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Innate and adaptive resistance to cancer therapies, such as chemotherapies, molecularly targeted therapies, and immune-modulating therapies, is a major issue in clinical practice. Subpopulations of tumor cells expressing the receptor tyrosine kinase AXL become enriched after treatment with antimitotic drugs, causing tumor relapse. Elevated AXL expression is closely associated with drug resistance in clinical samples, suggesting that AXL plays a pivotal role in drug resistance. Although several molecules with AXL inhibitory activity have been developed, none have sufficient activity and selectivity to be clinically effective when administered in combination with a cancer therapy. Here, we report a novel small molecule, ER-851, which is a potent and highly selective AXL inhibitor. To investigate resistance mechanisms and identify driving molecules, we conducted a comprehensive gene expression analysis of chemoresistant tumor cells in mouse xenograft models of genetically engineered human lung cancer and human triple-negative breast cancer. Consistent with the effect of AXL knockdown, cotreatment of ER-851 and antimitotic drugs produced an antitumor effect and prolonged relapse-free survival in the mouse xenograft model of human triple-negative breast cancer. Importantly, when orally administered to BALB/c mice, this compound did not induce retinal toxicity, a known side effect of chronic MER inhibition. Together, these data strongly suggest that AXL is a therapeutic target for overcoming drug resistance and that ER-851 is a promising candidate therapeutic agent for use against AXL-expressing antimitotic-resistant tumors.

Supplementary Figure S1-S7

Supplementary materials and methods

Combination therapies consisting of immune checkpoint inhibitors plus anti-VEGF therapy show enhanced antitumor activity and are approved treatments for patients with renal cell carcinoma (RCC). The immunosuppressive roles of VEGF in the tumor microenvironment are well studied, but those of FGF/FGFR signaling remain largely unknown. Lenvatinib is a receptor tyrosine kinase inhibitor that targets both VEGFR and FGFR. Here, we examine the antitumor activity of anti-PD-1 mAb combined with either lenvatinib or axitinib, a VEGFR-selective inhibitor, in RCC. Both combination treatments showed greater antitumor activity and longer survival in mouse models versus either single agent treatment, whereas anti-PD-1 mAb plus lenvatinib had enhanced antitumor activity compared with anti-PD-1 mAb plus axitinib. Flow cytometry analysis showed that lenvatinib decreased the population of tumor-associated macrophages and increased that of IFNγ-positive CD8+ T cells. Activation of FGFR signaling inhibited the IFNγ-stimulated JAK/STAT signaling pathway and decreased expression of its target genes, including B2M, CXCL10, and PD-L1. Furthermore, inhibition of FGFR signaling by lenvatinib restored the tumor response to IFNγ stimulation in mouse and human RCC cell lines. These preclinical results reveal novel roles of tumor FGFR signaling in the regulation of cancer immunity through inhibition of the IFNγ pathway, and the inhibitory activity of lenvatinib against FGFRs likely contributes to the enhanced antitumor activity of combination treatment comprising lenvatinib plus anti-PD-1 mAb.Significance:FGFR pathway activation inhibits IFNγ signaling in tumor cells, and FGFR inhibition with lenvatinib enhances antitumor immunity and the activity of anti-PD-1 antibodies.

Supplementary Data Figure S1-S7

Supplementary Table S1

Innate and adaptive resistance to cancer therapies, such as chemotherapies, molecularly targeted therapies, and immune-modulating therapies, is a major issue in clinical practice. Subpopulations of tumor cells expressing the receptor tyrosine kinase AXL become enriched after treatment with anti-mitotic drugs, causing tumor relapse. Elevated AXL expression is closely associated with drug resistance in clinical samples, suggesting that AXL plays a pivotal role in drug resistance. Although several molecules with AXL inhibitory activity have been developed, none have sufficient activity and selectivity to be clinically effective when administered in combination with a cancer therapy. Here, we report a novel small molecule, ER-001259851-000, which is a potent and highly selective AXL inhibitor. To investigate resistance mechanisms and identify driving molecules, we conducted a comprehensive gene expression analysis of chemo-resistant tumor cells in mouse xenograft models of genetically engineered human lung cancer and human triple-negative breast cancer. Consistent with the effect of AXL knockdown, co-treatment of ER-001259851-000 and antimitotic drugs produced an anti-tumor effect and prolonged relapse-free survival in the mouse xenograft model of human triple-negative breast cancer. Importantly, when orally administered to BALB/c mice, this compound did not induce retinal toxicity, a known side effect of chronic MER inhibition. Together, these data strongly suggest that AXL is a therapeutic target for overcoming drug resistance and that ER-001259851-000 is a promising candidate therapeutic agent for use against AXL-expressing anti-mitotic-resistant tumors.

T cell invigoration is an essential step for eliminating pathogens and cancer cells. Co-stimulatory molecules, such as CD80, CD86, and ICOSLG reinforce TCR stimuli for T cell activations. Despite identifying multiple co-stimulatory molecules, the differences of those in downstream signaling have remained unclear. Here, we unravel the differences in avidity of co-stimulatory molecules with T cells cause distinct T cell fates. Specially, CD80 + TCR stimulus promotes induction of multiple T cell effector genes based on prolonged and magnified activation of ERK and AKT compared with other combined stimuli. Long-term and robust activation of these signaling pathways leads to T cell impairment by induction of PD1, and exhausted T cells are vulnerable to disrupt effector functions by interactions with PDL1. Collectively, we reveal the quantitative differences in binding activities of co-stimulatory molecules to T cells cause qualitative differences in downstream signals and gene expressions, thereby branching T cell fates.

Axl and Mer are members of the TAM (Tyro3-Axl-Mer) family of receptor tyrosine kinases. Previously, we reported that enzyme-mediated inhibition of Mer by an Axl/Mer dual inhibitor led to retinal toxicity in mice, whereas selective Axl inhibition by compound 1 did not. On the other hand, compound 1 showed low membrane permeability. Here, we designed and synthesized a novel series of 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine derivatives and evaluated their Axl and Mer inhibitory activities, leading to identification of ER-001259851-000 as a potent and selective Axl inhibitor with drug-likeness and a promising pharmacokinetic profile in mice.

