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Vascular endothelial growth factor receptor ( VEGFR) inhibitors are approved for the treatment of several tumor types; however, some tumors show intrinsic resistance to VEGFR inhibitors, and some patients develop acquired resistance to these inhibitors. Therefore, a strategy to overcome VEGFR inhibitor resistance is urgently required. Recent reports suggest that activation of the hepatocyte growth factor ( HGF) pathway through its cognate receptor, Met, contributes to VEGFR inhibitor resistance. Here, we explored the effect of the HGF/ Met signaling pathway and its inhibitors on resistance to lenvatinib, a VEGFR inhibitor. In in vitro experiments, addition of VEGF plus HGF enhanced cell growth and tube formation of HUVECs when compared with stimulation by either factor alone. Lenvatinib potently inhibited the growth of HUVECs induced by VEGF alone, but cells induced by VEGF plus HGF showed lenvatinib resistance. This HGF-induced resistance was cancelled when the Met inhibitor, golvatinib, was added with lenvatinib. Conditioned medium from tumor cells producing high amounts of HGF also conferred resistance to inhibition by lenvatinib. In s.c. xenograft models based on various tumor cell lines with high HGF expression, treatment with lenvatinib alone showed weak antitumor effects, but treatment with lenvatinib plus golvatinib showed synergistic antitumor effects, accompanied by decreased tumor vessel density. These results suggest that HGF from tumor cells confers resistance to tumor endothelial cells against VEGFR inhibitors, and that combination therapy using VEGFR inhibitors with Met inhibitors may be effective for overcoming resistance to VEGFR inhibitors. Further evaluation in clinical trials is warranted. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem (iPS) cells are widely used as a research tool in regenerative medicine and embryology. In studies related to lens regeneration in the eye, iPS cells have been reported to differentiate into lens epithelial cells (LECs); however, to the best of our knowledge, no study to date has described their formation of three-dimensional cell aggregates. Notably, in vivo studies in newts have revealed that iris cells in the eye can dedifferentiate into LECs and regenerate a new lens. Thus, as basic research on lens regeneration, the present study investigated the differentiation of human iris tissue-derived cells and human iris tissue-derived iPS cells into LECs and their formation of three-dimensional cell aggregates using a combination of two-dimensional culture, static suspension culture and rotational suspension culture. The results revealed that three-dimensional cell aggregates were formed and differentiated into LECs expressing αA-crystallin, a specific marker protein for LECs, suggesting that the cell-cell interaction facilitated by cell aggregation may have a critical role in enabling highly efficient differentiation of LECs. However, the present study was unable to achieve transparency in the cell aggregates; therefore, we aim to continue to investigate the degradation of organelles and other materials necessary to make the interior of the formed cell aggregates transparent. Furthermore, we aim to expand on our current work to study the regeneration of the lens and ciliary body as a whole in vitro, with the aim of being able to restore focusing function after cataract surgery. [ABSTRACT FROM AUTHOR]

In antigen (Ag) cross-presentation, dendritic cells (DCs) take up extracellular Ag and translocate them from the endosome to the cytosol for proteasomal degradation. The processed peptides can enter the conventional MHC I pathway. The molecules responsible for the translocation of Ag across the endosomal membrane into the cytosol are unknown. Here we demonstrate that heat shock protein 90 (HSP90) is critical for this step. Cross-presentation and -priming were decreased in both HSP90a-null DCs and mice. CD8a+ DC apoptosis mediated by translocation of exogenous cytochrome c to the cytosol was also eliminated in HSP90a-null mice. Ag translocation into the cytosol was diminished in HSP90a-null DCs and in DCs treated with an HSP90 inhibitor. Internalized Ag was associated with HSP90 and translocated to the cytosol, a process abrogated by the HSP90 inhibitor. Ag within purified phagosomes was released in an HSP90-dependent manner. These results demonstrate the important role of HSP90 in cross-presentation by pulling endosomal Ag out into the cytosol. [ABSTRACT FROM AUTHOR]

The deficiency of programmed cell death 1 (PD-1, Pdcd1), a negative immuno-receptor belonging to the CD28/cytotoxic T lymphocyte antigen 4 (CTLA-4) family, can support various tissue-specific autoimmune conditions. Here, we analyzed the effect of PD-1 deficiency in MRL mice that is genetically predisposed to systemic autoimmunity. MRL-Pdcd1−/− mice developed a fatal myocarditis, which is reminiscent of CTLA-4-deficient (Ctla4−/−) mice. Massive infiltration of CD4+ and CD8+ T cells and myeloid cells was found in hearts of MRL-Pdcd1−/− mice concomitant with the production of high-titer auto-antibodies against cardiac myosin. In contrast to Ctla4−/− mice in which most of the CD4+ T cells are non-specifically activated and invade various organs, T cells in the heart but not in the spleen and lymph nodes are activated in MRL-Pdcd1−/− mice, suggesting that myocarditis is mediated by antigen-specific autoimmune response. Heart infiltrating myeloid cells strongly suppressed the allogenic response of T cells in vitro, suggesting that these Mac1+Gr1+ myeloid cells are phenotypically similar to myeloid suppressor cells, which can be found in tumor-bearing hosts. These findings unravel the hidden heart-specific autoimmune predisposition of MRL mice and provide MRL-Pdcd1−/− mice as a useful animal model of lymphocytic myocarditis. [ABSTRACT FROM PUBLISHER]

