[Thrombosis during thrombopoietin receptor agonist treatment for immune thrombocytopenia. A French multicentric observational study]. / Thromboses sous agonistes du récepteur de la thrombopoïétine au cours du purpura thrombopénique immunologique. Étude rétrospective multicentrique en France.

INTRODUCTION: Thrombopoietin-receptor agonists (TPO-RA) are marketed for immune thrombocytopenia (ITP). They have been associated to thrombosis occurrence in randomized controlled trials. However, the characteristics of these thromboses in the real-life practice as well as their management are poorly known. The objectives of this study were to determine the risk factors, circumstances and management of thrombosis occurring during exposure to TPO-RA in ITP. METHODS: We carried out a multicentre retrospective study in France. Moreover, all cases reported to the French pharmacovigilance system were also analyzed. RESULTS: Overall, 41 thrombosis (13 arterial) in 36 ITP patients (14 males and 22 females, mean age: 59 years) were recorded between January 2009 and October 2015. Twenty patients were treated with romiplostim, 15 with eltrombopag and 1 was treated by both medications. Thirty-three (92%) of the patients had another risk factor for thrombosis. Ten (28%) had an history of thrombosis and 13 (36%) received immunoglobulin in the month preceding the thrombotic event. Three had antiphospholipid antibodies; congenital low-risk thrombophilia was found in 4 cases; 18 patients (50%) were splenectomized. Median platelet count at the time of thrombosis was 172G/l (1-1049G/l). In 22 patients (56%), a good prognosis was associated with the thrombosis and was not linked with TPO-RA withdrawal. Bleeding events occurred in 14% of the patients treated with antiplatelet or anticoagulant drug, including 5% serious events (1 death of intracranial haemorrhage, 1 death of haemorrhagic shock). CONCLUSIONS: The thrombotic risk may be carefully assessed before starting TPO-RA in ITP patients. The impact of antiphospholipid antibodies and of congenital thrombophilia remains to be defined. Thrombosis evolution seems independent of TPO-RA management. Bleeding manifestations seem rare. Poor prognosis was mainly due to ischemic sequelae.

Pulmonary embolism and deep jugular venous thrombosis resulting from compression by a lipoma.

Lipomas usually extend in subcutaneous tissues and rarely may be compressive. We report a case of neck lipoma resulting in jugular vein thrombosis and pulmonary embolism in a patient treated by clozapine. Clozpine may be considered an associated risk factor for thrombosis. This case suggests that performing a regional evaluation may be particularly important when thrombophlebitis occurs.

Tuberculosis due to Mycobacterium bovis after alemtuzumab administration.

We describe a patient with relapsing B chronic lymphocytic leukemia who developed systemic bacille Calmette-Guérin infection (BCGitis) after administration of alemtuzumab (Campath-1H).

Acute-phase response, interleukin-6, and alteration of cyclosporine pharmacokinetics.

OBJECTIVE: Administration of interleukin-6 partially reproduces the inhibitory effects of the acute-phase response on cytochrome P450-dependent drug metabolism. The aim of the study was to determine whether endogenous cytokine has such an effect in patients treated by cyclosporine, which is metabolized by the cytochrome P4503A subfamily. METHODS: Blood cyclosporine and serum interleukin-6 levels were determined in six patients undergoing bone marrow transplantation, as long as they received cyclosporine by continuous infusion. Two serum acute-phase proteins, C-reactive protein and alpha 1-acid glycoprotein, and two cyclosporine metabolites, AM1 and AM9, were also determined. RESULTS: At the time of marrow infusion, levels of specific markers of inflammation were low. A peak in interleukin-6 level was then observed a mean of 10.8 days after transplantation, closely associated with variations in C-reactive protein levels. A parallel twofold increase in AM1 concentrations was observed, followed by a three-fold increase in cyclosporine levels, which peaked 4.8 days after interleukin-6. The times of peak cyclosporine and AM1 levels correlated with the time of peak interleukin-6 levels. AM9 was detectable in three patients but concentrations fell when interleukin 6 became detectable. CONCLUSIONS: An inflammatory reaction could be an important source of intraindividual variability in cyclosporine pharmacokinetics, possibly through an inhibition of cytochrome P4503A-dependent enzyme activities by endogenous interleukin-6. Blood AM1 accumulation might be explained by a secondary metabolic step that is highly sensitive to the inhibitory effect of interleukin-6.

Increase in tumor necrosis factor-alpha production linked to the toxicity of indomethacin for the rat small intestine.

