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Purpose To determine the effect of foveal vitreous cortex removal during pars plana vitrectomy (PPV) for rhegmatogenous retinal detachment (RRD) on the prevention of postoperative epiretinal membrane (ERM) development without internal limiting membrane (ILM) peeling. Design Case-control study. Participants A total of 105 consecutive eyes of 105 patients who underwent primary PPV for RRD between September 2018 and August 2019 and were followed for at least 6 months. Methods The presence of foveal vitreous cortex during PPV was determined by examining the images obtained by a widefield viewing system in 52 eyes (WF group) operated in the first half of the study period and obtained by a high-magnification floating lens in 53 eyes (FL group) operated in the latter half of the study period. Triamcinolone acetonide was used to make the vitreous cortex more visible during PPV. The foveal vitreous cortex was removed if detected by forceps with a high-magnification floating lens without ILM peeling or use of dye staining. The presence of postoperative ERM was examined by using OCT. Main Outcome Measures The rate of detected and removed foveal vitreous cortex during PPV and the incidence of postoperative ERM of each group. Results The rate of detected and removed foveal vitreous cortex during PPV was significantly higher in the FL group than in the WF group (41.5% vs. 15.4%, P = 0.004). The incidence of postoperative ERM was significantly lower in the FL group than in the WF group (1.9% vs. 13.5%, P = 0.03). None of the eyes required additional surgery for the postoperative ERM during the follow-up period. The retinal reattachment rate was not significantly different (98.1% vs. 100%, P = 0.99), and the final retinal attachment rate was 100% in both groups. A dissociated optic nerve fiber layer appearance and a temporal macular thinning were not detected postoperatively in any of the eyes with removal of the foveal vitreous cortex during PPV. Conclusions The detection and removal of foveal vitreous cortex with the high-magnification floating lens during PPV for RRD significantly reduce the incidence of postoperative ERM without adverse findings.

Patients who undergo renal replacement therapy often exhibit a high plasma linezolid concentration. Linezolid is metabolized via oxidation. Nafamostat mesilate has antioxidant effects and is frequently used as an anticoagulant during renal replacement therapy. We aimed to investigate the effect of nafamostat mesilate on plasma linezolid concentration. We examined whether the co-administration of linezolid and nafamostat had any effect on plasma linezolid concentration. Mice were randomly allocated to two groups (n = 18/group): linezolid (100 mg kg-1 , subcutaneous injection) + nafamostat (30 mg kg-1 , intraperitoneal injection) and linezolid + saline. At 5 hours, the linezolid concentration was significantly higher in the linezolid + nafamostat co-administration group than that in the linezolid + saline group (20.6 ± 9.8 vs 3.6 ± 1.2 μg/mL, respectively P < .001). The antioxidant effects of nafamostat may inhibit linezolid metabolism, resulting in the adverse event of high linezolid concentration if both are administered concurrently during renal replacement therapy.

Introduction: Renal replacement therapy (RRT) is widely used in the treatment of septic acute kidney injury. However, little is known about how the adsorption properties of hemofilters used in RRT affect antibiotic concentration. Because a cytokine-adsorption membrane is frequently used in RRT, it is important to determine the antibiotic adsorption capacity of this membrane. Objective: The present study aimed to investigate the antibiotic adsorption capacity of different hemofilter membranes by in vitro experiments using 2 antibacterial agents (linezolid and doripenem). Methods: We performed experimental hemofiltration in vitro using polyacrylonitrile (AN69ST), polymethylmethacrylate (PMMA), and polysulfone (PS) hemofilters for 1,440 min. The test solution was a 1,000-mL substitution fluid containing 30 µg/mL linezolid and 120 µg/mL doripenem. We measured drug concentrations at the inlet, outlet, and filtrate ports of the hemofilters for 1,440 min and calculated the sieving coefficient (SC) and adsorption rate (Ra) of the drugs onto the hemofilters. Results: The amount of linezolid adsorbed onto AN69ST, PMMA, and PS membranes was decreased relative to that in the control group at 15 min (p < 0.05). However, no SC for linezolid was obtained thereafter. The Ra of linezolid onto AN69ST, PMMA, and PS membranes was higher than that in the control group (p < 0.05). In contrast, no significant differences were observed in the concentrations and Ra values of doripenem adsorbed onto AN69ST, PMMA, and PS membranes compared with those in the control group. Conclusions: Doripenem was not adsorbed onto PMMA, PS, and AN69ST membranes. Linezolid was adsorbed onto PMMA, PS, and AN69ST membranes, but only temporarily, and this did not affect drug bioavailability.

Metastasis-associated macrophages (MAM) promote persistent growth of breast cancer cells at the metastatic site and are, thus, an attractive therapeutic target to treat breast cancer metastasis, a leading cause of cancer-related death in women. However, the precise mechanisms behind MAM-mediated metastatic tumor outgrowth have not been fully elucidated. Using mouse models of metastatic breast cancer, we showed that MAMs uniquely expressed hepatocyte growth factor (HGF) in metastatic tumors. We also demonstrated that a selected population of cancer cells with high metastatic potential (cancer cells that can establish metastatic tumors in mice with higher number and incidence than parental cells) had higher expression of HGF receptor, MNNG HOS transforming gene (MET), and were more responsive to HGF released from macrophages compared with the parental cells. Blockade of MET signaling in cancer cells suppressed metastatic tumor expansion, in part, through activation of natural killer cells. Results from this study suggest an approach to prevent life-threatening metastatic tumor formation using blockade of MAM-induced MET signal activation in metastatic cancer cells.

