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Notch signaling regulates cell-fate decisions during development and postnatal life. Little is known, however, about the role of Delta-like-4 (Dll4)-Notch signaling between cancer cells, or how this signaling affects cancer metastasis. We, therefore, assessed the role of Dll4-Notch signaling in cancer metastasis. We generated a soluble Dll4 fused to the IgG1 constant region (Dll4-Fc) that acts as a blocker of Dll4-Notch signaling and introduced it into human small cell lung cancer (SCLC) cell lines expressing either high levels (SBC-3 and H1048) or low levels (SBC-5) of Dll4. The effects of Dll4-Fc on metastasis of SCLC were evaluated using a mouse model. Although Dll4-Fc had no effect on the liver metastasis of SBC-5, the number of liver metastasis inoculated with SBC-3 and H1048 cells expressing Dll4-Fc was significantly lower than that injected with control cells. To study the molecular mechanisms of the effects of Dll4-Fc on liver metastasis, a PCR array analysis was conducted. Because the expression of NF-κB target genes was affected by Dll4-Fc, we conducted an electrophoretic mobility shift assay and observed that NF-κB activities, both with and without stimulation by TNF-α, were downregulated in Dll4-Fc–overexpressing SBC-3 and H1048 cells compared with control cells. Moreover, Dll4-Fc attenuates, at least in part, the classical and alternative NF-κB activation pathway by reducing Notch1 signaling. These results suggest that Dll4-Notch signaling in cancer cells plays a critical role in liver metastasis of SCLC by regulating NF-κB signaling. Mol Cancer Ther; 11(12); 2578–87. ©2012 AACR.

PDF file - 330K, Dll4-Fc reduced NF-κB/DNA binding in SBC-3 and H1048 cells

PDF file - 245K, Dll4-Fc promoted excessive angiogenesis of liver metastasis without affecting VEGFA expression and HUVECs proliferation

PDF file - 50K, The effect of recombinant Dll4-Fc on the NF-κB activity of SBC-3-vector cells

PDF file - 116K, Extracellular domain of murine Dll4-fused to Fc (Dll4-Fc) expressed in H1048 and SBC-5 cells

PDF file - 83K

PDF file - 174K, Dll4-Fc suppressed TNF-α induced NF-κB activity without attenuation of the cytoplasmic sequestration of NF-κB subunit

PDF file - 69K, Dll4-Fc suppresses genes-associated with metastasis in SCLC cells

PDF file - 47K, Taqman gene expression assays

The sensing of various extrinsic stimuli triggers the receptor-interacting protein kinase-3 (RIPK3)-mediated signaling pathway, which leads to mixed-lineage kinase-like (MLKL) phosphorylation followed by necroptosis. Although necroptosis is a form of cell death and is involved in inflammatory conditions, the roles of necroptosis in acute pancreatitis (AP) remain unclear. In the current study, we administered caerulein to Ripk3- or Mlkl-deficient mice (Ripk3−/− or Mlkl−/− mice, respectively) and assessed the roles of necroptosis in AP. We found that Ripk3−/− mice had significantly more severe pancreatic edema and inflammation associated with macrophage and neutrophil infiltration than control mice. Consistently, Mlkl−/− mice were more susceptible to caerulein-induced AP, which occurred in a time- and dose-dependent manner, than control mice. Mlkl−/− mice exhibit weight loss, edematous pancreatitis, necrotizing pancreatitis, and acinar cell dedifferentiation in response to tissue damage. Genetic deletion of Mlkl resulted in downregulation of the antiapoptotic genes Bclxl and Cflar in association with increases in the numbers of apoptotic cells, as detected by TUNEL assay. These findings suggest that RIPK3 and MLKL-mediated necroptosis exerts protective effects in AP and caution against the use of necroptosis inhibitors for AP treatment.