Neutrophil extracellular trap (NET) formation, an innate immune system response, is associated with thrombogenesis and vascular endothelial injury. Circulatory disorders due to microvascular thrombogenesis are one of the principal causes of organ damage. NET formation in organs contributes to the exacerbation of sepsis, which is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. We have previously reported that recombinant human soluble thrombomodulin (rTM) reduces lipopolysaccharide (LPS)-induced NET formation in vitro. Here, we aimed to show that thrombomodulin (TM)-mediated suppression of NET formation protects against organ damage in sepsis. Mice were injected intraperitoneally (i.p.) with 10 mg/kg LPS. rTM (6 mg/kg/day) or saline was administered i.p. 1 h after LPS injection. In the LPS-induced murine septic shock model, extracellular histones, which are components of NETs, were observed in the liver and lungs. In addition, the serum cytokine (interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), macrophage chemotactic protein-1 (MCP-1), and interleukin-10 (IL-10)) levels were increased. The administration of rTM in this model prevented NET formation in the organs and suppressed the increase in the levels of all cytokines except IL-1β. Furthermore, the survival rate improved. We provide a novel role of TM in treating inflammation and NETs in organs during sepsis. [ABSTRACT FROM AUTHOR]

How antigen valency affects B cells in vivo during immune responses is not well understood. Here, using HIV immunogens with defined valencies ranging from 1 to 60, we investigated the role of antigen valency during different phases of B cell responses in vivo. Highly multimerized immunogens preferentially rapidly activated cognate B cells, with little affinity discrimination. This led to strong early induction of the transcription factors IRF4 (interferon regulatory factor 4) and Bcl6, driving both early extrafollicular plasma cell and germinal center responses, in a CD4+ T-cell-dependent manner, involving B cells with a broad range of affinities. Low-valency antigens induced smaller effector B cell responses, with preferential recruitment of high-affinity B cells. Thus, antigen valency has multifaceted effects on B cell responses and can dictate affinity thresholds and competitive landscapes for B cells in vivo , with implications for vaccine design. • Antigen valency dictates the magnitude and composition of B cell responses • High valency enables robust activation and effector differentiation of B cells • Antigen valency alters breadth of B cell affinities recruited Detailed mechanistic understanding of how differing antigen valencies influence cognate B cells in vivo is lacking. Kato et al. show antigen valency dictates the breadth of affinity range of B cells that are recruited and the ability of cognate B cells to differentiate into effector cells. This had profound impacts on the magnitude of germinal center and extrafollicular plasmablast responses. The study also highlights the need to carefully consider valency in vaccine design. [ABSTRACT FROM AUTHOR]

Background: To compare the quality of the ultra-widefield images acquired by Optos® 200Tx to those acquired by Optos® California.Methods: Images of the posterior surface of a Gullstrand's model eye obtained by Optos® 200Tx were compared to those obtained by Optos® California in terms of the angular field of view and the symmetry of the image, i.e., vertical and horizontal aspect ratios at the center (0°) and at the periphery (40° and 80°) in each direction. In addition, we compared the enlargement of the image on the posterior surface as the square ratio, and the differences in the contrasts.Results: No significant differences were detected in the angular field of view between the two instruments. The aspect ratios showed that the Optos® California had more symmetrical images than the Optos® 200Tx at the center (0.98 vs 0.89, P < 0.01) and at the 40° periphery (0.93-1.04, △0.11 vs 0.79-1.01, △0.22) and 80° periphery (0.81-1.25, △0.44 vs 0.42-1.12, △0.70) in each direction. The amplitude of the square ratio of the Optos® California was smaller at 40° periphery (1.16-1.28, △0.12 vs 1.06-1.37, △0.31) and 80° periphery (2.12-2.46, △0.34 vs 1.14-3.29, △2.15). The contrast of the Optos® California images was significantly higher in the posterior pole (0.09 vs 0.12, P < 0.01), upper (0.07 vs 0.03, P < 0.01), and right (0.12 vs 0.07, P < 0.01) peripheries.Conclusion: Optos® California can record equal angular widefield images to Optos® 200Tx and more symmetrical images with higher contrast in the posterior pole, upper and right peripheries. [ABSTRACT FROM AUTHOR]

NET components were identified through immunohistochemistry (IHC) using an anti-myeloperoxidase antibody and an anti-histone H2A antibody. Compared with non rTM group mice, rTM group mice released fewer extracellular histones within the lungs and the liver in response to LPS. B Conclusions: b rTM reduced the amount of extracellular histone and inhibited NET formation in the lungs and the liver of LPS-induced septic mice. [Extracted from the article]

