Cytokines may be crucially involved in the pathogenesis of inflammatory bowel diseases (IBD), but it remains controversial whether interferon (IFN)-gamma, a typical proinflammatory cytokine, is an essential mediator to cause the disorders. In the present study, IFN-gamma(-/-) and wild-type (WT) C57BL/6 mice were fed 2.5% dextran sodium sulphate (DSS) in drinking water for 7 days, in order to investigate DSS-induced intestinal inflammation. The DSS-treated WT mice exhibited a robust production of IFN-gamma in the gut, a remarkable loss of body weight, as well as high rate of mortality (60%). In striking contrast, IFN-gamma deficient mice did not develop DSS-induced colitis, as indicated by the maintenance of body weight and survival rate of 100%. Severe intestinal inflammation was demonstrated exclusively in WT animals in terms of the shortening of the bowel as well as the elevation of the disease activity index, myeloperoxidase (MPO) activity and serum haptoglobin level. Histological study of DSS-treated WT intestine revealed disruption of mucosal epithelium and massive infiltration of inflammatory cells, while the organ from IFN-gamma(-/-) mice remained virtually normal in appearance. Enzyme-linked immunosorbent assay (ELISA) analyses indicated abundant production of three chemokines, i.e. monokine induced by interferon-gamma (MIG), interferon-inducible protein 10 (IP-10) and monocyte chemoattractant protein-1 (MCP-1), in the DSS-irritated intestine of WT but not of IFN-gamma(-/-) mice. The present results demonstrate clearly that IFN-gamma plays indispensable roles in the initiation of DSS colitis, and some chemokines are produced in an IFN-gamma-dependent fashion.

SARS-CoV-2 has mutated during the global pandemic leading to viral adaptation to medications and vaccinations. Here we describe an engineered human virus receptor, ACE2, by mutagenesis and screening for binding to the receptor binding domain (RBD). Three cycles of random mutagenesis and cell sorting achieved sub-nanomolar affinity to RBD. Our structural data show that the enhanced affinity comes from better hydrophobic packing and hydrogen-bonding geometry at the interface. Additional disulfide mutations caused the fixing of a closed ACE2 conformation to avoid off-target effects of protease activity, and also improved structural stability. Our engineered ACE2 neutralized SARS-CoV-2 at a 100-fold lower concentration than wild type; we also report that no escape mutants emerged in the co-incubation after 15 passages. Therapeutic administration of engineered ACE2 protected hamsters from SARS-CoV-2 infection, decreased lung virus titers and pathology. Our results provide evidence of a therapeutic potential of engineered ACE2.

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Osteoblasts produce calcified bone matrix and contribute to bone formation and remodeling. In this study, we established a procedure to directly convert human fibroblasts into osteoblasts by transducing some defined factors and culturing in osteogenic medium. Osteoblast-specific transcription factors, Runt-related transcription factor 2 (Runx2), and Osterix, in combination with Octamer-binding transcription factor 3/4 (Oct4) and L-Myc (RXOL) transduction, converted ∼80% of the fibroblasts into osteocalcin-producing cells. The directly converted osteoblasts (dOBs) induced by RXOL displayed a similar gene expression profile as normal human osteoblasts and contributed to bone repair after transplantation into immunodeficient mice at artificial bone defect lesions. The dOBs expressed endogenous Runx2 and Osterix, and did not require continuous expression of the exogenous genes to maintain their phenotype. Another combination, Oct4 plus L-Myc (OL), also induced fibroblasts to produce bone matrix, but the OL-transduced cells did not express Osterix and exhibited a more distant gene expression profile to osteoblasts compared with RXOL-transduced cells. These findings strongly suggest successful direct reprogramming of fibroblasts into functional osteoblasts by RXOL, a technology that may provide bone regeneration therapy against bone disorders.O steoblasts play a central role in bone formation and remodeling by producing type I collagen, osteopontin, osteocalcin, and bone sialoprotein (BSP), and calcifying these bone matrixes (1). They are also involved in hematopoiesis, phosphate metabolism, and glucose metabolism (2). Osteoblasts are derived from mesenchymal progenitor cells that are common precursors shared by chondrocytes, adipocytes, and myoblasts (3). The differentiation of osteoblasts is regulated by various transcription factors, including Runt-related transcription factor 2 [Runx2, also known as core-binding factor subunit α-1 (Cbfα-1)] (4, 5), Osterix (6, 7), Distal-less homeobox 5 (Dlx5) (8), and activation transcription factor 4 (ATF-4) (8). A functional decline in osteoblasts relative to osteoclasts results in imbalance between bone formation and resorption and may cause osteolytic pathological conditions, such as osteoporosis (9), alveolar bone resorption associated with periodontitis (10), and bone lysing associated with bone tumors, including multiple myeloma (11).It has been demonstrated that forced expression of combinations of some transcription factors, such as Octamer-binding transcription factor 3/4 (Oct4), Sox2, Klf-4, and c-Myc (reprogramming factors), induces immortality and pluripotency in mammalian somatic cells (12, 13). The generation of induced pluripotent stem (iPS) cells clearly indicates that genome-wide epigenetic programming can be drastically changed in somatic cells by a small number of transcription factors that may have key regulatory roles in cell fate decisions (14,15).Recent studies have reported that direct conversion, or direct reprogramming, of somatic cells into another dif...

