ag-490 and Acute-Disease

Although pediatric patient and renal graft survival rates have shown marked improvements during the past decade, the persistent toxicities of immunosuppressive drugs and chronic allograft attrition remain major obstacles in transplant therapy. Results in adult patients suggest that complete steroid withdrawal is possible in the majority of recipients under treatment with a cyclosporin A-rapamycin (CsA RAPA) regimen. Furthermore, preliminary studies suggest that a marked reduction in the dose of CsA may be possible under the umbrella of RAPA coverage. The gain in immunosuppressive efficacy afforded by RAPA has not only been obtained without an increased morbidity owing to infectious or neoplastic causes, but also with the potential for reducing the incidence and/or progression of chronic rejection.

Topics: Acute Disease; Adult; Age Factors; Antibodies, Monoclonal; Basiliximab; Child; Cohort Studies; Cyclosporine; Drug Monitoring; Fingolimod Hydrochloride; Graft Rejection; Graft Survival; Humans; Immunosuppressive Agents; Kidney Transplantation; Propylene Glycols; Protein-Tyrosine Kinases; Recombinant Fusion Proteins; Sirolimus; Sphingosine; Time Factors; Tyrphostins

Although pediatric patient and renal graft survival rates have shown marked improvements during the past decade, the persistent toxicities of immunosuppressive drugs and chronic allograft attrition remain major obstacles in transplant therapy. Results in adult patients suggest that complete steroid withdrawal is possible in the majority of recipients under treatment with a cyclosporin A-rapamycin (CsA RAPA) regimen. Furthermore, preliminary studies suggest that a marked reduction in the dose of CsA may be possible under the umbrella of RAPA coverage. The gain in immunosuppressive efficacy afforded by RAPA has not only been obtained without an increased morbidity owing to infectious or neoplastic causes, but also with the potential for reducing the incidence and/or progression of chronic rejection.

Topics: Acute Disease; Adult; Age Factors; Antibodies, Monoclonal; Basiliximab; Child; Cohort Studies; Cyclosporine; Drug Monitoring; Fingolimod Hydrochloride; Graft Rejection; Graft Survival; Humans; Immunosuppressive Agents; Kidney Transplantation; Propylene Glycols; Protein-Tyrosine Kinases; Recombinant Fusion Proteins; Sirolimus; Sphingosine; Time Factors; Tyrphostins

Acute heart failure (AHF) is a burden disease, with high mortality and re-hospitalisations. Using an ex-vivo model of AHF, we have previously reported that sphingosine-1-phosphate (S1P) confers cardioprotection. However, the mechanisms remain to be elucidated. In the present study, we aimed to examine the role of the cardioprotective signal transducer and activator of transcription 3 (STAT3) in S1P mediated improved functional recovery in AHF.. Isolated hearts from male Long-Evans rats were subjected to hypotensive AHF for 35 min followed by a recovery phase of 30 min (n ≥ 4/group). S1P (10 nM) was given during either the hypotensive or the recovery phase with/without an inhibitor of STAT3, AG490. Functional parameters were recorded throughout the experiment.. Following an AHF insult, S1P, given during the recovery phase, improved the heart rate (HR) compared to the control (175.2 ± 30.7 vs. 71.6 ± 27.4 beats per minute (BPM); p < 0.05), with no changes in the left ventricular developed pressure. This effect was associated with an increase in phosphorylated STAT3 levels in the nucleus. Addition of AG490 with S1P abolished the cardioprotective effect of S1P (42.3 ± 17.1 vs. 148.8 ± 26.4 BPM for S1P; p < 0.05).. Our data suggest that S1P protects in an ex-vivo rat heart model of AHF by activation of STAT3 and provide further evidence for the usage of S1P as a potential therapy in patients suffering from AHF.