The patients with respiratory failure need high tidal volume by mechanical ventilation, which lead to the ventilator-induced lung injury. We developed an extracorporeal lung and renal assist device (ELRAD), comprising acid infusion, membrane lung, continuous hemodiafiltration and alkaline infusion. To evaluate this system, we conducted in vivo studies using experimental swine which were connected to the new system. In vivo experiments consist of four protocols; baseline = hemodiafiltration only (no O2 gas flow to membrane lung); membrane lung = “Baseline” plus O2 gas flow to membrane lung; “Acid infusion” = “Membrane lung” plus continuous acid infusion; ELRAD = “Acid infusion” plus continuous alkaline infusion. We changed the ventilatory rate of the mechanical ventilation to maintain PCO2 at 50–55 mmHg during the four protocols. The results showed that there was statistically no significant difference in the levels of pH, HCO3−, and base excess when each study protocol was initiated. The amount of CO2 eliminated by the membrane lung significantly increased by 1.6 times in the acid infusion protocol and the ELRAD protocol compared to the conventional membrane lung protocol. Minute ventilation in the ELRAD protocol significantly decreased by 0.5 times compared with the hemodiafiltration only protocol (P

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 immunostimulatory complexes (ISCOMs) formed by the self-assembly of saponin and phospholipids in the absence or presence of the Toll like receptor (TLR) 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 non-human primates. Altogether, these findings shed new light on mechanisms by which saponin adjuvants act to promote the immune response and suggest SMNP may be a promising adjuvant in the setting of HIV, SARS-CoV-2, and other pathogens.

S1PR5 impairs TRM cell differentiation by limiting T cell entry and promoting T cell egress from peripheral tissues. Local TGF-β signaling coordinates suppression of the T-bet–ZEB2–S1PR5 emigration axis, thus enforcing tissue residency., Tissue-resident memory T (TRM) cells provide long-lasting immune protection. One of the key events controlling TRM cell development is the local retention of TRM cell precursors coupled to downregulation of molecules necessary for tissue exit. Sphingosine-1-phosphate receptor 5 (S1PR5) is a migratory receptor with an uncharted function in T cells. Here, we show that S1PR5 plays a critical role in T cell infiltration and emigration from peripheral organs, as well as being specifically downregulated in TRM cells. Consequentially, TRM cell development was selectively impaired upon ectopic expression of S1pr5, whereas loss of S1pr5 enhanced skin TRM cell formation by promoting peripheral T cell sequestration. Importantly, we found that T-bet and ZEB2 were required for S1pr5 induction and that local TGF-β signaling was necessary to promote coordinated Tbx21, Zeb2, and S1pr5 downregulation. Moreover, S1PR5-mediated control of tissue residency was conserved across innate and adaptive immune compartments. Together, these results identify the T-bet–ZEB2–S1PR5 axis as a previously unappreciated mechanism modulating the generation of tissue-resident lymphocytes., Graphical Abstract

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.

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

Purpose: To determine the changes in the foveal architecture before and after internal limiting membrane (ILM) peeling with and without fovea-sparing ILM peeling (FSIP) during vitrectomy for high myopic retinoschisis by intraoperative optical coherence tomography (iOCT). Methods: Qualitative assessments were made of the alterations of the retinal architecture imaged by iOCT following complete ILM peeling (peeled group) or fovea-sparing ILM peeling (FSIP group). A microscope-integrated real-time iOCT device was used to analyze the retinal architectures. The alterations included a worsening of the retinoschisis, development of a foveal detachment, and development of a full thickness macular hole. The changes in the retinal architecture, clinical characteristics, and outcomes were compared between the 2 groups. Results: The medical records of 15 eyes of 15 patients were studied. Complete ILM peeling was performed on 9 of 15 eyes, and 8 of the 9 eyes (89%) showed a worsening of the retinal architectures following the ILM peeling. FSIP was performed on 6 of the 15 eyes, and none of these eyes developed any retinal architectural changes in the iOCT images. The mean postoperative best-corrected visual acuity (BCVA) in eyes that underwent FSIP (20/38) was significantly better than that of preoperative BCVA (20/128; p < 0.05), although the differences in the preoperative BCVA (20/87) and postoperative BCVA (20/55) in the cases that underwent complete ILM peeling were not significant. Conclusions: The lack of alterations of the foveal architecture and significant improvements in the BCVA after FSIP indicate that FSIP peeling should be considered for eyes with retinoschisis. We recommend that iOCT be used to monitor the retinal architecture during intraocular surgery involving the retina.

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.

Background: Polymyxin B-immobilized fiber (PMX-F) columns are used as therapeutic interventions for septic shock. The clinical efficacy has been reported for 2-h applications, but their ability to adsorb endotoxin over longer treatments has not been fully elucidated. We hypothesized that PMX-F columns are capable of endotoxin removal for more than 2 h. Method: We designed closed circuits incorporating either a PMX-F column with an 8.5-mL priming volume (PMX-01R) or a sham-control column, and used inactivated fetal bovine serum as the circulating perfusate. Endotoxin was continuously injected at a fixed rate for 24 h, and perfusate endotoxin concentrations were measured at fixed time points. PMX-01R endotoxin adsorption was calculated from the difference in the endotoxin concentrations. Results: PMX-01R endotoxin adsorption increased continuously in a virtually linear manner. Conclusions: The PMX-01R column showed sustained endotoxin adsorption for at least 24 h. This indicated that PMX-F columns would be capable of clinical endotoxin removal for 24 h.