1. The toxic effects of nonsteroidal anti-inflammatory drugs for the lower gastrointestinal tract share certain features with inflammatory processes, suggesting that the release of inflammation cytokines such as TNF-alpha may damage the intestine. 2. Rats received a s.c. injection of indomethacin. Then, jejunum-ileum was taken up for the quantification of ulcerations, production of TNF-alpha, nitrites and PGE2 ex vivo and activity of calcium-independent NO synthase and myeloperoxydase. Activation of NO metabolism and myeloperoxydase were measured as potential effectors of TNF-alpha. 3. Jejunum-ileum from rats having received indomethacin (10 mg kg(-1)) produced TNF-alpha ex vivo. Cytokine production was associated with the onset of macroscopic ulcerations of the small intestine, and preceded nitrite production and tissue activity of myeloperoxidase. 4. Similar intestinal ulcerations and upregulation of TNF-alpha were obtained with flurbiprofen (30 mg kg(-1)), chemically unrelated to indomethacin. 5. TNF-alpha production was proportional to the indomethacin dose (from 3-20 mg kg(-1)) and correlated with the surface area of ulcerations and nitrite production, 24 h after indomethacin administration. 6. Pretreatment of rats with RO 20-1724, a type-IV phosphodiesterase inhibitor which inhibits TNF-alpha synthesis, substantially reduced jejunum-ileum ulcerations, TNF-alpha and nitrite production and tissue enzyme activities. 7. These findings provide evidence that TNF-alpha is increased in indomethacin-induced intestinal ulcerations and support suggestions that TNF-alpha is involved at an early stage of nonsteroidal anti-inflammatory drug toxicity for the small intestine.

Effects of interleukin-6 on cytochrome P450-dependent mixed-function oxidases in the rat.

Intravenous treatment of male rats with recombinant human interleukin-6 (rhIL6) at 50, 100 and 200 micrograms/kg (corresponding to 4, 8 and 16 x 10(4) U/animal, respectively) reduced the activities of hepatic microsomal cytochrome P450-dependent monoxygenases to varying degrees. Ethylmorphine-N-demethylase activity fell to 53% of control values, an effect similar to that induced by 2.5 mg/kg Escherichia coli lipopolysaccharide (LPS). Ethoxycoumarin-O-deethylase activity was also sensitive to inhibition, whereas IL6 had little effect on the activities of other P450-dependent enzymes, including ethoxyresorufin-O-deethylase. Pentoxyresorufin dealkylase activity, which is representative of the cytochrome P450 IIB 1/2 subfamily, was unaffected by IL6 whereas LPS reduced it to 33.7% of control values. Another hepatocyte-related parameter, serum concentration of alpha 1-acid glycoprotein (AGP), was increased by up to 3.5-fold over baseline by IL6 and 10-fold by LPS. Recombinant human interleukin-1 beta (rhIL1 beta) (10 micrograms/kg, corresponding to 5 x 10(4) U/rat) and recombinant human tumor necrosis factor alpha (rhTNF) (150 micrograms/kg corresponding to 24 x 10(4) U/rat) were both as potent as LPS (2.5 mg/kg) in increasing serum AGP levels and reducing hepatic microsomal monoxygenase activities. IL6 did not potentiate the effects of rhIL1 beta. Hepatic microsomal glucuronyltransferase activities were little affected by LPS and unaffected by rhIL6. Finally, rhIL6 was more potent after i.p. injection than after i.v. or s.c. injection. These results suggest that the effects of LPS, TNF and IL1 on the mixed-function oxidase system in vivo may be due partly to an induction of IL6 in vivo. The different sensitivities of the enzymes to IL6 but not to IL1 or TNF may be due to the involvement of two distinct mechanisms.

Effects of octreotide treatment on early TNF-alpha production and localization in experimental chronic colitis.

BACKGROUND: Colitis induced by trinitrobenzene sulphonic acid (TNB) is a model of Th1 disease, mainly explored from the third day of induction. It has recently been shown that octreotide and other somatostatin analogues can modify inflammatory/immune processes by acting on cytokines. AIM: To examine TNFalpha production and the effect of preventive treatment with octreotide, during the early phase of TNB-colitis. METHODS: Thirty milligrams TNB with 50% ethanol was instilled into the colon of male Wistar rats. Treated groups received octreotide (2x10 microg x day/rat) or dexamethasone (1x2 mg x day/kg), subcutaneously, with the first injection before TNB. Eight and 80 h later, the colon was excised and processed for histology, TNFalpha immunohistochemistry, quantification of cytokine release ex vivo and tissue-inducible NO synthase (iNOS) activity. RESULTS: Maximal TNFalpha production was observed at the 8th hour, associated with intense immunostaining of the external muscle layer. Octreotide treatment decreased TNFalpha expression (staining and activity) and iNOS activity. At the 80th hour, submucosal macrophages were positive for TNFalpha and colonic production of IL1beta and interferon gamma was increased; all these effects were reduced by octreotide treatment. CONCLUSIONS: TNFalpha was expressed early by resident muscle cells, before staining of infiltrated immune cells and increased production of interferon gamma. TNFalpha regulation by octreotide suggests that this drug might exert anti-inflammatory properties via smooth muscle cells.

