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Adalimumab (ADA) trough level and anti-ADA antibody (AAA) positivity influence mucosal healing and loss of response in patients with inflammatory bowel disease (IBD). In this study, we clarified the correlation between ADA monitoring, including non-trough level, and real-world IBD clinical outcomes. This retrospective, observational, single-center study involved patients with ulcerative colitis (19) and Crohn’s disease (33) treated with ADA from January 2007 to August 2018. Serum ADA and AAA levels were measured 4‒14 days after ADA administration. The AAA positivity rate was 23.1% (12/52). ADA continuity was higher in AAA-negative patients than in AAA-positive patients (P = 0.223). Receiver operating characteristic (ROC) analysis revealed that a serum AAA cut-off of 9.2 μg/mL was associated with ADA continuity. The ADA level was significantly higher in the endoscopic remission group than in the non-remission group (P = 0.02). Based on the ROC curve analysis results of serum ADA level and endoscopic remission, the cut-off value of the serum ADA level was set to 11.1 μg/mL. Under the combined use of ADA with immunomodulators and AAA positivity, ADA continuity was significantly higher when the serum AAA level at 4–14 days after ADA administration was ≥9.2 μg/mL. Furthermore, endoscopic remission can be expected with a serum ADA level of ≥11.1 μg/mL. Overall, to predict clinical outcomes, it would be useful to measure the blood level of ADA regardless of the timing of the trough.

Activation of c-jun N-terminal kinase (JNK) through c-kit-mediated phosphatidylinositol 3 (PI3) and Src kinase pathways plays an important role in cell proliferation and survival in mast cells. Gain-of-function mutations in c-kit are found in several human neoplasms. Constitutive activation of c-kit has been observed in human mastocytosis and gastrointestinal stromal tumor. In the present study, we demonstrate that an anthrapyrazole SP600125, a reversible ATP-competitive inhibitor of JNK inhibits proliferation of human HMC-1 mast cells expressing constitutively activated c-kit catalytic domain mutations. HMC-1 showed constitutive activation of JNK/c-Jun, and the inhibitory effect of SP600125 on cell proliferation was associated with cell cycle arrest at the G 1 phase and apoptosis accompanied by cleavage of caspase-3 and PARP. Caspase-3 inhibitor Z-DEVD-FMK almost completely inhibited SP600125-induced apoptosis of HMC-1 cells. In contrast, caspase-9 inhibitor Z-LEHD-FMK failed to block SP600125-induced apoptosis. Following SP600125 treatment, down-regulation of cyclin D3 protein expression, but not p53 was also observed. Thus, JNK/c-Jun is essential for proliferation and survival of HMC-1 cells. The results obtained from the present study suggest the possibility that JNK/c-Jun may be a therapeutic target in diseases associated with mutations in the catalytic domain of c-kit.

Objective Constitutive activation of nuclear factor (NF)-κB is a common feature of human T-cell leukemia virus type I (HTLV-I)–transformed T cells. Inhibition of NF-κB activity reduces cell growth and induces apoptosis of HTLV-I–transformed T cells, suggesting a central role of NF-κB in their proliferation and survival. In this study, we investigated whether MyD88, an adaptor protein of Toll-like receptor (TLR) signaling, contributes to constitutive NF-κB activation in HTLV-I–transformed T cells. Materials and Methods Activation status of MyD88 and interleukin (IL)-1R–associated kinase 1 (IRAK1) in HTLV-I–transformed human T cells, MT2, MT4, and HUT102 was examined by using Western blot and immunoprecipitation. TLR expression was evaluated with reverse transcription polymerase chain reaction. An expression vector encoding a dominant negative MyD88 with a deletion of its death domain (MyD88dn) was transfected into MT2 cells to evaluate roles of MyD88 in spontaneous activation of cytokine gene promoters and transcription factors, proliferation, and apoptosis in HTLV-I–transformed T cells. Results Constitutive association of MyD88 with IRAK1 was observed in all three of HTLV-I–transformed T cells, but not in HTLV-I–negative T cells, such as Jurkat, HUT78, and MOLT4. MT2 cells showed expression of TLR-1, -6, and -10 mRNAs. Constitutive activation of NF-κB and NF–IL-6 and cytokine gene promoters, such as IL-1α, interferon-γ, and tumor necrosis factor-α in MT2 cells was inhibited by MyD88dn expression. MyD88dn reduced proliferation and induced apoptosis of MT2 cells. HTLV-I Tax enhanced TLR expression and synergistically activated NF-κB with wild-type MyD88. Conclusion Our results show a novel pathway in NF-κB activation in HTLV-I–transformed T cells and further demonstrate a critical role of MyD88 in their dysregulated gene activation, survival, and proliferation.