Angiogenesis inhibitors such as lenvatinib and sorafenib, and an immune checkpoint inhibitor (ICI), nivolumab, are used for anticancer therapies against advanced hepatocellular carcinoma (HCC). Combination treatments comprising angiogenesis inhibitors plus ICIs are promising options for improving clinical benefits in HCC patients, and clinical trials are ongoing. Here, we investigated the antitumor and immunomodulatory activities of lenvatinib (a multiple receptor tyrosine kinase inhibitor targeting vascular endothelial growth factor receptor 1‐3, fibroblast growth factor receptor 1‐4, platelet‐derived growth factor receptor α, KIT and RET) and the combined antitumor activity of lenvatinib plus anti‐programmed cell death 1 (PD‐1) antibody in the Hepa1‐6 mouse HCC syngeneic model. We found that the antitumor activities of lenvatinib and sorafenib were not different in immunodeficient mice, but lenvatinib showed more potent antitumor activity than sorafenib in immunocompetent mice. The antitumor activity of lenvatinib was greater in immunocompetent mice than in immunodeficient mice and was attenuated by CD8+ T cell depletion. Treatment with lenvatinib plus anti‐PD‐1 antibody resulted in more tumor regression and a higher response rate compared with either treatment alone in immunocompetent mice. Single‐cell RNA sequencing analysis demonstrated that treatment with lenvatinib with or without anti‐PD‐1 antibody decreased the proportion of monocytes and macrophages population and increased that of CD8+ T cell populations. These data suggest that lenvatinib has immunomodulatory activity that contributes to the antitumor activity of lenvatinib and enhances the antitumor activity in combination treatment with anti‐PD‐1 antibody. Combination treatment of lenvatinib plus anti‐PD‐1 antibody therefore warrants further investigation against advanced HCC.

Axl and Mer are a members of the TAM (Tyro3-Axl-Mer) family of receptor tyrosine kinases, which, when activated, can promote tumor cell survival, proliferation, migration, invasion, angiogenesis, and tumor-host interactions. Chronic inhibition of Mer leads to retinal toxicity in mice. Therefore, successful development of an Axl targeting agent requires ensuring that it is safe for prolonged treatment. Here, to clarify whether enzyme inhibition of Mer by a small molecule leads to retinal toxicity in mice, we designed and synthesized Axl/Mer inhibitors and Axl-selective inhibitors. We identified an Axl/Mer dual inhibitor 28a, which showed retinal toxicity at a dose of 100 mg/kg in mice. Subsequent derivatization of a pyridine derivative led to the discovery of a pyrimidine derivative, 33g, which selectively inhibited the activity of Axl over Mer without retinal toxicity at a dose of 100 mg/kg in mice. Additionally, the compound displayed in vivo anti-tumor effects without influencing body weight in a Ba/F3-Axl isogenic subcutaneous model.

Combination therapies consisting of immune checkpoint inhibitors plus anti-VEGF therapy show enhanced antitumor activity and are approved treatments for patients with renal cell carcinoma (RCC). The immunosuppressive roles of VEGF in the tumor microenvironment are well studied, but those of FGF/FGFR signaling remain largely unknown. Lenvatinib is a receptor tyrosine kinase inhibitor that targets both VEGFR and FGFR. Here, we examine the antitumor activity of anti-PD-1 mAb combined with either lenvatinib or axitinib, a VEGFR-selective inhibitor, in RCC. Both combination treatments showed greater antitumor activity and longer survival in mouse models versus either single agent treatment, whereas anti-PD-1 mAb plus lenvatinib had enhanced antitumor activity compared with anti-PD-1 mAb plus axitinib. Flow cytometry analysis showed that lenvatinib decreased the population of tumor-associated macrophages and increased that of IFNγ-positive CD8+ T cells. Activation of FGFR signaling inhibited the IFNγ-stimulated JAK/STAT signaling pathway and decreased expression of its target genes, including B2M, CXCL10, and PD-L1. Furthermore, inhibition of FGFR signaling by lenvatinib restored the tumor response to IFNγ stimulation in mouse and human RCC cell lines. These preclinical results reveal novel roles of tumor FGFR signaling in the regulation of cancer immunity through inhibition of the IFNγ pathway, and the inhibitory activity of lenvatinib against FGFRs likely contributes to the enhanced antitumor activity of combination treatment comprising lenvatinib plus anti-PD-1 mAb. Significance: FGFR pathway activation inhibits IFNγ signaling in tumor cells, and FGFR inhibition with lenvatinib enhances antitumor immunity and the activity of anti-PD-1 antibodies.

Background: Aberrant activation of Wnt/β-catenin signaling plays an important role in both carcinogenesis and modulation of the tumor microenvironment. E7386, an investigational anti-cancer agent, inhibits the binding of β-catenin to its transcriptional co-activator, CREB binding protein (CBP), thereby modulating Wnt/β-catenin signaling. We previously reported that E7386 suppressed tumor growth in preclinical mouse models with aberrant activation of Wnt/β-catenin signaling. Moreover, the combination of E7386 plus lenvatinib, a multiple receptor tyrosine kinase inhibitor that mainly targets vascular endothelial growth factor receptors and fibroblast growth factor receptors, showed stronger antitumor and antiangiogenic activities than each treatment alone. Here, we investigated the molecular mechanism of the combination activities of E7386 plus lenvatinib. Methods: We examined antitumor activity of E7386 plus lenvatinib in multiple human tumor xenograft and mouse syngeneic models. Tumor samples underwent RNA sequencing (RNA-seq) followed by weighted gene correlation network analysis. Furthermore quantitative PCR was used to assess changes in the expression of some hypoxia-related genes. The protein interaction between β-catenin and HIF1 was analyzed by immunoprecipitation-western blotting. Results: RNA-seq analysis of the effects of E7386, lenvatinib, and E7386 plus lenvatinib on the tumor microenvironment in the Hepa1-6 hepatocellular carcinoma syngeneic tumor model in mice showed that lenvatinib suppressed the expression of angiogenesis-related genes and that E7386 plus lenvatinib enhanced this suppression. Although lenvatinib increased the expression of hypoxia-related genes, E7386 plus lenvatinib attenuated this phenomenon. According to these results, we hypothesized that E7386 inhibits the formation of the β-catenin/HIF-1 complex. To confirm the effect of E7386 on the hypoxic response, we conducted an in vitro quantitative PCR assay under hypoxic conditions in Hepa1-6 cells and the human hepatocellular carcinoma cell lines, HepG2 and Hep3B. In accordance with the RNA-seq result, E7386 significantly suppressed the expression levels of hypoxia-related genes in the HepG2 and Hep3B cells. Finally, data from immunoprecipitation-western blotting demonstrated that β-catenin formed a complex with HIF-1 and that E7386 clearly inhibited this complex formation in HepG2 cells. Conclusion: These results suggest that E7386 enhances angiogenesis inhibition when used in combination with lenvatinib and that E7386 suppresses the hypoxic response induced by angiogenesis inhibition. Further analysis is required to clarify the precise mechanisms of actions of the combination of E7386 plus lenvatinib. Citation Format: Mizuki Kimura, Yusaku Hori, Megumi Kuronishi, Takayuki Kimura, Ryoga Ishida, Kenji Ichikawa, Yoichi Ozawa, Yu Kato. E7386, a CREB binding protein (CBP)/β-catenin interaction inhibitor, suppresses the hypoxic response induced by angiogenesis inhibition in hepatocellular carcinoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1437.