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.

INTRODUCTION: Immunological memory is the basis for durable protective immunity after infections or vaccinations. Duration of immunological memory after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and COVID-19 is unclear. Immunological memory can consist of memory B cells, antibodies, memory CD4+ T cells, and/or memory CD8+ T cells. Knowledge of the kinetics and interrelationships among those four types of memory in humans is limited. Understanding immune memory to SARS-CoV-2 has implications for understanding protective immunity against COVID-19 and assessing the likely future course of the COVID-19 pandemic. RATIONALE: Assessing virus-specific immune memory over at least a 6-month period is likely necessary to ascertain the durability of immune memory to SARS-CoV-2. Given the evidence that antibodies, CD4+ T cells, and CD8+ T cells can all participate in protective immunity to SARS-CoV-2, we measured antigen-specific antibodies, memory B cells, CD4+ T cells, and CD8+ T cells in the blood from subjects who recovered from COVID-19, up to 8 months after infection. RESULTS: The study involved 254 samples from 188 COVID-19 cases, including 43 samples at 6 to 8 months after infection. Fifty-one subjects in the study provided longitudinal blood samples, allowing for both cross-sectional and longitudinal analyses of SARS-CoV-2–specific immune memory. Antibodies against SARS-CoV-2 spike and receptor binding domain (RBD) declined moderately over 8 months, comparable to several other reports. Memory B cells against SARS-CoV-2 spike actually increased between 1 month and 8 months after infection. Memory CD8+ T cells and memory CD4+ T cells declined with an initial half-life of 3 to 5 months. This is the largest antigen-specific study to date of the four major types of immune memory for any viral infection. Among the antibody responses, spike immunoglobulin G (IgG), RBD IgG, and neutralizing antibody titers exhibited similar kinetics. Spike IgA was still present in the large majority of subjects at 6 to 8 months after infection. Among the memory B cell responses, IgG was the dominant isotype, with a minor population of IgA memory B cells. IgM memory B cells appeared to be short-lived. CD8+ T cell and CD4+ T cell memory was measured for all SARS-CoV-2 proteins. Although ~70% of individuals possessed detectable CD8+ T cell memory at 1 month after infection, that proportion declined to ~50% by 6 to 8 months after infection. For CD4+ T cell memory, 93% of subjects had detectable SARS-CoV-2 memory at 1 month after infection, and the proportion of subjects positive for CD4+ T cells (92%) remained high at 6 to 8 months after infection. SARS-CoV-2 spike-specific memory CD4+ T cells with the specialized capacity to help B cells [T follicular helper (TFH) cells] were also maintained. The different types of immune memory each had distinct kinetics, resulting in complex interrelationships between the abundance of T cell, B cell, and antibody immune memory over time. Additionally, substantially heterogeneity in memory to SARS-CoV-2 was observed. CONCLUSION: Substantial immune memory is generated after COVID-19, involving all four major types of immune memory. About 95% of subjects retained immune memory at ~6 months after infection. Circulating antibody titers were not predictive of T cell memory. Thus, simple serological tests for SARS-CoV-2 antibodies do not reflect the richness and durability of immune memory to SARS-CoV-2. This work expands our understanding of immune memory in humans. These results have implications for protective immunity against SARS-CoV-2 and recurrent COVID-19.

Introduction 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 ≥ 1,000 ng/mL. Methods We retrospectively analyzed three patient groups based on the following three types of continuous hemofiltration: AN69ST membrane, polymethylmethacrylate membrane, and high-flow hemodiafiltration with increased dialysate flow rate using the polymethylmethacrylate membrane. Results There was no significant difference in clearance in continuous hemofiltration between AN69ST and polymethylmethacrylate membranes. However, the high-flow hemodiafiltration group showed the highest myoglobin clearance (p = 0.003). In the polymethylmethacrylate membrane, changing the treatment modality to high-flow hemodiafiltration increased clearance above the theoretical value, possibly due to internal filtration. Conclusion To remove myoglobin by kidney replacement therapy from patients with hypermyoglobinemia, a modality such as high-flow hemodiafiltration would be desirable. This article is protected by copyright. All rights reserved.

