of 100 nM rolipram and to 0.7 ng/ml in the presence of 10 nM cicaprost. The combination of both agents suppressed TNF synthesis more than 10-fold, to 0.3 ng/ml. Synergistic suppression was also demonstrated for TNF mRNA.

Suppression of tumor necrosis factor-alpha production by interleukin-10 is enhanced by cAMP-elevating agents.

The pro-inflammatory peptide tumor necrosis factor-alpha (TNF) stimulates production of the anti-inflammatory cytokine-interleukin-10 by monocytes which in turn inhibits the synthesis of TNF. This inhibitory effect of interleukin-10 may contribute to the balance of pro- and anti-inflammatory cytokines in several diseases, e.g., chronic inflammatory bowel disease. In the present study we addressed the question whether interleukin-10 in combination with other TNF-suppressing agents leads to enhanced suppression of TNF synthesis. We investigated the inhibitory potency of interleukin-10 in combination with rolipram, a specific type IV phosphodiesterase inhibitor, or with cicaprost, a stable prostacyclin analogue in lipopolysaccharide-stimulated human peripheral blood mononuclear cells. Peripheral blood mononuclear cells were stimulated with 10 ng/ml lipopolysaccharide in the absence or presence of interleukin-10 or one of the cAMP-elevating agents. First, we confirmed the TNF-suppressing effect of interleukin-10, rolipram and cicaprost alone and determined the IC50 for these substances. Second, for the combination of interleukin-10 with one of the cAMP-elevating substances we were able to demonstrate enhanced TNF inhibition. Of these, the combination of interleukin-10 and rolipram revealed an additive effect. The maximal TNF synthesis of 5.5 +/- 1.1 ng/ml after lipopolysaccharide stimulation alone was inhibited by 0.1 ng/ml interleukin-10 to 2.7 +/- 0.6 ng/ml TNF and by 100 nM rolipram to 3.1 +/- 0.6 ng/ml TNF. Both substances combined suppressed TNF synthesis to 1.5 +/- 0.3 ng/ml. After stimulation with Staphylococcus epidermidis we could demonstrate a more pronounced inhibition of TNF synthesis by interleukin-10 compared to rolipram which was more effective after stimulation with lipopolysaccharide. Finally, the additive inhibitory effect of interleukin-10 and rolipram could be confirmed on the level of TNF mRNA. The results obtained in the present investigation could form a prerequisite to study the combination of interleukin-10 and cAMP-elevating agents in in vivo models of acute or chronic inflammatory diseases.

Synthesis of tumour necrosis factor-alpha in tissue culture of rat caecum: lack of suppression by phosphodiesterase inhibitors and prostanoids.

Tumour necrosis factor-alpha (TNF) plays a pivotal role in acute and chronic inflammatory processes. It has been demonstrated that TNF is a mediator in inflammatory bowel disease. In the past different pharmacological approaches have been identified to suppress TNF synthesis in lipopolysaccharide-stimulated human mononuclear cells by cAMP-elevating agents. In the present study we examine whether TNF synthesis in the colon underlies similar regulatory mechanisms as in mononuclear cells. We therefore established a short-time organ culture of rat caecum. In this model we obtained maximal TNF formation of 159 pg/ml after a 7-h incubation period, as determined by L929 bioassay. The formation of bioactive TNF was confirmed by the application of neutralizing TNF antibody in the L929 bioassay and by immunodot blot. Lipopolysaccharide and pokeweed mitogen did not further enhance TNF synthesis. In contrast, TNF production was suppressed to 25% of control by 5 micrograms/ml hydrocortisone. Unexpectedly, the specific type IV phosphodiesterase inhibitor rolipram and cicaprost, a stable prostacyclin analogue, did not achieve significant suppression of TNF synthesis in this model. The present study defines an experimental model to investigate ex vivo TNF synthesis in rat colonic tissue. Applying this model, cAMP-elevating agents are identified as poor candidates for TNF-suppressing strategies in inflamed colon.