To clarify the physiological factors associated with the development of tomato leaf marginal necrosis associated with potassium deficiency, tomato leaf blades prior to development of the symptoms were collected, and profiles of water-soluble metabolites were analyzed using gas chromatography–mass spectrometry. Multivariate analysis was conducted to screen for a component that was related to potassium deficiency-induced leaf necrosis among the 60 metabolites detected in tomato leaves. Polyamines, sugars, and branched amino acids were ranked highly. Putrescine was most strongly and negatively correlated with potassium concentration and exhibited an exponential response, regardless of the site or plant body. In addition to putrescine, glucose, xylose and l -isoleucine are known to play important roles in stress response and ion balance control, and it is thought that changes in metabolite profiles are linked to tomato leaf marginal necrosis associated with a decrease in endogenous potassium concentration. These findings will be useful for cultivation management to mitigate this physiological disorder of tomato. [ABSTRACT FROM AUTHOR]

Background/aim: Sunitinib is used for the treatment of metastatic renal cell carcinoma (mRCC). Asian patients, including Japanese, tend not to tolerate long-term sunitinib therapy of 50 mg p.o. once daily for 4 weeks, followed by 2 week off treatment due to severe adverse events at this dosage level. The aim of this retrospective study was to investigate the optimal dose of sunitinib for long-term continuation in Asian patients with mRCC.Patients and Methods: The study cases were 50 patients with mRCC who were treated with sunitinib between June 2008 and December 2017. Risk analysis for "unacceptable" adverse events (depending on the physician, ranging from grade 2 to ≥ grade 3) leading to discontinuation of sunitinib was determined by time-dependent Cox proportional hazard regression analysis.Results: A total of 54 unacceptable adverse events leading to discontinuation occurred. Multivariable analysis indicated that a sunitinib dose of ≤ 37.5 mg/day significantly reduced the risk of discontinuation due to adverse events in comparison with 50 mg/day [hazard ratio (HR) 0.08, 95% confidence interval (CI) 0.03-0.21, p < 0.001). The progression-free survival (PFS) with a sunitinib dose ≤ 37.5 mg/day was longer than that associated with a dose of 50 mg/day, albeit not to a statistically significant degree (120 days for ≤ 37.5 mg/day vs 41 days for 50 mg/day, HR 0.39, 95% CI 0.10-1.44, p = 0.157).Conclusion: Our findings suggest that the optimal dose of sunitinib for Asian, including Japanese, patients with mRCC is ≤ 37.5 mg/day. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks (DSBs) trigger the recruitment of repair protein and promote signal transduction through posttranslational modifications such as phosphorylation. After DSB induction, ataxia telangiectasia mutated (ATM) phosphorylates H2AX on chromatin surrounds the mega-base pairs proximal to the DSBs. Advanced super-resolution microscopic technology has demonstrated the formation of γH2AX nano foci as a unit of nano domain comprised of multiple nucleosomes. The formation of γH2AX nano foci could be potentially affected by pre-existing chromatin structure prior to DSB induction; however, it remains unclear whether chromatin status around DSBs influences the formation of γH2AX nano foci. In this study, to investigate γH2AX nano foci formation in the context of chromatin relaxation, γH2AX nano foci were examined following the depletion of MeCP2, which is a factor promoting chromatin condensation. Remarkably, by using super-resolution imaging analysis, we found that the volume of γH2AX nano foci cluster in MeCP2-depleted cells was significantly greater than that in control cells, both 5 and 30 min after ionizing radiation (IR). Corresponding to the increased volume size, the number of γH2AX nano foci per cluster was greater than that in control cells, while the distance of each nano focus within foci clusters remained unchanged. These findings suggest that relaxed chromatin condition by MeCP2 depletion facilitates faster and more extensive γH2AX nano foci formation after IR. Collectively, our super-resolution analysis suggests that the chromatin status surrounding DSBs influences the expansion of γH2AX nano foci formation, thus, potentially influencing the DSB repair and signaling. • MeCP2 depletion promotes the speed of γH2AX foci formation in human cells. • MeCP2 depletion enhances the volume of γH2AX nano foci cluster. • MeCP2 depletion enhances the number of γH2AX nano foci per cluster. • Pre-existing chromatin prior to DSB may influence γH2AX nano foci formation. [ABSTRACT FROM AUTHOR]

Simple Summary: Human multicentric osteosarcoma (HMOS) is a variant of human osteosarcoma (HOS) in which multiple bone lesions occur simultaneously or asynchronously before lung metastasis. HOS is associated with a poor prognosis. Using the human antibody phage library, we obtained 95 types of antibody clones against HMOS cell lines and developed a fluorescence probe-based enzyme-linked immunosorbent assay (ELISA) that enables high-throughput screening of the reactivity of these antibodies. The lysate of the HMOS cell line was incubated with the selected antibody, and the antigen–antibody complex was recovered using magnetic beads. Liquid chromatography-mass spectrometry (LC/MS) analysis of the sample bands extracted from gel electrophoresis detected CAVIN1/PTRF specifically in HMOS cells. This novel high-throughput screening assay using antibody–drug conjugate targeting for these specific proteins is promising for clinical applications. Osteosarcoma is a malignant tumor that produces neoplastic bone or osteoid osteoma. In human multicentric osteosarcoma (HMOS), a unique variant of human osteosarcoma (HOS), multiple bone lesions occur simultaneously or asynchronously before lung metastasis. HMOS is associated with an extremely poor prognosis, and effective treatment options are lacking. Using the proteins in our previously generated HMOS cell lines as antigens, we generated antibodies using a human antibody phage library. We obtained antibody clones recognizing 95 independent antigens and developed a fluorescence probe-based enzyme-linked immunosorbent assay (ELISA) technique capable of evaluating the reactivity of these antibodies by fluorescence intensity, allowing simple, rapid, and high-throughput selection of antibody clones. These results were highly correlated with those using flow cytometry. Subsequently, the HMOS cell lysate was incubated with the antibody, the antigen–antibody complex was recovered with magnetic beads, and the protein bands from electrophoresis were analyzed using liquid chromatography-mass spectrometry (LC/MS). CAVIN1/polymerase I transcript release factor was specifically detected in the HMOS cells. In conclusion, we found via a novel high-throughput screening method that CAVIN1/PTRF is an HMOS-specific cell membrane biomarker and an antigen capable of producing human antibodies. In the future, antibody–drug conjugate targeting of these specific proteins may be promising for clinical applications. [ABSTRACT FROM AUTHOR]