Anti–vascular endothelial growth factor (VEGF) therapy shows antitumor activity against various types of solid cancers. Several resistance mechanisms against anti-VEGF therapy have been elucidated; however, little is known about the mechanisms by which the acquired resistance arises. Here, we developed new anti-VEGF therapy–resistant models driven by chronic expression of the mouse VEGFR2 extracellular domain fused with the human IgG4 fragment crystallizable (Fc) region (VEGFR2-Fc). In the VEGFR2-Fc–expressing resistant tumors, we demonstrated that the FGFR2 signaling pathway was activated, and pericytes expressing high levels of FGF2 were co-localized with endothelial cells. Lenvatinib, a multiple tyrosine kinase inhibitor including VEGFR and FGFR inhibition, showed marked antitumor activity against VEGFR2-Fc–expressing resistant tumors accompanied with a decrease in the area of tumor vessels and suppression of phospho-FGFR2 in tumors. Our findings reveal the key role that intercellular FGF2 signaling between pericytes and endothelial cells plays in maintaining the tumor vasculature in anti-VEGF therapy–resistant tumors.

The ERK pathway not only upregulates growth-promoting genes, but also downregulates anti-proliferative and tumor-suppressive genes. In particular, ERK signaling contributes to repression of the E-cadherin gene during epithelial-mesenchymal transition (EMT). The CtBP transcriptional co-repressor is also involved in gene silencing of E-cadherin. However, the functional relationship between ERK signaling and CtBP is unknown. Here, we identified an ERK substrate, designated MCRIP1, which bridges ERK signaling and CtBP-mediated gene silencing. CtBP is recruited to promoter elements of target genes by interacting with the DNA-binding transcriptional repressor ZEB1. We found that MCRIP1 binds to CtBP, thereby competitively inhibiting CtBP-ZEB1 interaction. When phosphorylated by ERK, MCRIP1 dissociates from CtBP, allowing CtBP to interact with ZEB1. In this manner, the CtBP co-repressor complex is recruited to, and silences, the E-cadherin promoter by inducing chromatin modifications. Our findings reveal a molecular mechanism underlying ERK-induced epigenetic gene silencing during EMT and its dysregulation in cancer.

Activating transcription factor 5 (ATF5) is a stress-response transcription factor that responds to amino acid limitation and exposure to cadmium chloride (CdCl2) and sodium arsenite (NaAsO2). The N-terminal amino acids contribute to the destabilization of the ATF5 protein in steady-state conditions and serve as a stabilization domain in the stress response after CdCl2 or NaAsO2 exposure. In this study, we show that interleukin 1β (IL-1β), a proinflammatory cytokine, increases the expression of ATF5 protein in HepG2 hepatoma cells in part by stabilizing the ATF5 protein. The N-terminal domain rich in hydrophobic amino acids that is predicted to form a hydrophobic network was responsible for destabilization in steady-state conditions and served as an IL-1β response domain. Furthermore, IL-1β increased the translational efficiency of ATF5 mRNA via the 5' UTRα and phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α). ATF5 knockdown in HepG2 cells up-regulated the IL-1β-induced expression of the serum amyloid A 1 (SAA1) and SAA2 genes. Our results show that the N-terminal hydrophobic amino acids play an important role in the regulation of ATF5 protein expression in IL-1β-mediated immune response and that ATF5 is a negative regulator for IL-1β-induced expression of SAA1 and SAA2 in HepG2 cells.

Lenvatinib mesilate (LEN) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that selectively inhibits the kinase activities of VEGFR1-3, in addition to other proangiogenic and oncogenic pathway-related RTKs including FGFR1-4; PDGFRα; KIT; and RET. LEN plus everolimus for advanced renal cell carcinoma (RCC) after one prior anti-VEGF therapy was approved in the US and EU in 2016. Currently, Phase1b/2 clinical trial of LEN in combination with pembrolizumab with selected solid tumors including RCC and Phase3 study of LEN in combination with everolimus or pembrolizumab for metastatic RCC patients are in progress. In this study, we investigated the antitumor and immunomodulatory activities of LEN alone and in combination with anti-PD1 Ab and explored the mechanism of action underlying the activities of LEN and PD1 blockade in preclinical syngeneic RCC models. We examined antitumor activity of combination treatment of LEN at 10 mg/kg (po, qd) and anti-PD1 Ab at 10 mg/kg (ip, twice weekly) in the murine subcutaneous RCC model using RAG cell line. Immune cell population analyses in tumor tissues were performed by flow cytometer. Immune-related gene expression profile in tumor tissues or cultured cells was analyzed by qPCR and RNA seq. Effects on FGFR and IFN gamma (IFNγ) signaling pathways and their downstream molecules in cultured cells were analyzed by western blot. In the murine syngeneic RAG tumor model, the combination treatment of LEN and anti-PD1 Ab showed tumor shrinkage, and some of tumors were regressed to nonpalpable sizes with long duration of response. LEN alone and in combination with anti-PD1 Ab significantly decreased the population of tumor associated macrophage in tumor tissues by FCM analysis. Gene expression analysis for immune cell responses in tumor tissues revealed the upregulation of IFNγ and its downstream genes including PD-L1 with combination of LEN and anti-PD1 Ab. Western blot analysis elucidated that FGFR signaling pathway inhibited the activation of IFNγ signaling pathway and the induction of PD-L1 expression in murine RCC cells, and LEN-treatment reactivated the IFNγ signaling pathway by inhibition of FGFR signaling. Combination of LEN and anti-PD1 Ab showed greater antitumor activity including tumor shrinkage with long duration of response in the murine RCC model. Modulation of immune microenvironment by LEN and combination of LEN with PD1 Ab may underlie the strong antitumor activity in preclinical RCC model. Inhibitory activity of LEN against FGFR reactivated the IFNγ pathway and PD-L1 expression suppressed by activation of FGFR signaling pathway, and this inhibitory activity of LEN may potentiate the antitumor activity of combination treatments with anti-PD1 Ab. These preclinical results provide one of the unique mechanisms of combinational effect of LEN with PD1 blockade in the preclinical RCC model. Citation Format: Yusuke Adachi, Kenji Ichikawa, Yu Kato, Yasuhiro Funahashi. Roles of FGFR inhibition on murine RCC cells in combination antitumor activity of lenvatinib with anti-PD1 antibody via regulatingIFN gamma signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2242.