Immunoglobulin class switch recombination (CSR) plays critical roles in controlling infections and inflammatory tissue injuries. Here, we show that AFF3 , a candidate gene for both rheumatoid arthritis and type 1 diabetes, is a molecular facilitator of CSR with an isotype preference. Aff3 -deficient mice exhibit low serum levels of immunoglobulins, predominantly immunoglobulin G2c (IgG2c) followed by IgG1 and IgG3 but not IgM. Furthermore, Aff3 -deficient mice show weak resistance to Plasmodium yoelii infection, confirming that Aff3 modulates immunity to this pathogen. Mechanistically, the AFF3 protein binds to the IgM and IgG1 switch regions via a C-terminal domain, and Aff3 deficiency reduces the binding of AID to the switch regions less efficiently. One AFF3 risk allele for rheumatoid arthritis is associated with high mRNA expression of AFF3 , IGHG2 , and IGHA2 in human B cells. These findings demonstrate that AFF3 directly regulates CSR by facilitating the recruitment of AID to the switch regions.

Pulmonary fibrosis is caused by the interplay between genetic and environmental factors. Recent studies have revealed various genes associated with idiopathic pulmonary fibrosis, as well as the causative genes for familial pulmonary fibrosis. Although increased death or dysfunction of type 2 alveolar epithelial (AT2) cells has been detected in lung specimens from pulmonary fibrosis patients, it remains unclear whether and how AT2 cell death or dysfunction is responsible for the progression of pulmonary fibrosis. A recent study showed that increased AT2 cell necroptosis is the initial event in pulmonary fibrosis by analyzing patients with familial pulmonary fibrosis and an animal model that harbors the same mutation as patients. The contribution of AT2 cell necroptosis to the pathogenesis of pulmonary fibrosis has not been identified in animal model studies, which validates the effectiveness of genetic analysis of familial diseases to uncover unknown pathogeneses. Thus, further extensive genetic studies of pulmonary fibrosis along with functional studies based on genetic analysis will be crucial not only in elucidating the precise disease process but also, ultimately, in identifying novel treatment strategies for both familial and non-familial pulmonary fibrosis.

Rapid, sensitive detection of biomolecules is important for improved testing methods for viruses as well as biomarkers and environmental hormones. For example, testing for SARS-CoV-2 is essential in the fight against the COVID-19 pandemic. Reverse-transcription polymerase chain reaction (RT-PCR) is the current standard for COVID-19 testing; however, it is hampered by the long testing process. Shortening the testing process while achieving high sensitivity would facilitate sooner quarantine and thus presumably prevention of the spread of SARS-CoV-2. Here, we aim to achieve rapid, sensitive detection of the SARS-CoV-2 nucleocapsid protein by enhancing the performance of optical biosensing with a dual-comb configuration of optical frequency combs. The virus-concentration-dependent optical spectrum shift is transformed into a photonic RF shift by frequency conversion between the optical and RF regions, facilitating mature electrical frequency measurements. Furthermore, active-dummy temperature-drift compensation enables very small changes in the virus-concentration-dependent signal to be extracted from the large, variable background signal. This dual-comb biosensing technique has the potential to reduce the COVID-19 testing time to 10 min while maintaining sensitivity close to that of RT-PCR. Furthermore, this system can be applied for sensing of not only viruses but also various biomolecules for clinical diagnosis, health care, and environmental monitoring., 42 pages, 6 figures, 2 tables

BNT162b2, a nucleoside-modified mRNA vaccine for SARS-CoV-2 spike glycoprotein (S), provides approximately 95% efficacy for preventing COVID-19. However, it remains unclear how effectively memory CD8+ T cells are generated and which genetic and environmental factors affect the generation and function of memory CD8+ T cells elicited by this vaccine. Here, we investigated the frequency and functions of memory CD8+ T cells 3 weeks after the second vaccination in the Japanese population. Using a peptide-MHC pentamer, we detected an increased number of memory CD8+ T cells together with increased serum anti-S protein antibody in females compared with that in males, but the frequency of pentamer-positive cells was not positively correlated with antibody titers. Memory precursor effector cells (KLRG1-CD127+) among both CD8+ cells and pentamer+ cells and effector cells (CD38-HLA-DR+) among pentamer+ cells were more abundant in females than in males. Upon S protein-mediated stimulation of T cells, the intensity of CD107a and granzyme B expression was increased in females compared with that in males, indicating stronger memory CD8+ T cell responses in females than in males. Our studies showed that the BNT162b2 vaccine elicits increased memory CD8+ T cell proliferation and secondary CTL responses in females compared with those in males in the Japanese population. These findings provide an important basis for the distinct sex difference in cellular immune responses to mRNA vaccination and suggest that memory precursor effector cells can be one of markers to evaluate and boost cellular immunity induced by BNT162b2.