Author(s): Dan, Jennifer M; Mateus, Jose; Kato, Yu; Hastie, Kathryn M; Yu, Esther Dawen; Faliti, Caterina E; Grifoni, Alba; Ramirez, Sydney I; Haupt, Sonya; Frazier, April; Nakao, Catherine; Rayaprolu, Vamseedhar; Rawlings, Stephen A; Peters, Bjoern; Krammer, Florian; Simon, Viviana; Saphire, Erica Ollmann; Smith, Davey M; Weiskopf, Daniela; Sette, Alessandro; Crotty, Shane | Abstract: Understanding immune memory to SARS-CoV-2 is critical for improving diagnostics and vaccines, and for assessing the likely future course of the COVID-19 pandemic. We analyzed multiple compartments of circulating immune memory to SARS-CoV-2 in 254 samples from 188 COVID-19 cases, including 43 samples at ≥ 6 months post-infection. IgG to the Spike protein was relatively stable over 6+ months. Spike-specific memory B cells were more abundant at 6 months than at 1 month post symptom onset. SARS-CoV-2-specific CD4 + T cells and CD8 + T cells declined with a half-life of 3-5 months. By studying antibody, memory B cell, CD4 + T cell, and CD8 + T cell memory to SARS-CoV-2 in an integrated manner, we observed that each component of SARS-CoV-2 immune memory exhibited distinct kinetics.

Purpose: To evaluate the outcome of vitrectomy combined with a subretinal injection of tissue plasminogen activator (tPA) to treat a massive subretinal hemorrhage. Methods: The medical records of 11 eyes of 11 patients (seven men, four women, mean age; 74.5 ± 9.6 years) with a massive (>10 disc area) subretinal hemorrhage were reviewed. The patients were treated with vitrectomy combined with a subretinal injection of tPA with or without external drainage. Results: The mean baseline visual acuity was 2.10 ± 0.45 logarithm of the minimum angle of resolution (logMAR) units. The hemorrhage was externalized in three eyes due to bullous hemorrhagic retinal detachment. The subretinal hemorrhage was displaced away from the fovea in 10 eyes (91%) and reduced in one eye. Retinal reattachment was achieved in all eyes however reoperations were needed in five eyes (45%) among the eight eyes (73%) with recurrent vitreous and/or subretinal hemorrhages. The postoperative vision at 3 months was significantly improved to 1.32 ± 0.65 logMAR units ( p = 0.0076). The vision in the two eyes without postoperative subretinal hyperreflective material at the fovea recovered to 0.4 logMAR units but none of the nine eyes with the foveal subretinal hyperreflective material had vision better than 0.1 logMAR units. Conclusion: Vitrectomy with subretinal injection of tPA with or without external drainage of the subretinal hemorrhage was effective in treating massive subretinal hemorrhages.

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.

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.

P-glycoprotein (encoded by MDR1) is a membrane transport protein expressed in the intestine, liver, kidney, placenta, and blood–brain barrier. It excludes various clinically important drugs from cells, such as verapamil, digoxin, tacrolimus, and vinblastine. Therefore, human P-glycoprotein plays important roles in drug absorption, distribution, and excretion. We reported previously that auraptene, a natural compound occurring widely in citrus fruit (e.g., grapefruit), inhibited P-glycoprotein-mediated drug transport. In this study, we investigated the effects of auraptene and other phenylpropanoids on P-glycoprotein expression using human intestinal epithelial LS174T cells and a reporter plasmid expressing 10.2 kbp of the upstream regulatory region of MDR1. Auraptene (7-geranyloxycoumarin), a prenylated coumarin, and several phenylpropanoids, such as 3-(4′-geranyloxy-3′-methoxyphenyl)-2-trans propenoic acid, derricidin [2′-hydroxy-4′-(prenyloxy)chalcone], and 3-(4′-geranyloxyphenyl)-propanoic acid, induced MDR1 promoter activity in LS174T cells. Overexpression of the nuclear receptor human pregnane X receptor gene (NR1I2) enhanced auraptene-induced MDR1 activation. Nuclear factor-kappaB inhibitors, Bay11-7082 and JSH-23, repressed MDR1 activation by auraptene. Western blot analyses showed the induction of P-glycoprotein expression in the auraptene-treated LS174T cells. The citrus phytochemical auraptene can induce the drug efflux transporter P-glycoprotein in human intestinal cells, and thus has the potential to cause food–drug interactions.

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.

Aim To investigate the effects of leuprorelin using a self-administered quality-of-life (QOL) questionnaire in patients with recurrent gynecological cancer. Methods Records of patients who received 3.75 mg leuprorelin every 4 weeks for the treatment of recurrent gynecological cancer were retrospectively reviewed. The physical domain of the QOL questionnaire, Care Notebook, was used to assess physical symptoms. Symptom deterioration was defined as a ≥10-point increase in baseline score; otherwise, symptoms were defined as controlled. Radiological and serological responses were evaluated according to the 2011 Gynecological Cancer Intergroup criteria. Results From 2007 to 2015, 25 patients were administered leuprorelin for the treatment of epithelial ovarian cancer, granulosa cell tumor, endometrial cancer, endometrial stromal sarcoma and clear cell cervical cancer (in 13, 3, 6, 2 and 1 patients, respectively). Twenty patients had received a median of three lines (range 1-12 lines) of chemotherapy. Ten patients had progressive disease during their previous round of chemotherapy. Twenty patients completed the questionnaire every 4 weeks. Following leuprorelin treatment for 8 weeks, the symptom and disease control rates were 65% (13/20) and 44% (11/25), respectively. Two patients, one each with granulosa cell tumor and endometrial cancer, had stable disease at 6 months. Among the 20 patients who completed the QOL questionnaire, symptom control and disease control at 8 weeks showed a significant correlation (P = 0.016). Conclusion Leuprorelin had minimal anticancer activity. The physical domain of the QOL questionnaire could be used to assess effects of hormonal treatment.