Galectins are a family member of animal lectins that function both extracellularly (by interacting with cell surface and extracellular matrix glycoproteins and glycolipids) and intracellularly (by interacting with cytoplasmic and nuclear proteins) to modulate signaling pathways. To date, 15 galectins have been cloned in mammals. The members can be classified into 3 subtypes according to their structure. The proto type and chimera type galectins have a single CRD. The tandem-repeat type galectins have two CRDs. In this regard, galectin-9 is a tandem repeat type galectin, which has been identified as a tumor antigen of unknown functions in patients with Hodgkin lymphoma. Galectin-9 has recently demonstrated to induce chemotaxis of eosinophils and apoptosis in a variety of cells, resulting in immunomodulation, inflammation and anti-metastatic potential of tumor cells. However, the role of galectin-9 in the inflammatory responses still remains unclear. In the present study, we investigated whether intracellular galectin-9 contributes to activation of the inflammatory cytokine genes such as IL-1α, IL-1β and IFNγin human monocytes. Protein expression of galectin-9 in human THP-1 monocytic cells was examined in western blot with anti-galectin-9 Ab and immunostaining studies using a confocal microscopy. A galectin-9 expression vector pBKCMV3-Galectin-9 was transiently cotransfected into THP-1 cells along with luciferase reporter plasmids for IL-1α, IL-1β and IFNγgene promoters. Galectin-9 was detected in the cytoplasm of untreated THP-1 cells, and the expression levels of galectin-9 protein was significantly enhanced in THP-1 cells treated with lipopolysaccharide (LPS). In transient transfection studies, overexpression of galectin-9 dose-dependently activated IL-1α, IL-1β and IFNγgene promoters in THP-1 cells, showing that intracellular galectin-9 has capacity to induce the inflammatory cytokine genes in monocytes. This argument was further supported by our reverse transcription-polymerase chain reaction (RT-PCR) data that galectin-9 overexpression enhanced mRNA levels of IL-1α and β in THP-1 cells. When luciferase reporter plasmids for transcription factors including NF-IL6, NF-κB and AP-1 were transiently cotransfected into THP-1 cells along with pBKCMV3-Galectin-9, NF-IL6 and AP-1 activities were induced by galectin-9 overexpression. Our coimmunoprecipitation study, using anti-galectin-9 Ab and anti-NF-IL6 Ab revealed that intracellular galectin-9 was physically associated with NF-IL6. On the other hand, galectin-9 failed to induce NF-κB activity. Additionally, in contrast to the data obtained from transient transfection studies using pBKCMV3-Galectin-9, no significant induction of IL-1α promoter activity was observed by adding galectin-9 protein to THP-1 cell culture medium, indicating that extracellular galectin-9 dose not affect the inflammatory cytokine gene regulation. Our results strongly suggested that intracellular galectin-9 has the capability to transactivate the inflammatory cytokine genes through the physical interaction with leucin zipper transcription factors such as NF-IL6 in monocytes.

Activation of c-jun N-terminal kinase (JNK) through c-kit-mediated phosphatidylinositol 3 (PI3) and Src kinase pathways plays an important role in cell proliferation and survival in mast cells. Gain-of-function mutations in c-kit are found in several human neoplasms. Constitutive activation of c-kit has been observed in human mastocytosis, acute myeloid leukemia, lymphoma, germ tumor and gastrointestinal stromal tumor. In the present study, we demonstrate that an anthrapyrazole SP600125, a reversible ATP-competitive inhibitor of JNK inhibits proliferation of human HMC-1 mast cells expressing constitutively activated c-kit mutant. We found that JNK/c-Jun was constitutively activated in HMC-1 cells without stimulation. When spontaneous activation of JNK/c-Jun was inhibited by treatment with SP600125, cell proliferation was suppressed. The concentration which effectively inhibited JNK/c-Jun activity in our experiment had no effect on SCF-induced phosphorylation of Akt or Erk, suggesting that SP600125 specifically inhibited JNK/c-Jun activity in HMC-1 cells. Moreover, we demonstrated that SP600125 induced HMC-1 cell apoptosis in dose- and time-dependent manner. Caspase-3 and PARP were cleaved as early as 12 h after treatment with SP600125, but caspase-9 was not. Also, cell cycle arrest in G1 phase was observed in SP600125 treated cells. Thus, the inhibitory effect of SP600125 on cell proliferation was associated with cell cycle arrest at the G1 phase and apoptosis accompanied by cleavage of caspase-3 and PARP. Caspase-3 inhibitor Z-DEVD-FMK almost completely inhibited SP600125-induced apoptosis of HMC-1 cells. In contrast, caspase-9 inhibitor Z-LEHD-FMK failed to block SP600125-induced apoptosis, suggesting that apoptosis induced by SP600125 was caspase-3 dependent. Following SP600125 treatment, down-regulation of cyclin D3 protein expression, but not p53 was also observed. Take together, JNK/c-Jun is essential for proliferation and survival of HMC-1 cells. The results obtained from the present study suggest the possibility that JNK/c-Jun may be a therapeutic target in diseases associated with c-kit mutant.