Topics: Acute Disease; Animals; Blood Pressure; Cardiotonic Agents; Heart Failure; Heart Rate; In Vitro Techniques; Lysophospholipids; Male; Phosphorylation; Rats; Rats, Long-Evans; Sphingosine; STAT3 Transcription Factor; Tyrphostins; Ventricular Dysfunction, Left

Studies have implied the positive association of JAK2/STAT3 signaling with the onset and severity of acute pancreatitis (AP). However, definitive functional study of JAK2/STAT3 signaling in the pathogenesis of acute pancreatitis in vivo is missing and its potential as a therapeutic target and the underlying mechanisms remain to be determined.. The aim of this study was to explore the role of JAK2/STAT3 signaling in the pathogenesis of hyperlipidemia-intensified caerulin-induced AP and its potential as a therapeutic target.. Using the caerulin-induced acute pancreatitis rat model, we showed that JAK2/STAT3 signaling was activated in pancreas and systemic inflammation was increased during AP. Pharmacological suppression of JAK2 by its inhibitor AG490 robustly protected against tissue damage, attenuated JAK2/STAT3 signaling and inflammatory responses. Local pancreatic tissue damage and phosphor- JAK2 in the pancreatic tissue were enhanced in animals fed with high fat diet compared to chow-diet fed animals. Interestingly, JAK2 inhibitor AG490 significantly inhibited pancreas necrosis and systemic inflammation in animals fed with high fat or chow-diet, but did not affect STAT3 signaling.. These results establish that JAK2 activation plays a significant role in the pathogenesis of caerulin-induced AP in animals on both chow and high-fat diets by regulating necrosis and systemic inflammation. Thus, our results not only clarify novel signaling mechanisms in AP but also suggest that JAK2 might constitute a target in the management of hyperlipidemia-intensified caerulin-induced AP.

Topics: Acute Disease; Animals; Ceruletide; Dietary Fats; Hyperlipidemias; Janus Kinase 2; Male; Pancreatitis; Rats; Rats, Sprague-Dawley; Signal Transduction; STAT3 Transcription Factor; Tyrphostins

Hepatic ischemia/reperfusion (I/R) injury is a serious complication of liver surgery and transplantation. Regulation of this injury response occurs at the cellular and molecular levels. Previous studies have shown that interleukin-6 (IL-6) is a negative regulator of the acute inflammatory injury occurring as a result of hepatic I/R. The signal transducer and activator of transcription-3 (STAT3) is a key target of receptor signaling for IL-6. Both IL-6 and STAT3 have been implicated in the protective effects of ischemic preconditioning of the liver. However, there have been no studies that have directly addressed the potential role of STAT3 in regulating acute inflammatory liver injury induced by I/R. In the current study, we investigated whether blockade of STAT3 phosphorylation altered the injury response to hepatic I/R injury.. Male Balb/c mice were subjected to 90 min of partial hepatic ischemia followed by reperfusion with or without treatment with specific inhibitors of STAT3 activation, AG490 (selective JAK2 inhibitor), or STATTIC (direct inhibitor of STAT3 phosphorylation). Mice were sacrificed at 8 and 24 h after reperfusion.. STAT3 activation was induced by I/R. This activation was partially inhibited by administration of AG490 and almost completely abrogated by treatment with STATTIC. Despite the blockade of STAT3, neither AG490 nor STATTIC had any effect on acute liver injury induced by I/R. Treatment with STATTIC did reduce hepatic neutrophil accumulation.. The data suggest that STAT3 is not a central regulator of acute liver injury induced by I/R.

Topics: Acute Disease; Animals; Cyclic S-Oxides; Enzyme Inhibitors; Hepatitis; Janus Kinase 2; Male; Mice; Mice, Inbred BALB C; Neutrophils; Phosphorylation; Reperfusion Injury; STAT3 Transcription Factor; Tyrphostins