Epithelial-mesenchymal transition (EMT) is associated with cancer malignancies such as invasion, metastasis, and drug resistance. In this study, HCT116 human colorectal cancer cells were transduced with SLUG or SNAIL retroviruses, and EMT cells with mesenchymal morphology were established. The EMT cells showed a high invasive activity and resistance to several anticancer agents such as methotrexate, SN-38, and cisplatin. Furthermore, they contained about 1-10% side population (SP) cells that were not stained by Hoechst 33342. This SP phenotype was not stable; the isolated SP cells generated both SP and non-SP cells, suggesting a potential for differentiation. Gene expression analysis of SP cells suggested the alteration of genes that are involved in epigenetic changes. Therefore, we examined the effect of 74 epigenetic inhibitors, and found that two inhibitors, namely I-BET151 and bromosporine, targeting the bromodomain and extra-terminal motif (BET) proteins, decreased the ratio of SP cells to50% compared with the control, without affecting the immediate efflux of Hoechst 33342 by transporters. In addition, compared with the parental cells, the EMT cells showed a higher sensitivity to I-BET151 and bromosporine. This study suggests that EMT development and SP phenotype can be independent events but both are regulated by BET inhibitors in SLUG- or SNAIL-transducted HCT116 cells.

We aimed to investigate the effects of blood purification and cold saline injection sites on the transpulmonary thermodilution values. We measured the cardiac output of eight pigs in every combination of cold saline injection (left jugular and femoral veins) and blood purification sites (right jugular and femoral veins), with or without blood purification. We examined the influence of the difference between the presence and absence of blood purification, vascular sites for blood purification, and sites for cold saline injection on the transpulmonary thermodilution values. Cardiac output measured during blood purification using transpulmonary thermodilution was underestimated; however, there was no difference between vascular sites. Cardiac output measured via injection of cold saline into the femoral vein was higher than that obtained through injection of cold saline into the jugular vein, with or without blood purification.

We describe an MHC class II (I-Ab)–restricted TCR transgenic mouse line that produces CD4+ T cells specific for Plasmodium species. This line, termed PbT-II, was derived from a CD4+ T cell hybridoma generated to blood-stage Plasmodium berghei ANKA (PbA). PbT-II cells responded to all Plasmodium species and stages tested so far, including rodent (PbA, P. berghei NK65, Plasmodium chabaudi AS, and Plasmodium yoelii 17XNL) and human (Plasmodium falciparum) blood-stage parasites as well as irradiated PbA sporozoites. PbT-II cells can provide help for generation of Ab to P. chabaudi infection and can control this otherwise lethal infection in CD40L-deficient mice. PbT-II cells can also provide help for development of CD8+ T cell–mediated experimental cerebral malaria (ECM) during PbA infection. Using PbT-II CD4+ T cells and the previously described PbT-I CD8+ T cells, we determined the dendritic cell (DC) subsets responsible for immunity to PbA blood-stage infection. CD8+ DC (a subset of XCR1+ DC) were the major APC responsible for activation of both T cell subsets, although other DC also contributed to CD4+ T cell responses. Depletion of CD8+ DC at the beginning of infection prevented ECM development and impaired both Th1 and follicular Th cell responses; in contrast, late depletion did not affect ECM. This study describes a novel and versatile tool for examining CD4+ T cell immunity during malaria and provides evidence that CD4+ T cell help, acting via CD40L signaling, can promote immunity or pathology to blood-stage malaria largely through Ag presentation by CD8+ DC.

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.

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. 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. 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

B cells are efficiently activated by antigens presented on cell membranes, which provide opportunity for receptor cross-linking and antigen capture. The two main cell types implicated in native antigen presentation to B cells are follicular dendritic cells (FDC), which reside in B cell follicles, and CD169+ macrophages, which line the antigen-exposed surfaces of these follicles in both the lymph nodes and the spleen. There is mounting evidence, however, that conventional dendritic cells (cDC) can also participate in native antigen presentation to B cells. This underappreciated role, largely hidden by the simultaneous need for cDC to participate in T cells priming, appears to be primarily mediated by the type 2 subset of cDC (cDC2), but may also be a function of cDC1. Better understanding of how cDC participate in B cell priming is likely to improve our capacity to develop effective humoral vaccines.

In this study, the results of our previously reported technique of quantitative analysis by using microscopic image analysis of tissue image slices to calculate the proportion of the area of the tunica media occupied by of elastic fibers was compared with Janzen et al.'s technique at the carotid bifurcation. This particularly analyzed the area of transition between the common carotid and the internal carotid, to observe the quantitative changes in elastic fiber content. The data obtained from our quantitative analysis of elastic fibers were clearly at variance with those obtained by counting the number of elastic fibers. The amount of elastic fibers in the tunica media (the elastic fiber ratio) decreased from the proximal carotid artery (the common carotid) to the bifurcation, then peaked in the internal carotid immediately after the bifurcation before declining again.

Summary 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.