We report a near-infrared surface plasmon resonance (SPR) system to achieve highly sensitive, unlabeled detection of the SARS-CoV-2 nucleocapsid protein antigen. Use of the near-infrared light in SPR makes the SPR dip of the angular spectrum sharp and causes a large change of the reflected light intensity at a fixed incident angle. The present SPR system achieves the resolution of 10−5 refractive index unit in the refractive index measurement of glycerol solution samples. Additionally, we measured the nucleocapsid protein antigen of SARS-CoV-2 down to a molar concentration of 1 fM by immobilizing its corresponding antibody on the SPR sensor surface. This demonstration indicates a high potential of the present system for highly sensitive biosensing in medical diagnostics.

We identify a homozygous mutation in SFTPA1 in patients with idiopathic pulmonary fibrosis (IPF). The mutation causes increased necroptosis of type II alveolar epithelial cells through the IRE1α–JNK axis, which highlights the necroptosis pathway as a therapeutic target for IPF., Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by scattered fibrotic lesions in the lungs. The pathogenesis and genetic basis of IPF remain poorly understood. Here, we show that a homozygous missense mutation in SFTPA1 caused IPF in a consanguineous Japanese family. The mutation in SFTPA1 disturbed the secretion of SFTPA1 protein. Sftpa1 knock-in (Sftpa1-KI) mice that harbored the same mutation as patients spontaneously developed pulmonary fibrosis that was accelerated by influenza virus infection. Sftpa1-KI mice showed increased necroptosis of alveolar epithelial type II (AEII) cells with phosphorylation of IRE1α leading to JNK-mediated up-regulation of Ripk3. The inhibition of JNK ameliorated pulmonary fibrosis in Sftpa1-KI mice, and overexpression of Ripk3 in Sftpa1-KI mice treated with a JNK inhibitor worsened pulmonary fibrosis. These findings provide new insight into the mechanisms of IPF in which a mutation in SFTPA1 promotes necroptosis of AEII cells through JNK-mediated up-regulation of Ripk3, highlighting the necroptosis pathway as a therapeutic target for IPF.

Inactivation technology for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is certainly a critical measure to mitigate the spread of coronavirus disease 2019 (COVID-19). A deep ultraviolet light-emitting diode (DUV-LED) would be a promising candidate to inactivate SARS-CoV-2, based on the well-known antiviral effects of DUV on microorganisms and viruses. However, due to variations in the inactivation effects across different viruses, quantitative evaluations of the inactivation profile of SARS-CoV-2 by DUV-LED irradiation need to be performed. In the present study, we quantify the irradiation dose of DUV-LED necessary to inactivate SARS-CoV-2. For this purpose, we determined the culture media suitable for the irradiation of SARS-CoV-2 and optimized the irradiation apparatus using commercially available DUV-LEDs that operate at a center wavelength of 265, 280, or 300 nm. Under these conditions, we successfully analyzed the relationship between SARS-CoV-2 infectivity and the irradiation dose of the DUV-LEDs at each wavelength without irrelevant biological effects. In conclusion, total doses of 1.8 mJ/cm2 for 265 nm, 3.0 mJ/cm2 for 280 nm, and 23 mJ/cm2 for 300 nm are required to inactivate 99.9% of SARS-CoV-2. Our results provide quantitative antiviral effects of DUV irradiation on SARS-CoV-2, serving as basic knowledge of inactivation technologies against SARS-CoV-2.