Anti-vascular endothelial growth factor (VEGF) therapies have been in clinical use to treat patients with multiple types of cancers, but benefits of anti-VEGF therapy are still limited because of the acquired resistance to the treatment. In this study, we developed a novel resistance model to anti-VEGF therapy by expressing VEGFR2-Fc in mouse Renca and B16F10 tumor models. We investigated activated signaling pathways in established resistant tumors by differentially expressed gene analysis and using pathway analysis using Reactome based on RNA-Seq data. Differentially expressed gene analysis identified 293 genes, which are altered commonly in both resistant tumors, and pathway analysis showed that those genes were enriched in FGF2-FGFR2 signaling pathway. IHC analysis by staining with anti-CD31 and αSMA antibodies demonstrated that tumor vessels in VEGFR2-Fc expressing resistant tumors were covered with pericytes, and further IHC analysis with anti-FGF2 antibody indicated that FGF2 was stained in pericytes, which covered endothelial cells in VEGFR2-Fc expressing tumor. Moreover, we isolated CD31 positive cells as endothelial cells, CD90.2 positive cells as pericytes, and CD45 positive cells as tumor infiltrated lymphocytes, respectively. Expression level of Fgf2 and Fgfr2 mRNA in these subsets from Mock tumors and VEGFR2-Fc expressing tumors were analyzed. As a result, Fgf2 and Fgfr2 mRNA expressions in VEGFR2-Fc expressing tumors were upregulated in pericytes and endothelial cells, respectively compared with Mock tumors. These results suggested that there is a spatially effective activation of FGF signaling pathway between endothelial cells and pericytes in tumor vessels that are resistant to anti-VEGF therapy. Indeed, by expression of FGFR2-Fc, in vivo tumor growth of anti-VEGF therapy-resistant tumors was suppressed as well as angiogenesis. Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that suppresses the kinase activities of VEGF receptors (VEGFR1-3), in addition to other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor receptors (FGFR1-4), the platelet-derived growth factor receptor (PDGFR) α, KIT, and RET. Lenvatinib suppressed phosphorylation of FGFR2 in VEGFR2-Fc expressing tumors, and showed antitumor activity accompanied with further decrease of tumor vessels. But, sorafenib and aflibercept, which are selective inhibitors against VEGF signaling pathway, didn’t show antitumor activity against anti-VEGF therapy resistant tumors. In conclusion, our findings revealed key roles of pericytes expressing FGF2 in anti-VEGF therapy acquired resistant tumor models, where lenvatinib showed antitumor activity, indicating an importance of inhibition of FGF signaling pathway to suppress escape from anti-VEGF therapy. Citation Format: Kenji Ichikawa, Saori Watanabe Miyano, Yukinori Minoshima, Junji Matsui, Yasuhiro Funahashi. Antitumor activity of lenvatinib against acquired resistance tumor to anti-VEGF therapy in mouse syngeneic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 196.

A prospective study was made to seek for a convenient biomarker to predict progression of bone destruction (PBD) in early stages of rheumatoid arthritis (ERA). All participated patients had definite RA and their radiographic stages were mild less than stage II of the Steinbrocker classification, naïve for treatment of any DMARDs or corticosteroids. After the entry, they were treated according to the 2002 ACR management guideline for RA. The candidate biomarkers (RF-IgM, RF-IgG, CARF, ACPA, CRP, ESR, NTx, MMP-3, IL-6 and osteopontin) were measured at the entry. PBD was assessed radiographically by interval changes in the modified Sharp scores (ΔSHS) for 24 months. The associations between ΔSHS and baseline biomarkers were assessed statistically by multivariate regression analyses. Both the baseline ACPA and IL-6 levels correlated with PBD, suggesting that they could predict PBD in ERA.

In this study, we enrolled early rheumatoid arthritis (RA) patients at multiple institutes who fulfilled the American Rheumatism Association 1987 revised criteria for the classification of RA, and followed the clinical results of disease-modifying anti-rheumatic drug (DMARD) treatment prospectively. With the aim of developing therapeutic guidelines using the disease activity score 28 (DAS28) as disease indices, we investigated the usefulness of bucillamine (BUC), one of the most widely used DMARDs in Japan. Eighty-one patients with early RA who had not previously been treated with DMARDs were suitable for BUC therapy as first-choice treatment. After 24 months of treatment, at least moderate improvement was seen in 87.5% of patients using the DAS28 erythrocyte sedimentation rate (ESR). After 24 months of BUC therapy, 7 patients (43.8%) met the remission criterion of DAS28 (ESR)

Anti-vascular endothelial growth factor (VEGF) therapies have been in clinical use to treat patients with multiple types of cancers, especially in renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). However, benefits of anti-VEGF therapies are still limited because of the intrinsic/acquired resistance to the treatment. Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that suppresses the kinase activities of VEGF receptors (VEGFR1-3), in addition to other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor receptors (FGFR1-4), the platelet-derived growth factor receptor (PDGFR) α, KIT, and RET. Lenvatinib has been approved as a monotherapy for the treatment of radioiodine-refractory differentiated thyroid cancer and in combination with everolimus for the treatment of patients with RCC treated with one prior anti-VEGF therapy in the US and EU. In this study, we established an anti-VEGF treatment resistant preclinical mouse tumor model. We also examined whether lenvatinib, which targeted multiple receptor tyrosine kinases, showed anti-tumor activity in this model. To establish model resistant to anti-VEGF treatment, an expression vector encoding VEGFR-Fc protein (VEGF decoy) was introduced into a mouse RCC cell line RENCA. First, we confirmed the RENCAVEGFR-Fc transfectants secreted VEGF decoy, which selectively interacted with recombinant VEGF protein but not with ligands to other RTKs in in vitro. Tumor growth in vivo of RENCAVEGFR-Fc was significantly slower than that of RENCAMock. RENCAMock transfectants took 16 days to reach a mean tumor volume (TV) of 500 mm3 whereas RENCAVEGFR-Fc transfectants required 72 days to reach same TV. Furthermore, 72 days after inoculation of RENCAVEGFR-Fc transfectants, we resected tumor tissues and confirmed the expression of VEGF decoy protein. Tumor microvessel density was decreased in resected RENCAVEGFR-Fc tumor compared to the same size of RENCAMock tumor. From the above results, we characterized RENCAVEGFR-Fc tumor was resistant to VEGF decoy. Finally, anti-tumor activity of lenvatinib was challenged against RENCAMock or RENCAVEGFR-Fc tumors. When tumor volume reached approximately 300mm3, 10 mg/kg of lenvatinib was orally administered once daily for 14 days. Treatment of lenvatinib led to almost complete inhibition of tumor growth in both RENCAMock and RENCAVEGFR-Fc models. In conclusion, we developed a mouse RENCAVEGFR-Fc model resistant to anti-VEGF treatment, and lenvatinib showed anti-tumor activity in the resistant model as with RENCAMock model. Additional preclinical studies will be investigated to elucidate the signaling pathways associated with resistance to anti-VEGF treatment, in which lenvatinib may inhibit such as FGFR1-4, PDGFRα, KIT and RET. Citation Format: Kenji Ichikawa, Saori Watanabe Miyano, Yukinori Minoshima, Junji Matsui. Antitumor activity of lenvatinib in the renal cell carcinoma cell line RENCA model resistant to a VEGF specific inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 11.