Background: Adenomyosis, a benign gynecological disease, causes cerebral infarction. Similar to Trousseau's syndrome, it elevates cancer antigen 125 (CA125) and D-dimer levels; causes hypercoagulability; and results in cerebral infarction. However, no case of adenomyosis causing major cerebral artery occlusion and requiring endovascular thrombectomy has yet been reported. We report on a woman with middle cerebral artery occlusion caused by adenomyosis progression with a benign gynecological tumor and recurrent cerebral infarction. She was successfully treated by endovascular thrombectomy and hysterectomy.Case Presentation: A 48-year-old woman with heavy uterine bleeding was transported by ambulance to our hospital. Upon arrival, she presented with impaired consciousness. Laboratory test results revealed decreased hemoglobin (8.2 g/dL) and elevated D-dimer (79.3 µg/mL) levels. Radiological imaging revealed adenomyosis, a left ovarian tumor, multiple uterine myomas, and old and new bilateral renal infarctions. She experienced repeated episodes of excessive menstruation caused by adenomyosis and was scheduled for hysterectomy in 2 months at another hospital. After hospital admission, uterine bleeding stopped. However, 5 days after initial bleeding, she had another episode of heavy uterine bleeding and developed left hemiparesis and dysarthria 20 min later. Brain magnetic resonance imaging revealed bilateral multiple cerebral infarctions indicating right middle cerebral artery occlusion. Thus, endovascular thrombectomy was performed, and anticoagulant therapy was administered. Laboratory test results after thrombectomy revealed elevated CA125 (3536 U/mL) and CA19-9 (892 U/mL) levels. She was at a risk of recurrent heavy uterine bleeding leading to repeated cerebral infarction because of anticoagulant treatment. Therefore, we performed hysterectomy and ovariectomy 11 days after initial bleeding. Histopathological assessment revealed no malignancy. Although she developed asymptomatic pulmonary thromboembolism 14 days after initial bleeding, D-dimer and tumor marker levels returned to normal soon after gynecological surgery. At 15 months post-surgery, she had not experienced further ischemic events.Conclusions: Adenomyosis with benign gynecological tumors may be associated with elevated D-dimer and tumor marker levels; excessive menstruation; and anemia. It may cause systemic thromboembolism, including cerebral infarction. To our knowledge, no other study has reported that adenomyosis causes major cerebral artery occlusion requiring endovascular thrombectomy. Hysterectomy may be an effective radical treatment of this condition. [ABSTRACT FROM AUTHOR]

JQ1 disrupts the binding of bromodomain and extra-terminal (BET) family of proteins to acetylated histones, modulates the expression of various genes, and inhibits the proliferation of cancer cells. We established two JQ1-resistant sublines from human colorectal cancer HCT116 cells. These resistant cells showed an 8- to 9-fold higher resistance to JQ1, and a 2- to 4-fold higher resistance to various anti-cancer agents, such as doxorubicin, etoposide, mitoxantrone, SN-38, cisplatin, and methotrexate than the parental HCT116 cells. The JQ1-resistant cells expressed higher levels of TRAF2 and NCK-interacting protein kinase (TNIK), cyclin D1 (CCND1), cyclin E1 (CCNE1), and their corresponding mRNAs than the parental cells. TNIK is a regulator of Wnt/β-catenin signaling and is known to transactivate CCND1. Transient transfection of HCT116 cells with a TNIK expression plasmid resulted in the upregulation of cyclin D1, cyclin E1, and their corresponding mRNAs, as well as an increase in CCNE1 promoter activity. Furthermore, luciferase assay revealed that the JQ1-resistant cells showed high CCNE1 promoter activity. These results suggest that TNIK also transactivates CCNE1. Three stable TNIK transfectant clones of HEK293 cells expressed 1.5- to 2-fold higher levels of TNIK, cyclin D1, and cyclin E1 than the parental cells. The 293/TNIK-6 cells, which expressed the highest level of TNIK among the transfectants, showed a 2.3-fold higher resistance to JQ1 than the parental cells. These results suggest the possible involvement of TNIK in cellular resistance to JQ1. • Cross-resistance of JQ1-resistant cells to DNA-damaging agents. • Upregulation of TNIK, cyclin D1, and cyclin E1 in JQ1-resistant cells. • Upregulation of cyclin D1 and cyclin E1 in TNIK -transfected cells. • Increased CCNE1 promoter activity in JQ1-resistant cells and TNIK -transfected cells. • JQ1 resistance of stable TNIK -transfected cells. [ABSTRACT FROM AUTHOR]