Introduction: The FGF family plays key roles in many developmental and physiological processes. Aberrant activation of FGF signals (receptor mutations, fusions, and amplifications of ligands or receptors) have been reported, and considered to cause malignant cancers. Targeting FGFR suppresses proliferation and survival of tumor cells with activated FGF signal. However, the role of tumor FGFR inhibition in tumor microenvironments (TMEs) is largely still unknown. Lenvatinib (LEN) is a multi-targeted tyrosine kinase inhibitor that mainly inhibits VEGFR1-3 and FGFR1-4. Previously we showed that LEN inhibited tumor FGF signal, and induced cancer cell death, which had a role in antitumor activity. Here, we investigated roles of targeting tumor FGFR in TMEs using LEN or FGFR1-3 specific inhibitor E7090 in tumor cells with activated FGF signals. Methods: To examine effects of FGF signal on gene expression profile (GEP) of tumor cells, mouse breast tumor 4T1 cell lines expressing Luc2 and AcGFP (4T1-Luc), human hepatocellular carcinoma (HCC) Hep3B2.1-7 and HuH-7 cells with FGF19 overexpression, and an endometrial cancer (EC) AN3CA cell line harboring FGFR2 mutation were treated with LEN or E7090. GEP was analyzed by RNA-Seq or qPCR in the 4T1-Luc model. Pathway enrichment analysis using RNA-Seq data was conducted by Ingenuity pathway analysis (IPA). Expression levels of type I IFN-related genes were analyzed in HCC and EC cell lines. Expressions of Cxcl10 and HMGB1 in culture supernatant were measured by ELISA. Cell surface expressions of PD-L1, MHC class I molecules and Calreticulin (CRT) were determined by flow cytometry. Results: Unbiased pathway analysis using RNA-Seq data (fold change >2 or 1) from 4T1-Luc cells treated with LEN or E7090 identified Interferon (IFN) signal as one of the top 3 most significantly regulated pathways with several genes known to regulate tumor immunity. qPCR analysis showed that LEN and E7090 increased expression of type I IFN-related genes in human HCC and EC cell lines. LEN significantly increased CXCL10 in culture supernatant and expression of MHC class I and PD-L1 on cell surfaces of 4T1-Luc and Hep3B2.1-7 cells. E7090 also increased Cxcl10 in culture sup and expressions of MHC class I and PD-L1 on cell surface of 4T1-Luc cells. In addition to type I IFN-response, we found that LEN and E7090 might regulate ICD evidenced by increased expression levels of cell surface CRT, and Len increased secreted HMGB1 in culture sup of Hep3B2.1-7 cells. Conclusion: Our results suggested that tumor FGF signals modulated immune response in TMEs through regulating interferon signal. Tumor FGF signal inhibition by LEN and E7090 may enhance antitumor immune response in the TMEs of that were immune suppressive with activated FGF signals. Further analyses will be warranted. Citation Format: Taisuke Hoshi, Yu Kato, Yasuhiro Funahashi. Targeting fibroblast growth factor (FGF) signals induced type I IFN regulated response and Immunogenic cell death (ICD) markers in tumor cells with activated FGF signals [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2976.

Endothelial Notch signaling is critical for tumor angiogenesis and growth. Inhibitors of Notch1 or its ligand Dll4 cause tumor vessel hypersprouting but interfere with vessel perfusion causing reduced tumor growth. Unfortunately, normal tissue vessels respond to these inhibitors causing tissue hypoxia or vascular neoplasms. Mice null for Notch4, unlike Notch1 mutants, exhibit reduced developmental and tumor angiogenesis. Notch4 expression is increased in the endothelium of some tumor types. These results suggest that specifically targeting Notch4 could reduce tumor angiogenesis with fewer effects on normal tissue and represents an unexplored therapeutic opportunity. We use a newly developed anti-Notch4 antibody, E7011, and show that in endothelial cells, inhibition of Notch4 signaling suppresses expression of a subset of canonical Notch target genes. Treatment with E7011 also suppresses expression of a unique set of genes not regulated by canonical Dll4-Notch1 signaling, which appear to be distinct Notch4 targets. We administered E7011 to neonatal mice to examine the effects on developmental retinal angiogenesis. A single dose of E7011 significantly reduced retinal vascular outgrowth, demonstrating that E7011 has anti-angiogenic activity in vivo. E7011 treatment did not alter capillary density within the vascular plexus or endothelial tip cell number, both of which are typically increased by loss of Notch1 signaling. Because the E7011 phenotype was markedly distinct from the Notch1 loss of function, we combined E7011 treatment with endothelial-specific loss of Notch1 (Notch1ECKO) to compare Notch1 to Notch4 function. E7011-treated Notch1ECKO retina showed a compound phenotype, exhibiting both the loss of vascular outgrowth characteristic of E7011/Notch4 and the hypersprouting characteristic of Notch1 loss of function. These results suggest that Notch1 and Notch4 have distinct roles in developmental angiogenesis, with Notch4 being a pro-angiogenic signaling pathway. We evaluated Notch4 expression in the breast cancer tumor microenvironment to determine if Notch4 functions in tumor vessels. Human triple negative breast cancer (TNBC) shows a range of expression of Notch4 in both tumor cells and tumor endothelium. The anti-angiogenic effects that we observe in the retina lead us to hypothesize that E7011 will show anti-tumor activity against tumors that induce Notch4 expression in neighboring endothelium, and that targeting endothelial Notch4 is a novel anti-angiogenic approach. Citation Format: L. A. Naiche, Bhairavi Swaminathan, Yu Kato, Sarita Das, Jason W. Eng, Qanber Raza, Krishna Thakkar, James H. Herts, Rajyasree Emmadi, Junji Matsui, Jan K. Kitajewski. A novel Notch4 neutralizing antibody inhibits angiogenesis and tumor growth via a distinct mechanism from endothelial Notch1 inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1488.