Activating transcription factor (ATF) 5 is a transcription factor belonging to the ATF/cAMP-response element-binding protein gene family. We previously reported that ATF5 mRNA expression increased in response to amino acid limitation. The ATF5 gene allows transcription of mRNAs with at least two alternative 5'-untranslated regions (5'-UTRs), 5'-UTRalpha and 5'-UTRbeta, derived from exon1alpha and exon1beta. 5'-UTRalpha contains highly conserved sequences, in which the upstream open reading frames (uORFs) uORF1 and uORF2 are found in many species. This study was designed to investigate the potential role of 5'-UTRs in translational control. These 5'-UTRs differentially determined translation efficiency from mRNA. The presence of 5'-UTRalpha or 5'-UTRbeta represses translation from the downstream ATF5 ORF. Moreover, 5'-UTRalpha-regulated translational repression is released by amino acid limitation or NaAsO(2) exposure. This release was not seen for 5'-UTRbeta. Mutation of uAUG2 in the uORF2 of 5'-UTRalpha restored the basal expression and abolished the positive regulation by amino acid limitation or arsenite exposure. We demonstrated that phosphorylation of eukaryotic initiation factor 2alpha was required for amino acid limitation-induced translational regulation of ATF5. Furthermore, arsenite exposure activated the exogenously expressed heme-regulated inhibitor kinase and induced the phosphorylation of eukaryotic initiation factor 2alpha in nonerythroid cells. These results suggest that translation of ATF5 is regulated by the alternative 5'-UTR region of its mRNA, and ATF5 may play a role in protecting cells from amino acid limitation or arsenite-induced oxidative stress.

症例は48歳の女性で, 2006年1月初旬に腹痛,嘔吐,下痢が出現したため当院を受診した.右下腹部に軽度圧痛を認めたが,腹膜刺激症状は認めなかった.腹部単純X線・腹部CTでイレウス像を呈しており,イレウス管を挿入し保存的治療を開始した.イレウス管造影で右下腹部に回腸の狭窄部を認め,内ヘルニアを疑って入院9日目に開腹術を施行した.回腸末端より約2m口側の回腸が回腸終末部背側の腸間膜にRichter型に嵌頓・癒着していた.腸管に色調不良や器質的狭窄を認めなかったため癒着剥離のみ施行した.剥離後に観察すると小腸間膜後葉に3.5×2.0cmの欠損部を認めたが,前葉には穿通していなかった.腸間膜欠損部は縫合閉鎖した.手術所見からRichter型の小腸間膜内ヘルニアと診断した.術後経過は良好で,術翌日より経口摂取を開始し術後8日目に軽快退院した.

Introduction In rheumatoid arthritis (RA) patients receiving immunosuppressive treatments, vaccination against Streptococcus pneumoniae is recommended. The objective of the study was to evaluate the effects of tacrolimus (TAC) on immune response following administration of a 23-valent pneumococcal polysaccharide vaccine (PPSV23) in patients with established RA. Methods Patients with RA (n = 133) were vaccinated with PPSV23. Patients were classified into TAC (n = 29), methotrexate (MTX) (n = 55), control (n = 35), and TAC/MTX (n = 14) treatment groups. We measured the concentrations of pneumococcal serotypes 6B and 23F by using an enzyme-linked immunosorbent assay and determined antibody functionality by using a multiplexed opsonophagocytic killing assay, reported as the opsonization index (OI), before and 4 to 6 weeks after vaccination. A positive antibody response was defined as at least a twofold increase in the IgG concentration or as at least a 10-fold increase in the OI. Results IgG concentrations and OIs were significantly increased in all treatment groups after PPSV23 vaccination. The TAC treatment group appears to respond in a manner similar to that of the RA control group in terms of 6B and 23F serotype concentration and function. In contrast, the MTX group had the lowest immune response. Patients who received a combination of TAC and MTX (TAC/MTX) also had a diminished immune response compared with those who received TAC alone. Conclusions TAC monotherapy does not appear to impair PPSV23 immunogenicity in patients with RA, whereas antibody production and function may be reduced when TAC is used with MTX. Thus, PPSV23 administration during ongoing TAC treatment should be encouraged for infection-prone TAC-treated patients with rheumatic diseases. Trial registration University Hospital Medical Information Network Clinical Trials Registry: UMIN000009566. Registered 12 December 2012. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0662-x) contains supplementary material, which is available to authorized users.

Beta2-glycoprotein I (beta2-GPI) is a major antigen for anti-cardiolipin antibodies and their epitopes are cryptic. Conformation of each domain of beta2-GPI was optimized from its crystal structure by energy minimization and by molecular dynamics simulation. Three electrostatic interactions, i.e. D193-K246, D222-K317 and E228-K308, were observed between domains IV and V in the optimized structure that was constructed based on the consensus sequences obtained by the phage-displayed random peptide library. Antigenic structures determined by the epitope mapping mainly consisted of hydrophobic amino acids located on two discontinuous sequences in domain IV. These amino acid clusters, as an epitope, were covered by domain V and were of a hidden nature. A similar but incomplete counterpart to the epitopic clusters was found in domain I but was not in domains II or III. Binding of anti-beta2-GPI auto-antibodies to solid-phase beta2-GPI was significantly reduced either by L replacement for W235, a common amino acid component for the epitopes, or by V replacement for all of D193, D222 and E228. Structural analysis indicated a hypothesis that these electrostatic interactions between domains IV and V retained exposure to W235 and that epitope spreading occurred in the region surrounding W235. Thus, epitopic structures recognized by anti-beta2-GPI auto-antibodies are cryptic and inter-domain electrostatic interactions are involved in their in exposure.

Changes in the mechanical and thermal properties, as well as in the microstructure, of unburned MgO-C bricks containing Al and Si powders were investigated at selected temperatures. Specimens with heat treatments at 500°C shrank and exhibited higher apparent porosity than untreated specimens. The bending strength and elastic modulus at 500°C were much lower than those of untreated specimens, and the apparent porosity increased and the mechanical properties at 500°C decreased with each repeating heat treatment. It was predicted that when the volatile matter was no longer generated, microstructure shrinkage would stop and the mechanical properties become constant. The bending strength and static elastic modulus at 800°, 1000°, and 1300°C were higher than those at 500°C because of the binding effect of the reaction products (i.e., Al4C3, SiC, and MgAl2O4), although the apparent porosity was higher than at 500°C. Repeated heat treatment from room temperature (RT) to the respective temperature, however, degraded the properties to nearly the same level as at 500°C because of the increased apparent porosity and the cracks generated in magnesia particles by the reaction products. Plastic deformation appeared to occur at 1300°C just before bricks were fractured. In addition, the thermal expansion ratio decreased through repeated heating and cooling from RT to 500°, 1000°, or 1300°C, and finally decreased to a constant value, as predicted.