• ATM-, ATR-, or MRE11-deficiency enhances micronuclei formation after IR. • IR upregulates inflammatory gene expression under ATM-, ATR-, or MRE11-deficiency. • RIG-I, MDA-5, IRF7, and IL6 pathway is upregulated after IR. • The balance of STAT1/3 activation differs between ATM- and ATR-deficiency. Ionizing radiation (IR) induces DNA double-strand breaks (DSBs), leading to micronuclei formation, which has emerged as a key mediator of inflammatory responses after IR. This study aimed to investigate the signaling cascade in inflammatory gene expression using fibroblasts harboring DNA damage response deficiency after exposure to IR. Micronuclei formation was examined in human dermal fibroblasts derived from patients with deficiencies in ATM, ATR, MRE11, XLF, Artemis, or BRCA2 after IR. RNA-sequencing analysis was performed to assess gene expression, pathway mapping, and the balance of transcriptional activity using the transcription factor–based downstream gene expression mapping (TDEM) method developed in this study. Deficiencies in ATM, ATR, or MRE11 led to increased micronuclei formation after IR compared to normal cells. RNA-seq analysis revealed significant upregulation of inflammatory expression in cells deficient in ATM, ATR, or MRE11 following IR. Pathway mapping analysis identified the upregulation of RIG-I, MDA-5, IRF7, IL6, and interferon stimulated gene expression after IR. These changes were pronounced in cells deficient in ATM, ATR, or MRE11. TDEM analysis suggested the differential activation of STAT1/3–pathway between ATM and ATR deficiency. Enhanced micronuclei formation upon ATM, ATR, or MRE11 deficiency activated the cGAS/STING, RIG-I-MDA-5-IRF7-IL6 pathway, resulting in its downstream interferon stimulated gene expression following exposure to IR. Our study provides comprehensive information regarding the status of inflammation-related gene expression under DSB repair deficiency after IR. The generated dataset may be useful in developing functional biomarkers to accurately identify patients sensitive to radiotherapy. [ABSTRACT FROM AUTHOR]

Summary In recent years, various intervention strategies have reduced malaria morbidity and mortality, but further improvements probably depend upon development of a broadly protective vaccine. To better understand immune requirement for protection, we examined liver-stage immunity after vaccination with irradiated sporozoites, an effective though logistically difficult vaccine. We identified a population of memory CD8 + T cells that expressed the gene signature of tissue-resident memory T (Trm) cells and remained permanently within the liver, where they patrolled the sinusoids. Exploring the requirements for liver Trm cell induction, we showed that by combining dendritic cell-targeted priming with liver inflammation and antigen recognition on hepatocytes, high frequencies of Trm cells could be induced and these cells were essential for protection against malaria sporozoite challenge. Our study highlights the immune potential of liver Trm cells and provides approaches for their selective transfer, expansion, or depletion, which may be harnessed to control liver infections or autoimmunity. [ABSTRACT FROM AUTHOR]

[Display omitted] 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. [ABSTRACT FROM AUTHOR]

[Display omitted] 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. [ABSTRACT FROM AUTHOR]

Limited knowledge is available on the relationship between antigen-specific immune responses and COVID-19 disease severity. We completed a combined examination of all three branches of adaptive immunity at the level of SARS-CoV-2-specific CD4+ and CD8+ T cell and neutralizing antibody responses in acute and convalescent subjects. SARS-CoV-2-specific CD4+ and CD8+ T cells were each associated with milder disease. Coordinated SARS-CoV-2-specific adaptive immune responses were associated with milder disease, suggesting roles for both CD4+ and CD8+ T cells in protective immunity in COVID-19. Notably, coordination of SARS-CoV-2 antigen-specific responses was disrupted in individuals ≥ 65 years old. Scarcity of naive T cells was also associated with aging and poor disease outcomes. A parsimonious explanation is that coordinated CD4+ T cell, CD8+ T cell, and antibody responses are protective, but uncoordinated responses frequently fail to control disease, with a connection between aging and impaired adaptive immune responses to SARS-CoV-2. • Adaptive immune responses limit COVID-19 disease severity • Multiple coordinated arms of adaptive immunity control better than partial responses • CXCL10 may be a biomarker of impaired T cell responses in acute COVID-19 • Aging and scarcity of naive T cells may be linked risk factors for severe COVID-19 Analysis of SARS-CoV-2-specific adaptive immune responses during acute COVID-19 identifies coordination between SARS-CoV-2-specific CD4 T cells and CD8 T cells to limit disease severity. Aged individuals often exhibit uncoordinated adaptive responses, potentially tied to scarcity of naive T cells, highlighting immunologic risk factors linked to disease severity. [ABSTRACT FROM AUTHOR]