Dendritic cell-based cancer vaccination: quo vadis?

Dendritic cells (DCs) play a central role in the initiation and regulation of primary immune responses. DCs loaded with tumor-associated antigens induce anti-tumoral cytotoxic T cells in vitro and in vivo. However, clinical trials using ex vivo-generated DCs have failed to demonstrate clinical efficacy. This review summarizes recent advances in concepts and techniques that are providing new impulses to DC-based cancer vaccination. Improvements in protocols for ex vivo-generation of DCs, innovations in immunomonitoring, strategies to overcome tumor-induced immunosuppression and insights into the mutual beneficial effects of vaccines and chemotherapy are all considered. Furthermore, we highlight novel developments in cell-free vaccines targeting DCs in vivo.

Dendritic cell-based vaccination combined with gemcitabine increases survival in a murine pancreatic carcinoma model.

BACKGROUND: Tumour-specific cytotoxic T lymphocytes (CTLs) can be activated in vivo by vaccination with dendritic cells (DCs). However, clinical responses to DC-based vaccination have only been observed in a minority of patients with solid cancer. Combination with other treatment modalities such as chemotherapy may overcome immunoresistance of cancer cells. It has been shown previously that gemcitabine sensitises human pancreatic carcinoma cells against CTL-mediated lysis. Here, a murine pancreatic carcinoma model was used to investigate whether combination with gemcitabine increases therapeutic efficacy of DC-based vaccination. METHODS: Bone marrow-derived DCs from C57BL/6 mice were loaded with UV-irradiated, syngeneic Panc02 carcinoma cells and were administered subcutaneously. For prophylactic vaccination, mice were vaccinated three times at weekly intervals prior to tumour challenge with Panc02 cells. Therapeutic vaccination was started when tumours formed a palpable nodule. Gemcitabine was administered intraperitoneally twice weekly. RESULTS: Prophylactic DC-based vaccination completely prevented subcutaneous and orthotopic tumour development and induced immunological memory as well as tumour antigen-specific CTLs. In the subcutaneous tumour model, therapeutic DC-based vaccination was equally effective as gemcitabine (14% vs 17% survival at day 58 after tumour challenge; controls, 0%). Combination of the two strategies significantly increased survival of tumour-bearing mice (50% at day 58 after tumour challenge). DC-based vaccination also prevented death from pulmonary metastatisation after intravenous injection of Panc02 cells. CONCLUSION: DC-based immunotherapy may not only be successfully combined with gemcitabine for the treatment of advanced pancreatic carcinoma, but may also be effective in preventing local recurrence or metastatisation in tumour-free patients.

Sphingosine kinase and sphingosine-1-phosphate regulate migration, endocytosis and apoptosis of dendritic cells.

Dendritic cells (DC) are inducers of primary immune responses and represent an attractive vector for cancer immunotherapy. Sphingosine kinase (SphK) and its product sphingosine-1-phosphate (S1P) play an important role in the regulation of immune cells and cancer, affecting processes such as differentiation, growth or migration. We studied the role of SphK and S1P on migration of DC. RT-PCR showed mRNA expression of SphK in DC, declining from immature (iDC) to mature DC (mDC) to antigen-loaded mDC. Expression of S1P receptors was S1P(1) > S1P(2) = S1P(3), unrelated to maturation or antigen uptake. In transwell assays, iDC migrated towards SDF-1, MIP-1alpha, MCP and S1P, whereby S1P combined with a chemokine had a synergistic effect. mDC migrated towards 6Ckine and MIP-3beta, but not towards S1P. The SphK-inhibitor dihydro-sphingosine (DHS) reduced migration of iDC but not of mDC. In addition S1P(3)-inhibitor suramin inhibited DC migration in response to S1P. DHS had a reverse effect on endocytosis, enhancing the uptake of FITC dextran. We also observed an anti-apoptotic effect of S1P on mDC for the first time. This indicates that SphK/S1P may play a role in accumulation of peripheral iDC at the location of antigen and subsequent antigen-uptake. These findings may help to optimise DC-based cancer immunotherapy by modulation of SphK/S1P.