Our objective in this study was to explore the diagnostic value of antiagalactosyl IgG antibodies in rheumatoid arthritis (RA). The study comprised 266 Japanese patients with systemic autoimmune diseases, including 60 with RA. Human agalactosyl IgG was prepared enzymatically, and the serum levels of antiagalactosyl IgG antibodies were determined using a lectin enzyme immunoassay. Serum IgG and IgM rheumatoid factors (RF) were measured using laser nephelometry for IgM (LN-RF) and an enzyme-linked immunosorbent assay for IgG (IgG-RF). Antiagalactosyl IgG antibodies were significantly more common in patients with RA than in those without (78% vs. 18%, odds ratio (OR) 16.51, 95% confidence interval (CI) 8.12-33.58, p

The ability of immobilization of fluorine dissolved from the fluorine containing slag with the calcium compound was evaluated by the shaking test. In the calcium compounds which have the ability of immobilization of fluorine, the 12CaO.7Al 2 O 3 was found to be the largest ability. From the results of evaluating the stability of immobilization with 12CaO.7Al 2 O3, it was shown that the dissolved fluorine from slag was immobilized for 100 h in shaking time. It was confirmed from the experimental results of X-ray analysis and the equilibrium thermodynamic calculation for aqueous solution that the generation of fluorite which was hard to dissolve in aqueous solution enabled the immobilization of fluorine dissolved from slag. Moreover, the 12CaO.7Al 2 O 3 of 5-15 mass% could be used for the road construction material which consisted of fluorine containing slag and blast furnace slag and as a result the immobilization of fluorine was confirmed.

In this study, we tried to solder directly heat treatable aluminum alloy to oxygen free copper without metallizing of solder, and investigated the effects of flux and the application of ultrasonic vibration on the strength of soldered joints and the soldered layer structure. The following results were obtained. Aluminum can be soldered to copper by applying ultrasonic vibration and using flux. However, in this case, many voids of 30~35 volume % are formed in the soldered layer. At the interface between Cu and soldered layer, AlCu intermetallic compound phase is formed and it grows with the duration of the ultrasonic vibration. When the thickness of the intermetallic phase is over about 760nm, the strength of the joint decreases. Only by applying ultrasonic vibration aluminum can be also soldered to copper without flux. The soldered layer includes few voids, however, the strength of the joint is lower than that of the joint soldered using flux. In this case, the AlCu intermetallic compound phase is also formed at the interface between Cu and soldered layer, and it grows with the duration of the ultrasonic vibration. However, the intermetallic phase does not grow uniformly and continuously along the overall interface, resulting in the low strength of the joint.

Summary. β2-glycoprotein I (β2GPI) bears the epitope(s) for autoimmune anticardiolipin antibodies (aCL) frequently present in patients with antiphospholipid syndrome (APS). β2GPI is involved in coagulation and fibrinolytic systems, including inhibition of contact activation. Coagulation factor XII is an initiator of intrinsic coagulation and also of intrinsic fibrinolysis. We investigated the effect of aCL (= anti-β2GPI antibodies), regarding intrinsic fibrinolysis using autoimmune monoclonal anti-β2GPI antibodies derived from a patient with APS or from an NZW/BXSB-F1 mouse. We developed a chromogenic assay system to determine intrinsic fibrinolytic activity. The reaction was activated by kaolin in the euglobulin fraction. Exogenous β2GPI slightly suppressed intrinsic fibrinolytic activity of the euglobulin fraction from normal plasma. Human monoclonal anti-β2GPI antibody (EY2C9) and mouse monoclonal anti-β2GPI antibody (WBCAL-1) in the presence of β2GPI decreased the activity. In this system, the suppression remained significant in the presence of an excess of exogenous activated factor XII. Euglobulin fractions from APS patients' plasma paralleled low activities of intrinsic fibrinolysis compared with those from healthy subjects. Our results suggest that β2GPI and anti-β2GPI antibodies suppress intrinsic fibrinolytic activities. This suppression was not only due to inhibition of factor XII activation but was also related to function of activated factor XII (XIIa). These phenomena partly explain the mechanisms of thrombosis in APS.

The multielement determination of major-to-ultratrace elements in industrial waste incineration bottom ash was carried out by inductively coupled plasma atomic emission spectrometry (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS) after alkali fusion using LiBO2. The concentrations of 50 elements in bottom ash could be determined over the concentration range from 192 mg g−1 of Si to 0.072 μg g−1 of In. Silicon, Al, Fe, Ca, Na, and Ti, which except for Fe are lithophile elements, were major elements of industrial waste incineration bottom ash. The distributions of major-to-ultratrace elements in incineration bottom ash were evaluated by the enrichment factors. The enrichment factors of major elements in incineration bottom ash indicated that their relative contents were quite similar to those in the continental crust. It was also found that most minor and trace elements in the lithophile element group provided almost the same enrichment factors as those of major elements. On the other ha...

SUMMARYThe diagnosis of the antiphospholipid syndrome (APS) requires both a typical clinical event plus a persistently positive test in an assay for either anticardiolipin (aCL) antibodies or a lupus anticoagulant (LA). Enzyme linked immunosorbent assays (ELISA) specific for autoantibodies against β2-glycoprotein I (β2GPI) or prothrombin are also used, but none of the tests are adequately sensitive or specific. A chromogenic assay was developed that measures the effect of test antibody or plasma samples on in vitro thrombin formation. It is able to detect both LA and β2GPI-dependent aCL antibodies and may have greater specificity for APS than currently available tests. Using this method various monoclonal antibodies (MoAbs) were examined, from mice immunized with β2GPI, mice with a spontaneous animal model of APS, and from three humans with APS. Plasma and affinity purified antibodies from patients with APS and control groups were also examined. Thrombin inhibition was more sensitive to perturbation by MoAbs than a combination of tests for LA (P

SummaryDespite the widely recognized practical importance of anticardiolipin (aCL) ELISA, the reliability of this test has been recently discussed. In order to investigate this area on European scale, we sent to 30 experienced centers a questionnaire focusing on the diagnostic procedures applied to patients with antiphospholipid syndrome (APS) and on the detailed protocols used to perform aCL. Anticardiolipin ELISA was found to be the most frequently performed test in patients with suspected APS, but significant difference was shown among the various protocols. The cross-laboratory multiple examination of ten serum samples evaluated independently by the 24 centers pointed out the difficulty in getting comparable results. Therefore a “consensus” protocol was derived from the aCL methods giving the best performance. The materials and reagents necessary to perform the “consensus” method, including, as putative standards, one IgG and one IgM monoclonal antibody (HCAL and EY2C9) were distributed to 19 Centers. The results of one IgG and one IgM aCL high positive sera measured in serial dilutions were compared. A progressive decrease in the variability of the values obtained for a given sample appeared evident when all the laboratories used the same standard, in their own in-house ELISA and even more in the “consensus” ELISA.Our data show that aCL ELISA standardization is necessary in order to obtain comparable results in different laboratories.