Developmental toxicity testing is essential to identify substances that may harm embryonic development. This study aimed to establish a protocol for evaluating developmental toxicity using human induced pluripotent stem cells (iPSCs) by analyzing cellular activity and gene expression changes. Two ICH S5(R3) positive substances, valproic acid (VPA), which is a substance previously detected as positive by other test methods, and thalidomide (Thalido), were examined during early trichoderm differentiation without fetal bovine serum. RNA-seq analysis identified seven candidate genes, including TP63 , associated with altered expression following exposure to VPA or Thalido. These genes were implicated in pathways related to tissue development, cell growth, and molecular interactions. While the assay effectively detected VPA and Thalido, its limitations include testing only soluble substances and focusing on early differentiation stages. Nevertheless, the protocol demonstrates potential for the classification and evaluation of emerging modality drugs based on physical properties such as solubility, polarity, and pH. Integration with AI analysis may enhance its capacity to uncover genetic variations and evaluate previously uncharacterized substances. This study provides a foundation for alternative developmental toxicity testing methods, with further refinements in the culture method expected to improve accuracy and applicability in regulatory toxicology.

Combination therapy with anti-angiogenic drugs and immune checkpoint inhibitors has shown enhanced clinical activity and has been approved for the treatment of multiple tumor types. Despite extensive research, predictive biomarkers for combination therapy remain poorly understood. Microvessel density (MVD), a surrogate marker for aberrant angiogenesis measured by immunohistochemistry (IHC), has been associated with response to monotherapy with anti-angiogenesis inhibitors. However, obtaining tumor tissue with a sufficient mass for IHC analysis is not always practical, and IHC-based MVD measurements are unavailable in large public datasets. In this study, we developed an MVD gene score based on RNA-sequencing data that reflects MVD by using RNA-seq and MVD measured by IHC in 12 mouse syngeneic tumor models. We explored the relationship between the MVD gene score and a gene signature predicting the response to anti-PD-1 therapy in mouse and human tumor datasets. The MVD gene score correlated with the antitumor activity of lenvatinib, a multiple tyrosine kinase inhibitor mainly targeting VEGFRs and FGFRs, in mouse tumor models and MVD measured by IHC in commercially available human formalin-fixed, paraffin-embedded tumor samples. Tumor types in The Cancer Genome Atlas were classified into four subgroups based on the MVD gene score and T-cell inflamed gene expression profile (TcellinfGEP), which were correlated with clinical indications for treatment. In conclusion, the newly developed MVD gene score enables the estimation of MVD in large public datasets where IHC data are unavailable and has potential clinical utility together with the TcellinfGEP to characterize patients' tumors for precision medicine.

ICVB-1042 is an oncolytic adenovirus containing modifications to enhance replication, lysis, and viral spreading in tumor cells. The anti-tumor activity, immune activation, tropism, selectivity, and mechanism of action were evaluated in preparation for a first-in-human study. ICVB-1042 was at least 100-fold more cytotoxic in A549 cells than in normal primary cells tested, demonstrating its high tumor selectivity and a low likelihood of targeting primary tissues. ICVB-1042 administered to mice intravenously or intratumorally was effective in reducing tumor burden. Its intravenous administration also inhibited tumor growth in orthotopic models. ICVB-1042 was well tolerated in mice compared to HAdV-C5 (Wt Ad5), with reduced liver sequestration, supporting safety of the drug for systemic delivery. These preclinical data demonstrating the safety and potency of ICVB-1042 for treatment of various solid tumors support the ongoing clinical investigation (NCT05904236)., (© 2024. The Author(s).)

Endothelial Notch signaling is critical for tumor angiogenesis. Notch1 blockade can interfere with tumor vessel function but causes tissue hypoxia and gastrointestinal toxicity. Notch4 is primarily expressed in endothelial cells, where it may promote angiogenesis; however, effective therapeutic targeting of Notch4 has not been successful. We developed highly specific Notch4-blocking antibodies, 6-3-A6 and humanized E7011, allowing therapeutic targeting of Notch4 to be assessed in tumor models. Notch4 was expressed in tumor endothelial cells in multiple cancer models, and endothelial expression was associated with response to E7011/6-3-A6. Anti-Notch4 treatment significantly delayed tumor growth in mouse models of breast, skin, and lung cancers. Enhanced tumor inhibition occurred when anti-Notch4 treatment was used in combination with chemotherapeutics. Endothelial transcriptomic analysis of murine breast tumors treated with 6-3-A6 identified significant changes in pathways of vascular function but caused only modest change in canonical Notch signaling. Analysis of early and late treatment timepoints revealed significant differences in vessel area and perfusion in response to anti-Notch4 treatment. We conclude that targeting Notch4 improves tumor growth control through endothelial intrinsic mechanisms., Significance: A first-in-class anti-Notch4 agent, E7011, demonstrates strong antitumor effects in murine tumor models including breast carcinoma. Endothelial Notch4 blockade reduces perfusion and vessel area., (©2024 The Authors; Published by the American Association for Cancer Research.)