Intraocular pressure (IOP) elevation is an important cause of glaucoma. Animal models of ocular hypertension have been widely used to mimic glaucoma to investigate the mechanisms underlying retinal ganglion cell (RGC) death and search for possible cure. The aim of the present study was to examine the role of JAK/STAT pathway in RGC viability in normal condition or after acute IOP elevation. Retinal explants obtained from intact or IOP-elevated eyes were firstly used to examine the effect of the JAK/STAT pathway inhibitors, AG490 and Jak Inhibitor I, on RGC viability in vitro. The role of this signal pathway was further investigated and confirmed in vivo. AG490 and Jak Inhibitor I were applied into the left eye on days 3, 9, and 15 post 2-h IOP elevation at 110mmHg. Fluorescence dye Fluorogold was used to retrogradely label surviving RGCs. Because macrophage recruitment was seen in the IOP-elevated eyes after inhibition of this pathway, clodronate liposomes were used to remove phagocytic cells in the eye and examine the role of JAK/STAT pathway in RGC survival independent of macrophages. Activities and location of JAK/STAT pathway in the retina were examined using Western blotting and immunohistochemistry. We found that inhibition of JAK/STAT pathway did not affect RGC survival in the retinal explants derived from intact eye but caused RGC death in the retinal explants that were derived from IOP-elevated eye. Importantly, the detrimental effect of JAK/STAT pathway inhibition on RGC survival was also observed in vivo following acute IOP elevation, but not in intact eye. In addition, both in vitro and in vivo experiments confirmed a detrimental action of phagocytic cells following acute IOP elevation and the pathway inhibition. Compatible with what were observed in vivo, Western blotting and immunohistochemistry showed that JAK/STAT activities were not present in intact retina, but acute IOP elevation activated JAK/STAT pathway in the retina, in the regions of inner nuclear layer and ganglion cell layer, including RGCs. The IOP elevation-induced JAK/STAT activities were effectively abolished by intravitreal application of AG490. This study thus shows that (1) acute IOP elevation activates JAK/STAT pathway in RGCs, and (2) JAK/STAT pathway mediates RGC survival following IOP elevation but not under normal condition.

Topics: Acute Disease; Animals; Cell Survival; Disease Models, Animal; Enzyme Inhibitors; Eye Proteins; Glaucoma; Janus Kinases; Macrophages; Rats; Rats, Sprague-Dawley; Retinal Ganglion Cells; Signal Transduction; STAT Transcription Factors; Tissue Culture Techniques; Tyrphostins

Several cytokines and growth factors that stimulate the proliferation of acute myelogenous leukemia (AML) cells transduce their signals by activating the transcription factor Janus-activated kinase 2 (JAK2). Accordingly, the inhibition of JAK2 or of its downstream signaling pathways suppresses the proliferation of AML cells. Because (E)-3(6-bromopyridin-2-yl)-2-cyano-N-((S0-1-phenylethyl)acrylamide) (WP1066) is a novel analogue of the JAK2 inhibitor AG490, we tested its activity in AML cells and investigated its mechanism of action. Using clonogenic assays, we found that although WP1066 had a marginal effect on normal marrow progenitors, it inhibited the proliferation of AML colony-forming cells obtained from patients with newly diagnosed AML and that of the AML cell lines OCIM2 and K562. WP1066 inhibited OCIM2 cell multiplication by inducing accumulation of cells at the G(0)-G(1) phase of the cell cycle. Similar to its parent compound AG490, WP1066 inhibited the phosphorylation of JAK2, but unlike AG490, WP1066 also degraded JAK2 protein, thereby blocking its downstream signal transducer and activator of transcription (STAT) and phosphoinositide-3-kinase pathways. These effects resulted in the activation of the caspase pathway. Incubation of both OCIM2 and K562 cells with WP1066 activated caspase-3, induced cleavage of poly(ADP-ribose) polymerase, and caused caspase-dependent apoptotic cell death. Thus, WP1066 is a potent JAK2 inhibitor whose effects in AML and other hematologic malignancies merit further investigation.

Topics: Acute Disease; Adult; Aged; Apoptosis; Caspases; Cell Cycle; Cell Proliferation; Colony-Forming Units Assay; Enzyme Inhibitors; Female; Humans; Immunoblotting; Janus Kinase 2; Leukemia, Myeloid; Male; Middle Aged; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein-Tyrosine Kinases; Pyridines; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; STAT3 Transcription Factor; Trans-Activators; Tumor Cells, Cultured; Tyrphostins