β 2 -glycoprotein I (β 2 -GPI=apolipoprotein H) is an important autoantigen in patients with the antiphospholipid syndrome. It also plays a role in lipoprotein metabolism, such as anti-atherogenic property, triglyceride removal, and enhancement of lipoprotein lipase. Serum β 2 -GPI concentration of 812 apparently healthy Japanese individuals was measured by sandwich EIA. Two families with complete β 2 -GPI deficiency were identified. In one family, all affected had increased serum LDL-cholesterol levels or smaller particle sizes of LDL, while the other had no apparent abnormality in lipid metabolism. Individuals investigated had no history of thrombosis or overt abnormalities in hemostatic tests. A thymine corresponding to position 379 of the β 2 -GPI cDNA was deleted in every β 2 -GPI deficient individual. The incidence of this heterozygous deficiency determined by RFLP was 6.3% in Japanese and none in Caucasians. Heterozygotes had significantly lower concentrations of serum β 2 -GPI than did those without the mutation, yet no significantly different lipid profiles, such as total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol, apoA-I, apoB and Lp(a), were observed. A low concentration of β 2 -GPI seemed not to be associated with apparent abnormality in lipoprotein metabolism.

Objective To clarify the association of autoantibodies against prothrombin with the clinical manifestations of the antiphospholipid syndrome (APS) and with the presence of lupus anticoagulant (LAC). Methods We examined 265 patients who visited our autoimmune disease clinic. IgG and IgM antiprothrombin antibodies were tested by enzyme-linked immunosorbent assay (ELISA) as either antiphosphatidylserine–prothrombin complex (aPS/PT) antibodies or as antibodies against prothrombin coated on irradiated ELISA plates (as antigen) (aPT). IgG, IgM, and IgA anticardiolipin (aCL) antibodies and their β2-glycoprotein I (β2GPI) dependency were also evaluated by ELISA. LAC was tested by 3 different methods. Results The presence of aPS/PT, but not of aPT, significantly correlated with the clinical manifestations of APS (odds ratio [OR] 4.39, 95% confidence interval [95% CI] 2.06–9.38), and aPS/PT antibodies were as specific as β2GPI-dependent aCL for APS (93.1% for both). IgG aPS/PT strongly correlated with the presence of LAC as detected using the dilute Russell viper venom time test (OR 38.2, 95% CI 13.4–109.1). Conclusion Antiprothrombin antibodies are heterogeneous and their clinical relevance depends on the method of detection applied. Positive results on the aPS/PT test can serve as a marker of thrombotic events in patients with autoimmune diseases.

Antiphospholipid antibodies (aPLs) are associated with an increased incidence of thrombosis, but the mechanisms responsible for thrombosis are unclear. The present study investigated the effect of both beta2-glycoprotein I (beta2-GPI) and aPLs on the activity of extrinsic fibrinolysis. The remaining tissue-plasminogen activator (t-PA) of the sample consisting of beta2-GPI, two-chain recombinant t-PA, plasminogen activator inhibitor (PAI) -1 was measured by a chromogenic assay using synthetic substrate S-2251, Glu-plasminogen, and soluble fibrin monomer. Without PAI-1, beta2-GPI did not affect t-PA activity. When 14.3 ng/ml PAI-1 was added to 3.6 U/ml t-PA, the remaining t-PA activity was increased from 48.9% to 60.4% by the addition of beta2-GPI (190 microg/ml). The effect of beta2-GPI did not require phospholipids. The beta2-GPI seems to protect t-PA activity from the inhibition by PAI-1. When monoclonal anticardiolipin antibodies (aCLs), EY1C8, and EY2C9, which were established from a patient with antiphospholipid syndrome, were further added to the mixture with a diluted phospholipid (Platelin) to investigate the influence of aPL, the remaining t-PA activity decreased to 50.1 and 80.7%. Monoclonal aCLs appeared to inhibit the effect of beta2-GPI, that is, these monoclonals inhibited the fibrinolytic activity by an elevation in PAI-1 activity. These results suggest the possibility that the impairment of fibrinolytic activity by aCLs is one of reasons for the increased incidence in thrombosis in patients with aCLs.

Objective Thromboembolic manifestations or thrombocytopenia in association with anticardiolipin antibodies (aCL) or lupus anticoagulant are known as the antiphospholipid syndrome (APS). Efforts have been made to elucidate precise clinical features and adequate therapeutic options for treating patients with APS. However, the lack of a proper international standard for measurement of aCL makes it difficult to compare data derived from different laboratories. We attempted to design a chimeric antibody with human gamma constant regions and variable regions of WBCAL-1, a monoclonal antibody established from an APS-prone mouse which has a specificity similar to that of aCL in sera from humans with APS. Methods Variable-region genes of WBCAL-1, which were cloned using reverse transcription-polymerase chain reaction, were inserted into plasmids containing human gamma1 and kappa constant-region genes. The construct was transfected to a mouse myeloma cell line. Stable transfectants that secreted a chimeric antibody, HCAL, into the culture supernatant were obtained. The reactivity of HCAL to cardiolipin and to beta2-glycoprotein I (beta2GPI) was studied using a solid-phase enzyme immunoassay. The binding of HCAL was compared with the binding of standards for IgG aCL and anti-beta2GPI antibody assays done in 18 independent laboratories. Results In the presence of beta2GPI, HCAL bound to the wells of cardiolipin-coated microtiter plates in a dose-dependent manner and reacted with beta2GPI on oxygenated polystyrene plates. The aCL activity of HCAL can be converted into GPL units (IgG phospholipid units), which is widely used to quantify IgG aCL activity, using the following formula: 1 GPL unit = 32.9 x (concentration of HCAL [in microg/ml])(0.503). The reactivity of HCAL to cardiolipin or beta2GPI was similar to the reactivity of standards for IgG aCL or anti-beta2GPI antibody assays done in collaborative laboratories. Conclusion Because the reactivity of HCAL is similar to that of aCL in sera from humans with APS, HCAL will be useful as a standard for human IgG aCL and anti-beta2GPI antibody assays.

OBJECTIVE To clarify mechanisms of the thrombosis associated with anticardiolipin antibodies (aCL), we examined the effects on activated protein C (APC) of monoclonal aCL and beta2-glycoprotein I (beta2GPI), which is required for formation of the epitopes of aCL. METHODS We developed the chromogenic assay, in which the degradation of coagulation factor Va by APC is reflected in the reduced generation of thrombin from prothrombin, using soybean trypsin inhibitor to inhibit APC. APC activities were measured in the presence and absence of 3.4 microM beta2GPI and/or 2.5 microg/ml of IgM monoclonal aCL (EY2C9 and EY1C8) established from peripheral blood lymphocytes obtained from a patient with aCL. RESULTS Without APC, the formed thrombin activity decreased by the addition of 3.4 microM beta2GPI. When 12.8 nM APC was added, beta2GPI partially reversed the APC-induced inhibition of thrombin generation in a concentration-dependent manner. With 3.4 microM beta2GPI, the thrombin generation in monoclonal aCL (2.5 microg/ml) decreased to 77.1-80.2% by the addition of 12.8 nM APC, but the values were above that in the control IgM (72.7%). Without beta2GPI, the APC activity was unaffected by the addition of monoclonal aCL. CONCLUSION Beta2-glycoprotein I exhibits procoagulant activity by inhibiting APC activity and anticoagulant activity by inhibiting thrombin generation. Any further inhibition of APC activity was caused by monoclonal aCL and only in the presence of beta2GPI.