Ionizing radiation (IR)-induced double-strand breaks (DSBs) are primarily repaired by non-homologous end joining or homologous recombination (HR) in human cells. DSB repair requires adenosine-5'-triphosphate (ATP) for protein kinase activities in the multiple steps of DSB repair, such as DNA ligation, chromatin remodeling, and DNA damage signaling via protein kinase and ATPase activities. To investigate whether low ATP culture conditions affect the recruitment of repair proteins at DSB sites, IR-induced foci were examined in the presence of ATP synthesis inhibitors. We found that p53 binding protein 1 foci formation was modestly reduced under low ATP conditions after IR, although phosphorylated histone H2AX and mediator of DNA damage checkpoint 1 foci formation were not impaired. Next, we examined the foci formation of breast cancer susceptibility gene I (BRCA1), replication protein A (RPA) and radiation 51 (RAD51), which are HR factors, in G2 phase cells following IR. Interestingly, BRCA1 and RPA foci in the G2 phase were significantly reduced under low ATP conditions compared to that under normal culture conditions. Notably, RAD51 foci were drastically impaired under low ATP conditions. These results suggest that HR does not effectively progress under low ATP conditions; in particular, ATP shortages impair downstream steps in HR, such as RAD51 loading. Taken together, these results suggest that the maintenance of cellular ATP levels is critical for DNA damage response and HR progression after IR., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

Purpose: Recent radiation therapy (RT), such as intensity modulated radiation therapy and particle RT, has improved the concentration of the radiation field targeting tumors. However, severe adverse effects still occur, possibly due to genetic factors in patients. We aimed to investigate the mechanism of exacerbated inflammation during RT., Methods and Materials: Dermal fibroblasts derived from a patient with severe inflammatory adverse effects during RT were compared with 2 normal human dermal fibroblasts. Micronuclei formation, G2/M-checkpoint arrest, DNA damage signaling and repair, and inflammatory gene expression were comprehensively examined., Results: We found greater micronuclei formation in radiation-sensitive fibroblasts (RS-Fs) after ionizing radiation (IR). RS-Fs exhibited premature G2/M-checkpoint release after IR, which triggers micronuclei formation because RS-Fs undergo mitosis with unrepaired DNA double-strand breaks (DSBs). Additionally, we found that DSB end-resection and activation of the ATR-Chk1 pathway were impaired in RS-Fs after IR. Consistent with the increase in the formation of micronuclei, which can deliver cytosolic nucleic acids resulting in an innate immune response, the expression of genes associated with inflammatory responses was highly upregulated in RS-Fs after IR., Conclusions: Although this is a single case of RT-dependent adverse effect, our findings suggest that impaired G2/M-checkpoint arrest due to the lack of DSB end-resection and activation of the ATR-Chk1 pathway causes exacerbated inflammation during RT; therefore, genes involved in G2/M-checkpoint arrest may be a predictive marker for unexpected inflammatory responses in RT., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 Published by Elsevier Inc. on behalf of American Society for Radiation Oncology.)

Objectives: Sepsis is a life-threatening condition characterized by multi-organ dysfunction due to host immune system dysregulation in response to an infection. During sepsis, neutrophils release neutrophil extracellular traps (NETs) as part of the innate immune response. However, excessive NETs play a critical role in the development of organ failure during sepsis. Although recombinant human soluble thrombomodulin (rTM) can inhibit NET formation in the lungs and liver of a mouse model of endotoxin shock, its effects on the kidneys are unclear., Methods: The specific effects of NETs and rTM on the renal cortex and renal medulla were examined in a mouse model of endotoxin shock generated by intraperitoneal (i.p.) injection of lipopolysaccharide (LPS), followed by i.p. injection of rTM or an identical volume of saline 1 h later., Results: LPS injection increased serum creatinine, blood urea nitrogen, and histone H3 levels. However, rTM administration significantly decreased histone H3 and citrullinated histone H3 (citH3) levels. Immunohistochemical analysis revealed no significant changes in citH3 quantity in the renal cortex of any group. However, in the renal medulla, the increase in citH3 induced by LPS was abolished in the LPS+rTM group., Conclusions: Our findings demonstrate that rTM can suppress NETs in the renal medulla of mice with endotoxin-induced acute kidney injury.

Some oncoproteins along with stress kinase general control non-derepressible 2 (GCN2) can ensure the induction of activating transcription factor 4 (ATF4) to counteract amino acid deprivation; however, little is known regarding the role of the oncogenic EGFR-PI3K pathway. In this study, we demonstrate that both mutated EGFR and PIK3CA contribute to ATF4 induction following GCN2 activation in NSCLC cells. The inhibition of EGFR or PI3K mutant proteins, pharmacologically or through genetic knockdown, inhibited ATF4 induction without affecting GCN2 activation. A downstream analysis revealed that the oncogenic EGFR-PI3K pathway may utilize mTOR-mediated translation control mechanisms for ATF4 induction. Furthermore, in NSCLC cells harboring co-mutations in EGFR and PIK3CA, the combined inhibition of these oncoproteins markedly suppressed ATF4 induction and the subsequent gene expression program as well as cell viability during amino acid deprivation. Our findings establish a role for the oncogenic EGFR-PI3K pathway in the adaptive stress response and provide a strategy to improve EGFR-targeted NSCLC therapy., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

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., (©2022 American Association for Cancer Research.)