Tyrphostin AG490 (AG490) potently and selectively inhibits gammac/Janus kinase 3-dependent signaling pathways, including downstream Stat5a/b activation and subsequent T cell proliferation by alloantigen stimulation. We evaluated the effects of AG490 on acute rat lung allograft rejection.. A 7-day course of an intraperitoneal (IP) injection with 10 mg/kg, 15 mg/kg, or 20 mg/kg AG490 was administered to inhibit the rejection of orthotopically transplanted Brown Norway (RT1n) rat lung allografts in Fischer 344 (RT1(1vl)) rat recipients. The progression of allograft rejection was evaluated by X-ray with a semi-quantitative scoring system and was evaluated histologically with a semi-quantitative rejection scoring system for acute lung allograft rejection. Moreover, to determine whether AG490 regulates CD4+ T cell differentiation during acute rejection, flow cytometry was used to investigate Th1 (interferon-gamma) and Th2 (interleukin [IL]-4, IL-10) intracellular cytokine profiles and the CD4+CD25+ T cell population in recipient splenocytes.. Results of radiology and histology confirmed that treatment with AG490 significantly suppressed acute lung allograft rejection. Furthermore, the splenocytes of the AG490-treated recipients had significantly lower production of interferon-gamma and relatively higher production of IL-10, implying that a Th2 shift was induced by AG490. In addition, AG490-treated recipients had a significantly increased population of CD4+CD25+ T cells in their splenocytes on Day 6 after transplantation.. These findings suggest that treatment with AG490 prevents acute lung allograft rejection in rats. The effects of AG490 may contribute to development of CD4+CD25+ T cells and a Th2 shift of CD4+ T cells.

Topics: Acute Disease; Animals; CD4 Antigens; Cell Differentiation; Cell Proliferation; Disease Models, Animal; Disease Progression; Graft Rejection; Injections, Intraperitoneal; Interferon-gamma; Interleukin-10; Interleukin-4; Janus Kinase 3; Lung Transplantation; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Rats; Rats, Inbred BN; Rats, Inbred F344; Receptors, Interleukin-2; Signal Transduction; Spleen; STAT5 Transcription Factor; T-Lymphocytes; Tyrphostins

High levels of cytokines are associated with a poor prognosis in acute myeloid leukemia (AML). However, cytokines may induce, on one hand, survival factor expression and cell proliferation and, on the other hand, expression of inhibitory signals such as up-regulation of suppressors of cytokine signaling (SOCS) and induce apoptotic cell death. Because blasts from patients with AML express high procaspase protein levels, we asked whether granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances procaspase protein production in AML cells. In the GM-CSF-responsive OCIM2 AML cell line, GM-CSF induced signal transducer and activator of transcription 5 (Stat 5) phosphorylation, up-regulated cyclin D2, and stimulated cell cycle progression. Concurrently, GM-CSF stimulated expression of SOCS-2 and -3 and of procaspases 2 and 3 and induced caspase 3 activation, poly(ADP[adenosine 5'-diphosphate]-ribose) polymerase (PARP) cleavage, and apoptotic cell death. The Janus kinase (Jak)-Stat inhibitor AG490 abrogated GM-CSF-induced expression of procaspase 3 and activation of caspase 3. Under the same conditions GM-CSF up-regulated production of BAX as well as Bcl-2, Bcl-XL, survivin, and XIAP. GM-CSF also increased procaspase 3 protein levels in OCI/AML3 and Mo7e cells, suggesting that this phenomenon is not restricted to a single leukemia cell line. Our data suggest that GM-CSF exerts a dual effect: it stimulates cell division but contemporaneously up-regulates Jak-Stat-dependent proapoptotic proteins. Up-regulation of procaspase levels in AML is thus a beacon for an ongoing growth-stimulatory signal.