The NLRC4 inflammasome assembles in response to detection of bacterial invasion, and NLRC4 activation leads to the production of IL-1β and IL-18 together with pyroptosis-mediated cell death. Missense activating mutations in NLRC4 cause autoinflammatory disorders whose symptoms are distinctly dependent on the site of mutation and other aspects of the genetic background. To determine the involvement of IL-1β and IL-18 in the inflammation induced by NLRC4 mutation, we depleted IL-1β, IL-18, or both cytokines in Nlrc4-transgenic mice in which mutant Nlrc4 is expressed under the MHC class II promoter (Nlrc4-H443P-Tg mice). The deletion of the Il1b or Il18 gene in Nlrc4-H443P-Tg mice reduced the neutrophil numbers in the spleen, and mice with deletion of both genes had an equivalent number of neutrophils compared to wild-type mice. Deletion of Il1b ameliorated but did not eliminate bone marrow hyperplasia, while mice deficient in Il18 showed no bone marrow hyperplasia. In contrast, tail bone deformity remained in the presence of Il18 deficiency, but Il1b deficiency completely abolished bone deformity. The decreased bone density in Nlrc4-H443P-Tg mice was counteracted by Il1b but not Il18 deficiency. Our results demonstrate the distinct effects of IL-1β and IL-18 on NLRC4-induced inflammation among tissues, which suggests that blockers for each cytokine should be utilized depending on the site of inflammation.

Immunoproteasomes degrade ubiquitin-coupled proteins and play a role in creating peptides for presentation by MHC class I proteins. Studies of gene-deficient mice, in which each immunoproteasomal subunit was affected, have demonstrated that dysfunction of immunoproteasomes leads to immunodeficiency, i.e. reduced expression of MHC class I and attenuation of CD8 T-cell responses. Recent studies, however, have uncovered a new type of autoinflammatory syndrome characterized by fever, nodular erythema and progressive partial lipodystrophy that is caused by genetic mutations in immunoproteasome subunits. These mutations disturbed the assembly of immunoproteasomes, which led to reduced proteasomal activity and thus accumulation of ubiquitin-coupled proteins. Those findings suggest that immunoproteasomes function as anti-inflammatory machinery in humans. The discovery of a new type of autoinflammatory syndrome caused by dysregulated immunoproteasomes provides novel insights into the important roles of immunoproteasomes in inflammation as well as the spectrum of autoinflammatory diseases.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and spread globally to cause severe damage to public health and economics worldwide as coronavirus disease 2019 (COVID-19). A deep ultraviolet light-emitting diode (DUV-LED) would be a promising candidate for the inactivation technology of SARS-CoV-2 to mitigate the spread of COVID-19. The DUV-LED has the potential to provide energy-efficient and contact-less virus inactivation technology with no residual substances after treatment, little effect on targets and the applicability of various viruses, including virus variants. This paper introduces an overview of the virus inactivation technology using DUV-LED and discusses the effect of the DUV-LED irradiation on the SARS-CoV-2.

Inactivation technology for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is certainly a critical measure to mitigate the spread of coronavirus disease 2019 (COVID-19). A deep ultraviolet light-emitting diode (DUV-LED) would be a promising candidate to inactivate SARS-CoV-2, based on the well-known antiviral effects of DUV on microorganisms and viruses. However, due to variations in the inactivation effects across different viruses, quantitative evaluations of the inactivation profile of SARS-CoV-2 by DUV-LED irradiation need to be performed. In the present study, we quantify the irradiation dose of DUV-LED necessary to inactivate SARS-CoV-2. For this purpose, we determined the culture media suitable for the irradiation of SARS-CoV-2 and optimized the irradiation apparatus using commercially available DUV-LEDs that operate at a center wavelength of 265, 280, or 300 nm. Under these conditions, we successfully analyzed the relationship between SARS-CoV-2 infectivity and the irradiation dose of the DUV-LEDs at each wavelength without irrelevant biological effects. In conclusion, total doses of 1.8 mJ/cm