Vascular ectasia of the whole intestine as a cause of recurrent gastrointestinal bleeding after high-dose chemotherapy.

We present the first case in the literature of vascular ectasia of the whole intestine as a cause of recurrent and profuse gastrointestinal bleeding in a patient with relapsing Hodgkin's disease. The 17-year-old patient experienced early relapse of his Hodgkin's disease after first-line chemotherapy. Salvage chemotherapy was followed by high-dose chemotherapy and autologous stem cell transplantation. Complete remission was achieved after another relapse by means of a second transplant. The patient presented with profuse gastrointestinal bleeding 5 months later, however. Gastric antral vascular ectasia following hematopoietic stem cell transplantation was diagnosed by endoscopy, with histological confirmation. Similar lesions were found in the duodenum, the ileum, and throughout the entire colon. In conclusion, vascular ectasia of the whole intestine should be considered as cause of acute gastrointestinal bleeding after stem cell transplantation. Physicians should be aware of this complication because its onset is typically delayed. Importantly, this disease is not limited to patients who have undergone allogeneic transplantation, but can also occur after autologous transplantation.

Nitric oxide-releasing agents enhance cytokine-induced tumor necrosis factor synthesis in human mononuclear cells.

In septic shock tumor necrosis factor (TNF) leads to increased nitric oxide (NO) production by induction of NO synthase. An inverse regulatory effect, the influence of NO on cytokine synthesis, has rarely been investigated. The present study assessed the influence of NO-releasing agents on TNF production from interleukin-1 alpha (IL-1 alpha)-stimulated human peripheral blood mononuclear cells (PBMC). 3-Morpholino-sydnonimine (SIN-1) enhanced IL-1 alpha-induced TNF synthesis to a maximum of 272% (mean of n = 5 donors), with 100% set as TNF production by stimulation with IL-1 alpha alone. This finding was confirmed using another NO-donor, i.e., sodium nitroprusside (SNP). The effect was specific for TNF compared to the uninfluenced synthesis of IL-1 beta. Kinetic analysis showed the most pronounced increase in TNF synthesis when SIN-1 was added during the first 60 min after IL-1 alpha addition. These data reveal an enhancing effect of NO on cytokine-induced TNF synthesis. It may contribute to the regulation of TNF synthesis in pathological processes such as microbicidal activity, tumor cell lysis or endothelium-mediated hypotension.

Exogenous and endogenous nitric oxide attenuates tumor necrosis factor synthesis in the murine macrophage cell line RAW 264.7.

Effects of TNF on nitric oxide (NO) production have been well documented in a variety of experimental and clinical settings, as for example, in septic shock. Investigations focusing on an inverse relation of NO on TNF synthesis are rare. Previously we could demonstrate that exogenous NO-releasing agents suppress LPS-induced TNF production in human PBMC. We now investigate whether a regulatory role on TNF synthesis could also be ascribed to endogenous NO. This was studied in the murine macrophage cell line RAW 264.7, which is able to express both TNF and the inducible NO synthase. No production was determined by measuring nitrite with Griess reagents. TNF formation was quantified by L929 cytotoxicity. We found a suppression of LPS-induced TNF synthesis by the exogenous addition of NO-releasing agents in the murine cell line, as previously observed in human cells. The application of NO synthase inhibitors led to a decrease in NO production, associated with an increase in TNF synthesis. TNF production increased from a base line (stimulation with 1 microgram/ml LPS alone) of 20.8 ng/ml to 36.3 ng/ml (means of six experiments) in the presence of the NO synthase inhibitor NG-monomethyl L-arginine (100 microM). Similar results were obtained with another NO synthase inhibitor, NG-nitro L-arginine-methylester. Lack of L-arginine in the medium resulted in a threefold increase in LPS-stimulated TNF synthesis compared with medium containing the usual concentration of 1 mM L-arginine. Restitution of L-arginine but not of D-arginine reversed this increase in TNF synthesis in a dose-dependent manner. To our knowledge these results indicate for the first time a negative feedback by endogenous NO on TNF synthesis in vitro. This finding may be relevant in pathophysiologic processes in which both TNF and NO are formed and in experimental therapies aiming at changes of NO concentrations.