Anti-tumor necrosis factor properties of non-peptide drugs in acute-phase responses.

Dexamethasone (sodium phosphate), pentoxifylline, fusidic acid (sodium salt), pentamidine (isethionate) and R-phenylisopropyladenosine (R-PIA) were tested for their anti-tumor necrosis factor (TNF) activities in an endotoxin-induced shock rat model. All the drugs reduced serum TNF concentrations in a dose-dependent manner, whereas their effects on serum interleukin-6 levels differed. Doses that reduced TNF levels by 50% were 0.012 mg/kg for dexamethasone, 0.06 mg/kg for R-PIA, 0.24 mg/kg for pentamidine, 6.5 mg/kg for fusidic acid and 15 mg/kg for pentoxifylline. Administration of the drugs to rats before intraplantar injection of carrageenan reduced paw edema by 50-70%. Injection of a monoclonal anti-TNF antibody reproduced the inhibitory effect. Moreover, the time course of tissue-associated TNF following carrageenan injection was compatible with mediation of edema by TNF. Results obtained for this acute, non-immunological inflammatory reaction strongly suggest that the model is TNF-dependent. Our results reinforce the idea that TNF is a crucial target in the therapeutics of inflammatory reactions. These drugs, which are able to cross cell barriers, might have clinical applications in localized and/or chronic diseases in which TNF is involved.

Role of tumour necrosis factor-alpha and inducible nitric oxide synthase in the prevention of nitro-flurbiprofen small intestine toxicity.

The present study compares the intestinal toxicity of nitro-flurbiprofen and flurbiprofen in order to determine their differential properties on tumour necrosis factor-alpha production and inducible nitric oxide synthase induction. Rats received one s.c. injection of flurbiprofen, nitro-flurbiprofen at equimolar dose of solvent. Twenty-four hours later, the rats were sacrificed and small intestine tissue was taken up for macroscopical quantification of ulceration, ex vivo production of tumour necrosis factor-alpha and nitrites, and determination of tissue inducible nitric oxide synthase and myeloperoxidase activities. Anti-inflammatory activity was examined in the carrageenan-induced paw edema model. We demonstrated that flurbiprofen induced dose-dependently small intestine production of tumour necrosis factor-alpha, nitrites, myeloperoxidase and inducible nitric oxide synthase activities. On the other hand, nitro-flurbiprofen did neither induce tumour necrosis factor-alpha nor nitrite production. Concurrently, no small intestine ulceration was observed with nitro-flurbiprofen whereas flurbiprofen induced dose-dependent ulceration. Nitro-flurbiprofen is devoid of intestinal toxicity despite inhibiting cyclooxygenase activity. This is associated with the absence of tumour necrosis factor-alpha and inducible nitric oxide synthase induction in normal rats. Nitro-flurbiprofen is an anti-inflammatory drug with a much more favorable gastro-intestinal toxicity profile than flurbiprofen.

Leukemia inhibitory factor involvement in human ulcerative colitis and its potential role in malignant course.

The pleiotropic cytokine leukemia inhibitory factor (LIF) possesses proinflammatory properties in common with tumor necrosis factor (TNF-alpha), interleukine (IL) -1 and -6, such as the induction of acute phase protein synthesis. LIF may have chemotactic activity through the induction of IL-8 production. LIF is produced by normal and tumoral cells and appears to facilitate in vivo rat colon carcinoma cells growth. Inflammatory bowel diseases, ulcerative colitis (UC) in particular, are histologically characterized by the infiltration of the colonic mucosa with activated neutrophils, macrophages and lymphocytes. Cytokines with their inflammatory as well as their regulatory activities may play a role in the perpetuation and possibly the initiation of inflammation in this disease and its local and/or systemic complications. Moreover, colorectal cancer is a late well identified complication in patients with long standing inflammatory bowel disease, UC in particular. Taken together, these results suggest that LIF could be involved in tumorigenic and/or metastatic processes of colorectal cells in UC patients. The aims of the present study was to quantify and to compare the colonic and systemic productions of LIF in UC patients. We showed for the first time in patients with UC, a high local production of LIF well correlated with IL-8 production. We also analyzed the effect of LIF on a human colon carcinoma cell line HT29. We demonstrated that LIF stimulated HT29 cell growth in a dose dependent-manner. These results suggest that LIF may play a critical role in the susceptibility of colonic host cells to tumor growth in patients with UC.