Topics: Acute Disease; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Caspases; Cell Division; DNA-Binding Proteins; Enzyme Induction; Enzyme Inhibitors; Enzyme Precursors; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inhibitor of Apoptosis Proteins; Leukemia, Erythroblastic, Acute; Leukemia, Myeloid; Microtubule-Associated Proteins; Neoplasm Proteins; Poly(ADP-ribose) Polymerases; Protein Biosynthesis; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Repressor Proteins; Signal Transduction; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Survivin; Trans-Activators; Transcription Factors; Tumor Cells, Cultured; Tumor Stem Cell Assay; Tyrphostins; X-Linked Inhibitor of Apoptosis Protein

Cells from patients with MDS-derived AML display heterogeneous proliferative responses to transforming growth factor beta (TGF beta). We analyzed growth inhibition and SMAD2 phosphorylation by TGF beta in CD34+ cells from nine patients, as compared to normal controls. While TGF beta consistently inhibited thymidine incorporation of normal cells (41% of control, P < 0.05), cells from patients with AML were growth-inhibited in only four of seven cases (40%), whereas TGF beta stimulated thymidine incorporation in the three other samples (166%). Remarkably, TPO reverted the stimulatory effect of TGF beta to profound growth inhibition. Upon exposure to TGF beta, SMAD2 protein was phosphorylated in normal CD34+ cells (n = 3), CD34+ leukemic blasts from all examined patients with AML (n = 4), and in the myeloid leukemic cell lines M-07e and HEL. TGF beta inhibited TPO-mediated thymidine incorporation, cell proliferation and survival in all samples analyzed. In M-07e cells and CD34+ cells from healthy donors, this inhibition was enhanced by an antagonist of JAK2 (AG490), but not a MEK-1 antagonist (PD098059). Conversely, in CD34+ cells from a patient with AML, both AG490 and PD098059 significantly enhanced TGF beta-mediated suppression of TPO-induced thymidine incorporation. Thus, in MDS-derived AML, altered responses to TGF beta may be due to defects downstream of SMAD2 and may involve MAPK activation.

Topics: Acute Disease; Adult; Antigens, CD34; Cell Division; Disease Progression; DNA Replication; DNA-Binding Proteins; DNA, Neoplasm; Enzyme Inhibitors; Flavonoids; Hematopoietic Stem Cells; Humans; Interleukin-3; Janus Kinase 2; Leukemia, Myeloid; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Myelodysplastic Syndromes; Neoplasm Proteins; Neoplastic Stem Cells; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Reverse Transcriptase Polymerase Chain Reaction; Smad2 Protein; Thrombopoietin; Thymidine; Trans-Activators; Transforming Growth Factor beta; Tyrphostins

Hematopoietic growth factors (HGF) are essential for proliferation and differentiation of hematopoietic precursors and activate a distinct set of JAK-STAT (Janus kinases-signal transducers and activators of transcription) proteins. Previous results from our group have shown a strong expression of JAK-STAT proteins in primary acute myelogenous leukemia (AML) blasts and AML cell lines. Here, we asked whether a constitutive activation of the JAK-STAT pathway might be involved in the pathogenesis of AML. We could demonstrate a constitutive activation of STAT1, 3 and 5 by immunoprecipitation of the tyrosine phosphorylated proteins in different human AML cell lines. Three patterns of STAT activation were found: (I) activation of only STAT1, (II) activation of STAT1 in combination with STAT3, and (III) activation of STAT1, 3 and 5. Furthermore, STAT1 and 3 formed stable heterodimers only in cell lines with constitutive STAT3 activation. In all cell lines analyzed, tyrosine phosphorylation of the four known Janus kinases could not be detected, although JAK1 was stably associated with STAT3. To further analyze whether a constitutive activation of tyrosine kinases might contribute to the autonomous growth of AML blasts, inhibitor studies were performed. The tyrphostin AG490, an inhibitor of the JAK-STAT pathway, but not A1, an inactive tyrphostin induced a time- and dose-dependent growth arrest without overt morphological signs of differentiation in AML cell lines. Our results show that STAT transcription factors are constitutively activated in human AML cell lines and might contribute to the autonomous proliferation of AML blasts. Inhibition of this pathway might be of interest for the establishment of more specific antileukemic strategies.

Topics: Acute Disease; Alternative Splicing; Cell Division; Dimerization; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Leukemic; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; HL-60 Cells; Humans; Interleukin-3; Interleukin-6; Janus Kinase 1; K562 Cells; Leukemia, Myeloid; Milk Proteins; Neoplasm Proteins; Phosphorylation; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Proteins; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; STAT5 Transcription Factor; Trans-Activators; Transcription, Genetic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; TYK2 Kinase; Tyrphostins; U937 Cells