Background: The development of memory B cells after asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is not well understood., Methods: We compared spike antibody titers, pseudovirus neutralizing antibody titers, and memory B-cell responses among SARS-CoV-2 PCR-positive Marine recruits who either reported asymptomatic or symptomatic infection., Results: Thirty-six asymptomatic participants exhibited similar spike IgG titers, spike IgA titers, and pseudovirus neutralization titers compared to 30 symptomatic participants. Pseudovirus neutralization and spike IgG titers showed significant positive correlations with frequency of memory B cells., Conclusions: Among young adults, asymptomatic SARS-CoV-2 infection induced antibody and memory B-cell responses comparable to mild symptomatic infection., Competing Interests: Potential conflicts of interest. S. C. has consulted for GSK, JP Morgan, Citi, Morgan Stanley, Avalia NZ, Nutcracker Therapeutics, University of California, California State Universities, United Airlines, Adagio, and Roche. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2022.)

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., (©2021 The Authors; Published by the American Association for Cancer Research.)

We have experienced an enormous cohesive effort of the scientific community to understand how the immune system reacts to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and how to elicit protective immunity via vaccination. This effort resulted in the development of vaccines in record time with high levels of safety, efficacy, and real-life effectiveness. However, the rapid diffusion of viral variants that escape protective antibodies prompted new studies to understand SARS-CoV-2 vulnerabilities and strategies to guide follow-up actions to increase, and maintain, the protection offered by vaccines. In this review, we report the main findings on human immunity to SARS-CoV-2 after natural infection and vaccination; we dissect the immunogenicity and efficacy of the different vaccination strategies that resulted in products widely used in the population; and we describe the impact of viral variants on vaccine-elicited immunity, summarizing the main discoveries and challenges to stay ahead of SARS-CoV-2 evolution. SIGNIFICANCE STATEMENT: This study reviewed findings on human immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), analyzed the immunogenicity and efficacy of the various vaccines currently used in large vaccination campaigns or candidates in advanced clinical development, and discussed the challenging task to ensure high protective efficacy against the rapidly evolving SARS-CoV-2 virus. This manuscript was completed prior to the emergence of the Omicron variant and to global vaccine boosting efforts., (Copyright © 2022 The Author(s).)

Saponins are potent and safe vaccine adjuvants, but their mechanisms of action remain incompletely understood. Here, we explored the properties of several saponin formulations, including immune-stimulatory complexes (ISCOMs) formed by the self-assembly of saponin and phospholipids in the absence or presence of the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA). We found that MPLA self-assembles with saponins to form particles physically resembling ISCOMs, which we termed saponin/MPLA nanoparticles (SMNP). Saponin-containing adjuvants exhibited distinctive mechanisms of action, altering lymph flow in a mast cell–dependent manner and promoting antigen entry into draining lymph nodes. SMNP was particularly effective, exhibiting even greater potency than the compositionally related adjuvant AS01 B in mice, and primed robust germinal center B cell, T FH , and HIV tier 2 neutralizing antibodies in nonhuman primates. Together, these findings shed new light on mechanisms by which saponin adjuvants act to promote the immune response and suggest that SMNP may be a promising adjuvant in the setting of HIV, SARS-CoV-2, and other pathogens.

Adaptive immune responses to SARS-CoV-2 infection have been extensively characterized in blood; however, most functions of protective immunity must be accomplished in tissues. Here, we report from examination of SARS-CoV-2 seropositive organ donors (ages 10 to 74) that CD4 + T, CD8 + T, and B cell memory generated in response to infection is present in the bone marrow, spleen, lung, and multiple lymph nodes (LNs) for up to 6 months after infection. Lungs and lung-associated LNs were the most prevalent sites for SARS-CoV-2–specific memory T and B cells with significant correlations between circulating and tissue-resident memory T and B cells in all sites. We further identified SARS-CoV-2–specific germinal centers in the lung-associated LNs up to 6 months after infection. SARS-CoV-2–specific follicular helper T cells were also abundant in lung-associated LNs and lungs. Together, the results indicate local tissue coordination of cellular and humoral immune memory against SARS-CoV-2 for site-specific protection against future infectious challenges.

Myoglobin, which can cause acute kidney injury, has a relatively high molecular weight and is poorly cleared by diffusion. We compared and examined myoglobin clearance by changing the blood purification membrane and modality in patients with a myoglobin blood concentration ≥ 1000 ng/ml. We retrospectively analyzed three patient groups based on the following three types of continuous hemofiltration (CHF): AN69ST membrane, polymethylmethacrylate (PMMA) membrane, and high-flow hemodiafiltration (HDF) with increased dialysate flow rate using the PMMA membrane. There was no significant difference in clearance in CHF between AN69ST and PMMA membranes. However, the high-flow HDF group showed the highest myoglobin clearance (p = 0.003). In the PMMA membrane, changing the treatment modality to high-flow HDF increased clearance above the theoretical value, possibly due to internal filtration. To remove myoglobin by kidney replacement therapy from patients with hypermyoglobinemia, a modality such as high-flow HDF would be desirable., (© 2021 International Society for Apheresis, Japanese Society for Apheresis, and Japanese Society for Dialysis Therapy.)