Notch4 is selectively expressed on tumor endothelial cells and hypothesized to contribute to cancer malignancy through mechanisms including tumor angiogenesis regulation and promoting drug resistance. We developed E7011, a humanized IgG2κ antibody targeting Notch4 receptor signaling. To obtain anti-human Notch4 monoclonal antibody, human Notch4 3EGFR (epidermal growth factor repeat) domain - NRR (negative regulatory region) - SEAP (secreted alkaline phosphatase) - His protein was immunized to mice and 6698 hybridomas were obtained. By screening these clones with the Notch4 binding assay and Notch4-specific gal4-fusion luciferase reporter assay, mouse anti-human Notch4 mAb clone (6-3-A6) was selected. The CDRs (complementarity determining regions) of 6-3-A6 were grafted into the variable regions of human IgG2κ, and E7011, novel humanized anti-human Notch4 monoclonal antibody, was developed by screening in assays above. The biomolecular interaction analysis showed that E7011 is able to bind to human and mouse Notch4-NRR domain-SEAP-His, and inhibit human Notch4 receptor signaling in an antibody-dose dependent manner as well as 6-3-A6. IHC analysis showed that Notch4 was expressed on CD31 positive endothelial cells in Calu6 human non-small cell lung carcinoma xenograft tumor. Intravenous administration of E7011 at 1, 3, 10 mg/kg in twice a week significantly decreased tumor growth compared with control IgG treatment without severe body weight loss. We also investigated the functional tumor angiogenesis by hoechest dye-perfusion and staining with anti-CD31 antibody in the Calu6 xenograft model. E7011 treatment significantly decreased hoechst-positive perfusion area compared with vehicle treatment. In conclusion, E7011 is a novel antitumor agent that is a specific signal inhibitor targeting the Notch4 receptor and suppresses tumor growth through decreasing tumor vessel function in based on preclinical studies. Citation Format: Yusuke Adachi, Yoshimasa Sakamoto, Youya Nakazawa, Sho Tachino, Ken Ito, Takanori Abe, Yukinori Minoshima, Kana Hoshino-Negishi, Hideaki Ogasawara, Tomomi Kawakatsu, Miyuki Nishimura, Masashi Shimizu, Kazuhiro Tahara, Toshitaka Sato, Katsuhisa Suzuki, Kishan Agarwala, Masao Iwata, Yasuhiro Funahashi, Kenichi Nomoto, Yoichi Ozawa, Junji Matsui, Toshio Imai, Yu Kato. E7011, a novel anti-angiogenesis inhibitor targeting Notch4 receptor signal pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1485.

CD4+ T cell responses are crucial for the control of many intracellular pathogens, yet the requirements for their induction are not fully understood. To better understand the role that various dendritic cell (DC) subtypes play in CD4+ T cell priming, we compared in vivo T cell responses to skin inoculation of mice with infectious or UV-inactivated HSV type 1. Localized infection elicited a Th1 response that was primed in skin-draining lymph nodes involving Ag presentation by migratory dermal and lymph node–resident DC. However, expansion and Th1 differentiation was impaired in response to UV-inactivated virus (UV-HSV), and this defect correlated with a restriction of Ag presentation to migratory CD103– dermal DC. A similar differentiation defect was seen in infected mice lacking CD8α+ and CD103+ classical type 1 DC (cDC1). Finally, Th1 differentiation after UV-HSV inoculation was rescued by targeted Ag delivery to CD8α+ and CD103+ cDC1 using an anti-Clec9A Ab construct. This suggests that Ag presentation by cDC1 is crucial for optimal Th1 immunity to HSV type 1 infection and potentially other pathogens of the skin.

Purpose To report the clinical characteristics of asymptomatic cases with RP1L1 gene mutations in four families with occult macular dystrophy (OMD). Methods Four asymptomatic cases from four families were selected from a cohort of 40 subjects (16 families) with RP1L1 pathogenic variants. Clinical data of the four asymptomatic cases and three symptomatic patients in the same families were reviewed. The three asymptomatic cases did not have any visual symptoms in either eye, and one was unilaterally affected. Ophthalmologic examinations, including spectral-domain optical coherence tomography (OCT) were performed, and the morphologic characteristics of the photoreceptor layer of the asymptomatic cases were compared to those of the symptomatic patients within the same family. Results The OCT images demonstrated photoreceptor abnormalities in the parafoveal regions in all of the four asymptomatic cases (i.e., absence of the interdigitation zone and blurring of the ellipsoid zone). However, these microstructures were preserved at the foveal center. The longitudinal reflectivity profiles clearly identified this distinct pattern in the asymptomatic cases. In contrast, no distinct abnormalities were detected by other examinations including perimetry, fundus autofluorescence images, and multifocal electroretinograms (ERGs). Conclusions The sparing of the central foveal photoreceptor layer accounts for the well-preserved visual acuity in the asymptomatic patients. The sparing may represent either the initial phase of typical OMD or a subtype of macular lesion associated with OMD. It is necessary to examine asymptomatic subjects in families with OMD because some of them may progress to the typical phenotype of OMD.