Dendritic cells (DCs) are important for adaptive immune responses through the activation of T cells. The molecular interplay between DCs and T cells determines the magnitude of T cell responses or outcomes of functional differentiation of T cells. In this study, we demonstrated that DCs in mice that are Rbpj deficient in CD11c+ cells (Rbpj−/− mice) promoted the differentiation of IL-17A–producing Th17 cells. Rbpj-deficient DCs expressed little Aldh1a2 protein that is required for generating retinoic acid. Those DCs exhibited a reduced ability for differentiating regulatory T cells induced by TGF-β. Rbpj protein directly regulated Aldh1a2 transcription by binding to its promoter region. The overexpression of Aldh1a2 in Rbpj-deficient DCs negated their Th17-promoting ability. Transfer of naive CD4+ T cells into Rag1-deficient Rbpj−/− mice enhanced colitis with increased Th17 and reduced induced regulatory T cells (iTreg) compared with control Rag1-deficient mice. The cotransfer of iTreg and naive CD4+ T cells into Rag1-deficient Rbpj−/− mice improved colitis compared with transfer of naive CD4+ T cell alone. Furthermore, cotransfer of DCs from Rbpj−/− mice that overexpressed Aldh1a2 or Notch-stimulated DCs together with naive CD4+ T cells into Rbpj−/− Rag1-deficient mice led to reduced colitis with increased iTreg numbers. Therefore, our studies identify Notch signaling in DCs as a crucial balancer of Th17/iTreg, which depends on the direct regulation of Aldh1a2 transcription in DCs.

MicroRNAs have broad roles in tumorigenesis and cell differentiation through regulation of target genes. Notch signaling also controls cell differentiation and tumorigenesis. However, the mechanisms through which Notch mediates microRNA expression are still unclear. In this study, we aimed to identify microRNAs regulated by Notch signaling. Our analysis found that microRNA-449a (miR-449a) was indirectly regulated by Notch signaling. Although miR-449a-deficient mice did not show any Notch-dependent defects in immune cell development, treatment of miR-449a-deficient mice with azoxymethane (AOM) or dextran sodium sulfate (DSS) increased the numbers and sizes of colon tumors. These effects were associated with an increase in intestinal epithelial cell proliferation following AOM/DSS treatment. In patients with colon cancer, miR-449a expression was inversely correlated with disease-free survival and histological scores and was positively correlated with the expression of MLH1 for which loss-of function mutations have been shown to be involved in colon cancer. Colon tissues of miR-449a-deficient mice showed reduced Mlh1 expression compared with those of wild-type mice. Thus, these data suggested that miR-449a acted as a key regulator of colon tumorigenesis by controlling the proliferation of intestinal epithelial cells. Additionally, activation of miR-449a may represent an effective therapeutic strategy and prognostic marker in colon cancer.

Background In a previous study, we demonstrated that deficiency of microRNA-449a (miR-449a) promoted colorectal tumorigenesis. In this study, the significance of miR-449a in the prognosis and relationship with HDAC1 in colorectal cancer was examined. Materials and methods Seventy-two patients with colorectal cancer and 16 patients with colorectal liver metastasis who underwent surgery were included. miR-449a expression in tumor tissue of resected specimen was investigated by real-time polymerase chain reaction and histone deacetylase 1 (HDAC1) expression was examined by immunohistochemistry. Results Lymphovascular invasion and increased serum carcinoembryonic antigen levels were seen more frequent in patients with low miR-449a expression. Patients with low miR-449a expression were found to have a poorer prognosis than those with high expression. Vascular invasion, increased serum carbohydrate antigen 19-9 level and low miR-449a were associated with poorer disease-free survival. miR-449a expression was lower in metastatic liver tumor compared to primary tumor. HDAC1 positivity was higher in patients with low miR-449a. Conclusion miR-449a level might be a prognostic indicator for colorectal cancer and miR-449a might regulate HDAC1 expression.