Objective To investigate the effect of human monoclonal anticardiolipin antibodies (aCL) on platelet interaction with the subendothelium under flow conditions and on tissue factor (TF) expression on normal monocytes. Methods Three monoclonal IgM aCL (TM1B3, GR1D5, and EY2C9) and 2 affinity-purified IgM aCL were studied. Immunoglobulins were added to normal blood and perfused through chambers containing denuded vascular segments. Platelet interactions were morphometrically evaluated by determining the percentage of total surface covered by platelets (PCS) or by large aggregates of thrombi platelets (TP). Expression of TF on monocytes was measured after immunoglobulin incubation with normal mononuclear cells. Results Significant increases in the total PCS and expression of TF were observed using all aCL. Increased levels of TP were induced by all aCL except EY2C9 (obtained from a patient without thrombosis). Previous incubations of these aCL with subendothelial surfaces did not increase platelet interaction. Conclusion The effects of aCL on platelet function may help to explain the pathophysiology of thrombosis in the antiphospholipid syndrome.

SUMMARYIt is known that antiphospholipid antibodies (aPL) hamper the anticoagulant activity of the protein C system, but the mechanism is still obscure. In this study, we demonstrate that anticardiolipin antibodies (not anti-protein C autoantibodies) can bind protein C via β2-GPI, which bears their binding epitope, in a fashion dependent on negatively charged phospholipids. We studied the binding of IgG from aPL to protein C in the presence of β2-GPI by ELISA (anti-‘protein C’ antibody ELISA), and compared their binding with those obtained in the absence of β2-GPI. In the anti-‘protein C’ antibody ELISA system, 47% of 78 aPL+ patients had a positive titre in the presence of cardiolipin (CL) and β2-GPI, but binding was not found in the absence of β2-GPI. Highly significant correlations were found between the titre of anti-‘protein C’ antibody in the presence of β2-GPI and that of anti-β2-GPI antibody (r = 0·802, P = 0·0001). We further analysed the interaction between protein C, phospholipids, β2-GPI and human aCL MoAbs established from patients with antiphospholipid syndrome. In a first set of experiments, the binding of β2-GPI to protein C and its phospholipid dependency were investigated. β2-GPI bound to protein C in the presence of CL or phosphatidylserine, but not in the presence of phosphatidylcholine or phosphatidylethanolamine. In a second group of experiments, the binding of three human monoclonal aCL recognizing the cryptic epitope of β2-GPI (virtually anti-β2-GPI antibodies) was evaluated in the presence of cardiolipin and β2-GPI. All three human monoclonal aCL bound to protein C in the presence of CL and β2-GPI, whereas they did not in the absence of either β2-GPI or CL. These data suggest that protein C could be a target of aCL by making a complex with CL and β2-GPI, leading to protein C dysfunction.

Objective To explore a possible correlation between endothelin 1 (ET-1), the most potent endothelium-derived contracting factor that modulates vascular smooth muscle tone, and arterial disease in patients with the antiphospholipid syndrome (APS). Methods Plasma levels of ET-1 were measured in APS patients with (n = 16) and without (n = 11) arterial thrombosis and in non-APS patients with arterial thrombosis (n = 9). In addition, steady-state prepro-ET-1 messenger RNA (mRNA) levels were determined in endothelial cells treated with a range of human monoclonal anticardiolipin antibodies (aCL) (as anti-β2-glycoprotein I antibodies) by semiquantitative 32P-dCTP-labeled reverse transcription-polymerase chain reaction. Results Compared with healthy controls, markedly increased plasma levels of ET-1 were found in APS patients with arterial thrombosis (2.00 ± 0.87 versus 0.96 ± 0.37 pg/ml; P = 0.0001) but not in other groups. Three human monoclonal aCL induced prepro-ET-1 mRNA levels significantly more than did control monoclonal antibody lacking aCL activity. Conclusion Plasma ET-1 levels correlated significantly with a history of arterial thrombosis in patients with APS. Prepro-ET-1 mRNA was induced by human monoclonal aCL in the in vitro experimental system. The induction of ET-1 by antiphospholipid antibodies might contribute to increased arterial tone, leading to vasospasm and, ultimately, to arterial occlusion.

Anticardiolipin antibodies (aCL) found in sera from patients with antiphospholipid syndrome recognize a cryptic epitope that appears on the beta2-glycoprotein I (beta2-GPI) molecule when beta2-GPI interacts with a lipid membrane composed of negatively charged phospholipid or when beta2-GPI is adsorbed on a polyoxygenated polystyrene plate. A homology based model of beta2-GPI was constructed based on the NMR coordinates of sushi domains of human factor H. The conformation was like a cylinder consisting of five domains, its IV and V domains being glued by electrostatic interaction. We used phage-displayed random peptide libraries to search the epitopes of human aCL. Structures similar to consensus sequences selected by a biopanning method was found on domain IV of beta2-GPI.

The relationship between presence of anti-β2-glycoprotein I autoantibodies (aβ2-GPI) and history of thrombosis is now widely known. However, differences in the methodology of aβ2-GPI detection have made the comparison of data from different laboratories extremely difficult. We discuss the significance of aβ2-GPI of the IgG, IgM and IgA isotypes, and our approach to developing an easier and more reproducible method for the detection of this autoantibody. In addition, we present data that shows that commercially available enzyme immunoassay plates differ regarding detectability of aβ2-GPI. Since the clinical significance of this heterogeneity is presently unclear, the set-up of the detection systems and interpretation of data need great care.

We analyzed the mRNA expression of somatostatin receptor subtypes 1 to 5 (SSTR1-5) in human lymphoid cell lines, human peripheral blood lymphocytes (PBL), and human lymphatic leukemia cells, using the reverse transcription-polymerase chain reaction method. In human lymphoid cell lines, SSTR2 mRNA expression was clearly detectable, and there was no evidence of SSTR1 mRNA expression. SSTR2 mRNA was barely detectable in PBL from healthy individuals but was clearly detectable in EB virus-transformed lymphocytes. Lymphocytes from some of the leukemic patients showed elevated SSTR2 mRNA expression. SSTR2 mRNA expression in PBL was upregulated upon stimulation by PHA. SSTR3 mRNA was also observed in all the cell lines examined, although in one cell line, the expression was weak. Some cell lines showed little or no SSTR4 or 5 mRNA expression. The expression pattern of SSTR2 mRNA suggests that this receptor may have some important roles in lymphocyte activation, development, and/or tumorgenesis.