Promising results have been reported with blood purification as adjuvant treatment; however, the immunological mechanisms remain unclear. We have been developing a new blood purification system for regulating excessive immune reactions in severe sepsis and septic shock using a granulocyte adsorbing column (Adacolumn [Ada]), and a cytokine-adsorbing hemofilter (AN69ST hemofilter [AN69]). Fresh porcine blood was circulated for 6 h in five experimental groups including Ada and AN69 to assess the effects of leukocyte adsorption, phagocytic activity and adhesiveness of granulocytes. In the present study, we found that Ada mainly adsorbed granulocytes and monocytes, but not lymphocytes. The phagocytic activity level of granulocytes decreased, and adhesiveness increased, but the number of CD11b-positive cells markedly decreased in the current system. Elevated cytokine levels (IL-1β, IL-8 and IL-10) at the outlet of Ada were significantly lower than at the outlet of AN69 due to cytokine adsorption. Further studies are needed to better understand cellular interactions.

Aim To investigate the effects of the c-Jun N-terminal kinase (JNK1/2) on the inflammation cytokine tumour necrosis factor-alpha (TNF-α)-enhanced production of matrix metalloproteinase-3 (MMP-3) in human dental pulp fibroblast-like cells (HPFs). Methodology HPFs were grown from pulp explants from healthy donors. Primary cultures were established by culturing the cells for 20 to 30 days. The experiments with HPFs were performed between passages 3 and 10. The HPFs were incubated in serum-free medium containing TNF-α for 24 h. The medium in each well was prepared in SDS sample buffer and was analysed for MMP-3 by Western blotting. Results JNK inhibitor SP601245 markedly inhibited the production of MMP-3 in TNF-α-stimulated human dental pulp fibroblasts. MMP-3 production was enhanced by TNF-α in HPFs; silencing JNK1 and JNK2 expression inhibited this activation. cAMP response element-binding protein (CREB) was activated by TNF-α in HPFs; silencing JNK1 and JNK2 expression inhibited this activation. Conclusion The activation of CREB via JNK pathways in the presence of TNF-α occurred with enhancement of MMP-3 production in dental pulp fibroblasts.

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.

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell's arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

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.

Lenvatinib is a multiple receptor tyrosine kinase inhibitor targeting mainly vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) receptors. We investigated the immunomodulatory activities of lenvatinib in the tumor microenvironment and its mechanisms of enhanced antitumor activity when combined with a programmed cell death-1 (PD-1) blockade. Antitumor activity was examined in immunodeficient and immunocompetent mouse tumor models. Single-cell analysis, flow cytometric analysis, and immunohistochemistry were used to analyze immune cell populations and their activation. Gene co-expression network analysis and pathway analysis using RNA sequencing data were used to identify lenvatinib-driven combined activity with anti-PD-1 antibody (anti-PD-1). Lenvatinib showed potent antitumor activity in the immunocompetent tumor microenvironment compared with the immunodeficient tumor microenvironment. Antitumor activity of lenvatinib plus anti-PD-1 was greater than that of either single treatment. Flow cytometric analysis revealed that lenvatinib reduced tumor-associated macrophages (TAMs) and increased the percentage of activated CD8+ T cells secreting interferon (IFN)-γ+ and granzyme B (GzmB). Combination treatment further increased the percentage of T cells, especially CD8+ T cells, among CD45+ cells and increased IFN-γ+ and GzmB+ CD8+ T cells. Transcriptome analyses of tumors resected from treated mice showed that genes specifically regulated by the combination were significantly enriched for type-I IFN signaling. Pretreatment with lenvatinib followed by anti-PD-1 treatment induced significant antitumor activity compared with anti-PD-1 treatment alone. Our findings show that lenvatinib modulates cancer immunity in the tumor microenvironment by reducing TAMs and, when combined with PD-1 blockade, shows enhanced antitumor activity via the IFN signaling pathway. These findings provide a scientific rationale for combination therapy of lenvatinib with PD-1 blockade to improve cancer immunotherapy.

The 26S proteasome is a large, complex multisubunit protease involved in protein quality control and other critical processes in eukaryotes. More than 110 post-translational modification (PTM) sites have been identified by a mass spectrometry of the 26S proteasome of Saccharomyces cerevisiae and are predicted to be implicated in the dynamic regulation of proteasomal functions. Here, we report that the N-myristoylation of the Rpt2 subunit controls the intracellular localization of the 26S proteasome. While proteasomes were mainly localized in the nucleus in normal cells, mutation of the N-myristoylation site of Rpt2 caused diffusion of the nuclear proteasome into the cytoplasm, where it formed aggregates. In mutant cells, the level of accumulation of cytoplasmic proteasomes was significantly increased in the nonproliferating state. Although the molecular assembly and peptidase activity of the 26S proteasome were totally unchanged in the nonmyristoylated mutants of Rpt2, an increased level of accumulation of polyubiquitinated proteins and a severe growth defect were observed in mutant cells induced for protein misfolding. In addition, polyubiquitinated protein and the nuclear protein Gcn4 tended not to colocalize with the proteasome in normal and mutant cells. Our results suggest that N-myristoylation is involved in regulating the proper intracellular distribution of proteasome activity by controlling the nuclear localization of the 26S proteasome.