Effects of murine recombinant interleukin-10 on the inflammatory disease of rats transgenic for HLA-B27 and human beta 2-microglobulin.

Rats transgenic for HLA-B27 and human beta 2-microglobulin develop a spontaneous, multisystem, inflammatory disease that resembles human B27-associated disease and that involves the gut mucosa. This model predominantly affects the colon and is characterized by an extensive infiltration of the mucosa by inflammatory cells, largely composed of mononuclear cells. In addition, an increased plasma level of nitric oxide (NO)-derived metabolites was described in this model. Deficiency in the anti-inflammatory cytokine, interleukin-10 (IL-10), leads to the development of colitis in IL-10 knockout mice, suggesting that IL-10 plays a major role in the control of gut inflammation. The objectives of this work were to study the mechanisms of the inflammatory bowel disease (IBD) in HLA-B27 rats and to determine the effects of treatment with IL-10. The 33-3 line of HLA-B27 recombinant rats with established disease was treated in two consecutive experiments with murine recombinant IL-10 for five weeks. Assessment of the effect of this treatment was performed, based on clinical, histological and biological (myeloperoxidase and inducible NO synthase activities; tumor necrosis factor-alpha, interferon-delta, CD3, iNOS and beta-actin mRNA expression. In 33-3 rats with established disease, mesenteric lymph nodes were hyperplastic, and colonic cellularity and MPO and iNOS activities in the colonic mucosa were increased without any detectable effects of IL-10 administration. IFN-gamma and iNOS mRNA were only detected in the colon of transgenic rats. Despite a lack of effect on disease expression, IL-10 strikingly reduced the level of IFN-gamma mRNA in gut mucosa. Up-regulation of IFN-gamma mRNA suggests that the IBD of HLA-B27 rats is mediated by T-helper 1 lymphocytes. Sustained administration of IL-10, in HLA-B27 rats with established disease, efficiently inhibited IFN-gamma mRNA expression but did not influence disease expression: these results indicate that IFN-gamma may exert a critical role at an earlier stage of the disease rather in the maintenance of the lesions.

Inhibition of human monocyte TNF production by adenosine receptor agonists.

Adenosine receptor agonists and agents enhancing pericellular concentrations of adenosine possess antiinflammatory properties. In the present study, we found that R-phenylisopropyladenosine (R-PIA), 5'-N-ethylcarboxamido adenosine (NECA), other agonists of adenosine receptors and dipyridamole, an adenosine uptake inhibitor, inhibited tumor necrosis factor (TNF) production by endotoxin-stimulated human monocytes in a concentration-dependent manner with no inhibition of interleukin-6. The rank order of agonist potency is characteristic of neither A1 nor A2 receptors and suggests the involvement of another receptor subtype. The effect of R-PIA on TNF was in part abolished by the antagonist 8-sulfophenyltheophylline. In endotoxin-treated rats, R-PIA pretreatment (2.5 mg/kg) reduced serum TNF levels by 98%, with no modification of serum IL6 levels. TNF inhibition could be an important mechanism by which adenosine analogs exert their antiinflammatory action.

Blood polymorphonuclear behavior in patients with ankylosing spondylitis.

The oxidative metabolism and chemotaxis of polymorphonuclear leukocytes (PMNs) collected from patients with ankylosing spondylitis and healthy subjects were studied in parallel. The responses to opsonized zymosan were significantly lowered considering oxygen consumption and release of superoxide anions, whereas no modification of these parameters to phorbol myristate acetate and calcium ionophore (A 23187) stimulations were observed. A seric factor was not involved but the characterization of a specific intrinsic abnormality of the PMNs needs further investigations. PMN chemotaxis, assessed by two methods performed in parallel, remained unchanged.

Nitric oxide mediates the depression of lymphoproliferative responses following burn injury in rats.

Among the multiple biological activities of nitric oxide (NO) an immunoregulatory role consisting of the mediation of macrophage suppressive activity, has recently been evidenced. In the present work, we investigated whether NO was implicated in immunosuppression following burn injury. Thermal injury affecting 20-25% of the total body surface area in Wistar rats, provoked a biphasic depression of spleen cell proliferative responses to phytohemagglutinin (PHA) and concanavalin A (Con A). We show that these responses are fully restored on day 4 after burn and only by 55% on day 10 when spleen cells were stimulated in the presence of NG-monomethyl-L-arginine (NMMA), a potent inhibitor of the macrophage inducible NO synthase. Nitrite content in culture supernatant, as an indicator of NO release (in the absence of NMMA), was significantly augmented in Con A-stimulated spleen cells from burned rats as compared to normal spleen cells. These results show for the first time that NO is implicated, at least in part, in an immunosuppression state which is not linked to an infectious disease.