Taming TNF: strategies to restrain this proinflammatory cytokine.

Recent studies have demonstrated the essential role of tumor necrosis factor alpha (TNF-alpha) in rheumatoid arthritis and Crohn's disease. This article discusses agents known to suppress the formation or activity of TNF-alpha, and summarizes clinical studies using anti-TNF-alpha antibodies.

Specific suppression of human tumor necrosis factor-alpha synthesis by antisense oligodeoxynucleotides.

Recent clinical studies using neutralizing antibodies point to a key role for tumor necrosis factor-alpha (TNF-alpha) in chronic inflammatory diseases. Antisense technique is a recent approach aiming at inhibition of single proteins. Previously, we described nonspecific induction of TNF by phosphorothioate oligonucleotides. In this study, we established an in vitro model that allows specific inhibition of TNF synthesis, bypassing TNF induction. Freshly isolated human monocytes were incubated with oligonucleotides and the cationic lipid lipofectin in different ratios. TNF synthesis was stimulated with lipopolysaccharide and quantified by a specific radioimmunoassay (RIA). Among all sequences tested, one of the antisense oligonucleotides complementary to the translation initiation region of TNF mRNA (5'-CAT GCT TTC AGT CAT-3') revealed highest efficacy. At 2 microM, the antisense oligonucleotide inhibited TNF synthesis by up to 79%. A concentration as low as 250 nM of the antisense oligonucleotide was effective. Scrambled controls and controls with different, defined degrees of mismatches confirmed a sequence-specific action. Examination with confocal fluorescence microscopy showed a marked difference comparing lipofectin-mediated vs. spontaneous uptake. This study defines criteria that from the prerequisite necessary for design and application of antisense oligonucleotides against TNF in vivo.

Endogenous adenosine curtails lipopolysaccharide-stimulated tumour necrosis factor synthesis.

Recent studies have demonstrated the inhibitory effect of exogenous adenosine on TNF production. During inflammation endogenous adenosine levels are elevated and may be one of several anti-inflammatory mediators that reduce TNF synthesis. In the present study the authors investigated this role of adenosine in freshly isolated human PBMC. The effect of endogenous adenosine on TNF formation was studied by four different approaches. First, adenosine deaminase was added to LPS-stimulated mononuclear cells. This enzyme specifically deaminates extracellular adenosine to the inactive metabolite inosine. TNF production was augmented from baseline stimulation (LPS alone) of 3.5 +/- 0.4 ng ml-1 -5.2 +/- 0.9 ng ml-1 in the presence of 10 U ml-1 adenosine deaminase. Second, TNF production was determined after stimulation in the presence of dipyridamole, an inhibitor of cellular re-uptake of adenosine which increases extracellular concentrations. TNF synthesis was reduced dose-dependently from 3.1 +/- 0.9 ng ml-1 -1.1 +/- 0.2 ng ml-1 by 10 microM dipyridamole. Third, the adenosine A2 receptor antagonist 8-(3-chlorostyryl)caffeine (100 nM) enhanced TNF synthesis from a baseline of 3.7 +/- 0.5 ng ml-1 -5.5 +/- 0.9 ng ml-1. In contrast, no increase resulted from the addition of 100 nM of the specific A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Finally, the authors were able to show that suppression of TNF formation by the specific type IV phosphodiesterase inhibitor rolipram can be completely reversed by adenosine deaminase or by the application of the A2 receptor antagonist. The authors conclude that endogenous adenosine controls TNF production. This effect of adenosine may not only have a physiological role but also appears to contribute to the pharmacological inhibition of TNF synthesis by exogenous agents such as the specific type IV phosphodiesterase inhibitor rolipram.