Consumption of brown rice and rice bran fermented with Aspergillus oryzae (FBRA) suppresses spontaneously occurring diabetes in female NOD mouse. While control diet-fed mice showed glucosuria and hyperglycemia at around 20 week of age and the ratio reached to 57% at 30 weeks of age, the ratio did not increase in the 0.5% FBRA-containing diet-fed group. The FBRA-fed group at 30 weeks of age kept higher ratio of intact islets and showed significantly lower insulitis score compared to the control diet group, with dose-dependency from 0.25% to 0.5% dietary concentration of FBRA. The percentage of diabetic mice was significantly lower at 24 weeks of age as compared to the control group (p = 0.01, log rank test). These results indicate that the suppressive effects of dietary administration of 0.5% FBRA in delaying the spontaneous onset of diabetes in NOD mice is probably achieved by maintaining the number of intact islets.

Atopic dermatitis (AD) is caused by both dysregulated immune responses and an impaired skin barrier. Although leukotriene B4 (LTB4) is involved in tissue inflammation that occurs in several disorders, including AD, therapeutic strategies based on LTB4 inhibition have not been explored. Here we demonstrate that progression of an AD-like skin disease in NC/Nga mice is inhibited when docosahexaenoic acid (DHA)/eicosapentaenoic acid (EPA) is administered together with FK506. Treatment with DHA/EPA and FK506 decreases the clinical score of dermatitis in NC/Nga mice and lowers local LTB4 concentrations. The treatment also suppressed the infiltration of T cells, B cells, eosinophils and neutrophils, and promoted reduced serum IgE levels. Secretion of IL-13 and IL-17A in CD4(+) T cells was lower in DHA/EPA- and FK506-treated mice than in mice treated with FK506 alone. The inhibition of disease progression induced by DHA/EPA was reversed by local injection of LTB4, suggesting that the therapeutic effect of DHA/EPA is LTB4-dependent. Our results demonstrate that treatment of AD with DHA/EPA is effective for allergic skin inflammation and acts by suppressing LTB4 production. J. Med. Invest. 63: 187-191, August, 2016.

An interlaboratory comparison of radiation dosimetry was conducted to determine the accuracy of doses being used experimentally for animal exposures within a large multi-institutional research project. The background and approach to this effort are described and discussed in terms of basic findings, problems and solutions.Dosimetry tests were carried out utilizing optically stimulated luminescence (OSL) dosimeters embedded midline into mouse carcasses and thermal luminescence dosimeters (TLD) embedded midline into acrylic phantoms.The effort demonstrated that the majority (4/7) of the laboratories was able to deliver sufficiently accurate exposures having maximum dosing errors of ≤5%. Comparable rates of 'dosimetric compliance' were noted between OSL- and TLD-based tests. Data analysis showed a highly linear relationship between 'measured' and 'target' doses, with errors falling largely between 0 and 20%. Outliers were most notable for OSL-based tests, while multiple tests by 'non-compliant' laboratories using orthovoltage X-rays contributed heavily to the wide variation in dosing errors.For the dosimetrically non-compliant laboratories, the relatively high rates of dosing errors were problematic, potentially compromising the quality of ongoing radiobiological research. This dosimetry effort proved to be instructive in establishing rigorous reviews of basic dosimetry protocols ensuring that dosing errors were minimized.

Homeostasis of Pdpn+ fibroblastic reticular cells (FRCs) is thought to be regulated by hematopoietic cells. However, the cellular and molecular mechanisms of such homeostasis have been poorly understood, especially under the steady-state condition in adults. We show that dendritic cells, particularly CD4+ conventional dendritic cells (cDCs), are crucial for the homeostasis of FRCs in the adult spleen under the steady-state condition. The production of TNF receptor ligands by CD4+ cDCs regulates such homeostasis of FRCs, with SIRPα and CD47 likely being indispensable for the production of TNFR ligands by CD4+ cDCs. The CD47–SIRPα interaction on CD4+ cDCs may thus control the homeostasis of FRCs, which in turn maintain homeostasis of T cells in the white pulp of the spleen.