Recombinant human interleukin 1 beta and tumor necrosis factor affect glycosylation of serum alpha 1-acid glycoprotein in rats.

Serum concentration and glycosylation of rat alpha 1-acid glycoprotein (alpha 1-AGP) were evaluated after the in vivo administration of recombinant human interleukin-1 beta (rhIL-1 beta) and tumor necrosis factor alpha (rhTNF-alpha), alone or associated. The effect of LPS and turpentine was also studied. In all models, serum alpha 1-AGP concentrations were increased and glycosylation was altered. The alpha 1-AGP levels reached 1.8 g/liter with cytokines alone, 2.1 g/liter with cytokines associated or LPS, and 3.4 g/liter with turpentine. Analysis by concanavalin A (Con A) affinoimmunoelectrophoresis (CAIE) revealed that the relative proportion of Con A unreactive form always decreased whatever the inducing agent. On the other hand, the resulting effect on the concentrations of Con A unreactive alpha 1-AGP concentrations was an increase with cytokines alone or LPS and a decrease with cytokines associated or turpentine. These results suggest a dissociation between the alteration in the level of alpha 1-AGP synthesis and in the pattern of its glycosylation in the various inflammatory models.

Modification of inflammatory processes by phenobarbital in rats.

Enzyme-inducing drugs such as phenobarbital (PB) increase serum concentrations of an acute-phase protein, alpha 1-acid glycoprotein (AGP), in man, dogs, and rats via an unknown mechanism. We studied the effects of PB on components of an acute inflammatory reaction in rats in order to determine if PB acts only on this biological marker of inflammation or is capable of altering the clinical course of inflammatory processes. Local carrageenan injection induces a similar time-dependent plantar edema and increases serum AGP levels in Sprague-Dawley (SD) and Dark Agouti (DA) rats. Pretreatment with PB for seven days modified neither parameter in SD rats while plantar edema was aggravated and serum AGP levels were increased in DA rats. The sedative-hypnotic properties of PB were not involved, since a single administration of this drug had no action in DA rats. On the other hand, chronic PB administration reduced the severity of an autoimmune disease, type II collagen-induced arthritis, in DA rats. These data indicate that PB, a potent inducer a cytochrome P-450-dependent enzymes, modifies the course of the inflammatory process. Preliminary results with macrophage transfer experiments suggest that this response to PB could be mediated by stimulated macrophages.

[Activity of adenosine in relation to tumor necrosis factor (TNF). Therapeutic outlook]. / Activité de l'adénosine vis-à-vis du facteur nécrosant les tumeurs (TNF). Perspectives thérapeutiques.

At physiological and pharmacological doses, adenosine protects tissues against a varieties of injuries: ischemia-reperfusion, convulsions, inflammation.... We tested the hypothesis that the antiinflammatory properties of adenosine occur via a down-regulation of TNF. Agonists of adenosine receptors (ARA) and agents potentiating endogenous adenosine (APA) were evaluated for their effects on TNF production by endotoxin-stimulated human monocytes. Additionally, one of the most potent agonists, R-phenylisopropyladenosine (R-PIA), was tested on two experimental models of acute phase response, endotoxin shock and carrageenan-induced plantar oedema. Several ARA and APA inhibited monocyte TNF production in a concentration-dependent manner. R-PIA and other ARA were active at micromolar concentrations. The property is pharmacologically relevant since rats receiving a lethal dose of endotoxins were protected by R-PIA and endotoxin-induced serum TNF levels were abolished by a pretreatment with R-PIA. Inhibitory effects on serum TNF production were obtained with similar doses of dexamethasone sodium phosphate and one hundred-fold higher doses of pentoxifylline. R-PIA was also found active on carrageenan-induced oedema. The anti-oedematous properties of R-PIA were associated with a marked reduction of locally-produced TNF and were also observed after the administration of dexamethasone, pentoxifylline and a neutralizing anti-TNF antibody. Our results indicate that adenosine is a potent inhibitor of TNF production induced by different stimuli. This property could lead to therapeutic applications in inflammatory diseases and other in which TNF is known to play a pathogenic or aggravating role. Comparison between ARA and APA in terms of tolerance and efficacy merits further attention.