Signal transducer and activator of transcription (STAT) proteins are activated in response to cytokine stimulation. STAT3 is activated in response to interleukin (IL)-6 family of cytokines, following activation of Janus family of tyrosine kinases JAK1 and JAK2, which in turn phosphorylate STAT3 on tyrosine 705 (tyr-705). On the other hand, STAT4 is activated in response to IL-12 following activation of Janus family of tyrosine kinases JAK2 and Tyk2, which in turn phosphorylate STAT4 on tyrosine 693 (tyr-693). p38/MKK6 signaling pathway activated by IL-12 further phosphorylates STAT4 on serine 721 (ser-721). STAT4 plays a crucial role in the development of type-1 helper T (Th1) cells and production of interferon (IFN)-γ in response to IL-12. STAT4 is activated by IFN-α as well as IL-12. Several STAT heterodimer complexes have been demonstrated. Selective interactions of individual STATs upon complex formation might contribute to the transcriptional target specificity of cytokine and growth factor signaling. In the present study, we demonstrated that STAT4 forms a heterodimer with STAT3 to synergistically transactivate the high-affinity sis-inducible element (hSIE). A wild-type STAT3 expression vector Rc/CMV-STAT3WT and/or a wild-type STAT4 expression vector Rc/CMV-STAT4WT were cotransfected along with a hSIE reporter pGLmfoshSIE into Hep3B cells, and cells were treated with IL-6 and/or IFN-α. In the absence of overexpression of STAT3 and STAT4, little increase in hSIE activities was observed following treatment with the cytokines, However, when Rc/CMV-STAT3WT and -STAT4WT were cotransfected, transcriptional activities were synergistically enhanced following simultaneous stimulation with IL-6 and IFN-α. γ-interferon activated site (GAS) activity was not enhanced by the two STATs. No synergistic activation of hSIE was detected between STAT1 and STAT4, when a wild-type STAT1 expression vector was used instead of Rc/CMV-STAT3WT. Our mutational analyses further revealed that the synergy between STAT3 and STAT4 was almost completely abrogated by either mutation of STAT4 at tyr-693 or STAT3 at tyr-705, suggesting direct interaction of STAT3 with STAT4 as well as the importance of STAT tyrosine phosphorylation. However, mutation of STAT4 at ser-721 caused only a little suppression of the synergy. Similar results were obtained from mutation of STAT3 at ser-727. Our immunoprecipitation (IP)/western blot (WB) analyses exhibited that STAT4 forms a heterodimer with STAT3 in response to stimulation with IL-6 and IFN-α. Moreover, in electrophoretic mobility shift assay (EMSA), hSIE preferentially bound STAT3/STAT4 heterodimer. Based upon our results, STAT3 and STAT4 can form a heterodimer, which exhibits significantly different transcripitional activities and sequence preferences to bind its target DNA. Thus, the specificity in STAT signaling is at least partly dependent on selective interactions between individual STATs upon complex formation. STAT3/STAT4 heterodimer may play an important role in immune and inflammatory reactions in NK and T-cells in response to IL-12 and IFN-α.

Human T-cell leukemia virus type I (HTLV-I) is etiologically associated with the development of an aggressive and fatal malignancy of CD4+ T lymphocytes called adult T-cell leukemia (ATL). Constitutive activation of nuclear factor-κB (NF-κB) is a common feature of ATL. Although the mechanism by which NF-κB is spontaneously activated in ATL cells still remains unclear, inhibition of NF-κB activity induces apoptosis, suggesting a central role of NF-κB in their proliferation. Toll like receptors (TLRs) are involved in innate cell activation by conserved structures expressed by microorganisms. Engagement of IL-1R or TLR with their cognate ligands causes an adaptor protein MyD88 to be recruited to the receptor complex, which in turn promotes its association with the IL-1R-associated kinase (IRAK) via an interaction between the respective death domains of each molecule. Several recent reports have indicated unique expression profiles of TLRs on different subsets of human T cells, and that some TLR ligands modulate the function of human T cells. We examined expression of the TLR mRNAs in primary ATL cells and ATL cell lines, MT2, MT4 and HUT102 by RT-PCR. Expression of TLR mRNAs, except of TLR7 and TLR8, was detected in all cell samples examined. We further demonstrated constitutive association of MyD88, an adaptor protein for the TLR signaling, with the IL-1R-associated kinase 1 (IRAK1) in ATL cell lines, MT2, MT4 and HUT102. In MT2 cells, constitutive activation of NF-κB and NF-IL6, but not Stat3 was significantly inhibited by expression of a dominant negative form of MyD88 protein (MyD88dn). Spontaneous transcriptional activation of IL-1α, IFN-γ and TNF-α gene promoters in MT2 cells was also suppressed by MyD88dn expression. MyD88dn inhibited cell proliferation and induced apoptosis of MT2 cells. In addition, overexpression of wild-type MyD88 and HTLV-I Tax induces synergistically transcriptional activity of NF-κB in 293T cells, showing interaction of Tax with MyD88. Thus, our results show a critical role of MyD88 in dysregulated gene activation and cell proliferation in HTLV-I-transformed T-cells, and further suggest the involvement of MyD88 in Tax-mediated intracellular signal transduction in HTLV-I-infected cells. Considering the fact that blocking NF-κB is a potential strategy to treat ATL, our argument raises a possibility that we may be able to find new treatment targets against ATL.