Naked plasmid DNA (pDNA) and short interfering RNA (siRNA) duplexes were transduced into adult murine heart by means of sonoporation using the third-generation microbubble, BR14. Plasmid DNAs carrying luciferase, b-galactosidase (b-gal), or enhanced green fluorescent protein (EGFP) reporter genes were mixed with BR14 and injected percutaneously into the left ventricular (LV) cavity of C57BL/6 mice while exposed to transthoracic ultrasound at 1 MHz for 60 s. Sonoporation at an output intensity of 2.0 W/cm 2 and a 50% pulse duty ratio resulted in the highest luciferase expression in the heart. Histological examinations revealed significant expression of the b-gal and EGFP reporters in the subendocardial myocardium of LV. Intraventricular co-injection of siRNA-GFP and BR14 with concomitant ultrasonic exposure resulted in substantial reduction in EGFP expression in the coronary artery in EGFP transgenic mice. The present method may be applicable to gain-of-function and loss-of-function genetic engineering in vivo of adult murine heart.

SummaryWe have recently revealed that the thymus is the organ showing the highest expression of thromboxane (TX) A2 receptor in mice. In this study, thymic cell populations expressing the receptor were identified, and the effects of a TXAz agonist on these ceUs were examined. Radioligand binding using a TXAz receptor-specific radioligand revealed a single class of binding sites in the thymocytes with an at~inity and specificity identical to those reported for the TXAu receptor. The receptor density in these cells was comparable to that seen in blood platdets. This receptor is most highly expressed in CD4-8-and CD4+8 + immature thymocytes, followed by CD4 +8-and CD4-8 + cells. The receptor density in splenic T cells was less than one fifth of that in CD4 + 8 + cells and no binding activity was detectable in splenic B cells. The addition of a TXA~ agonist, STA2, to thymocytes induced the disappearance of the CD4+8 + calls in a time-and concentration-dependent manner and caused DNA fragmentation. These changes were blocked by a specific TXA2 antagonist, S-145. These results demonstrate that TXA2 induces apoptotic cell death in immature thymocytes by acting on the TXAa receptor on their cell surface and suggest a role for the TXA2/TXA2 receptor system in the thymic microenvironment.p rostanoids such as prostaglandins (PGs), thromboxane (TX), and leukotrienes (LTs) are a family of oxygenated arachidomte metabolites that exit a variety of actions to maintain local homeostasis in the body (1, 2). Their actions as inflammatory mediators are well known; for example, PGE2 causes vasodilation to increase local blood flow, LTB4 induces chemotaxis and activates neutrophils, and LTC4 increases vascular permeability. Some of these compounds are produced upon immunologic challenge and are mediators of anaphylaxis. A recent report also suggests the participation of PGE2 to the apoptotic cell death of ovarian surface epithelial calls (3). However, little is known about their action in the thymus. TXA~ is a very unstable arachidonate metabolite with a half life of about 30 s under physiological conditions. Yet, it is a potent stimulator of platelets and a constrictor of vascular and respiratory smooth muscles, and is presumed to play a role in thrombosis, myocardial infarction, and bronchial asthma (4). This compound is synthesized by a specific enzyme, TX synthase, and functions through a cell surface receptor. We have cloned cDNAs for the human and mouse TXA2 receptors and revealed that they bdong to the family of rhodopsintype receptors (5, 6). Northern blot analysis in various mouse organs showed that the TXAz receptor mRNA is most abundantly expressed in the thymus, followed by the spleen and lung (6). On the other hand, TX synthase is richest in platdets, foUowed by macrophages (7). In the thymus, it is present in the cortical epithdial cells and dendritic cells of the medulla (8, 9). These findings urged us to investigate the localization of the TXAz receptor and its possible role in the thymus. In this study, using a radio...

Direct intratumoral transfection of cytokine genes was per-

Interleukin (IL)-27 is an IL-12 family cytokine playing a pivotal role in the induction of Th1 immune responses, although its action on natural killer (NK) cells has not been fully elucidated. Here, we show that IL-27 is capable of inducing phosphorylation of signal transducers and activators of transcription 1 and 3, as well as expression of T-bet and granzyme B in murine DX-5+ NK cells. IL-27 also enhances cytotoxic activity of NK cells both in vitro and in vivo, while the in vitro viability of NK cells is also improved by this cytokine. Therapeutic administration of the IL-27 gene drastically suppressed the growth of NK-unsusceptible SCCVII tumors that had been preestablished in syngenic mice, resulting in significant prolongation of the survival of the animals. This can likely be ascribed to the antibody-dependent cellular cytotoxicity machinery because IL-27 successfully induced tumor-specific IgG in the sera of the tumor-bearing mice, and supplementation of the sera enabled IL-27-activated NK cells to kill SCCVII cells in an Fc; receptor IIIdependent manner. These findings strongly suggest that IL-27 may offer a powerful immunotherapeutic tool to eradicate head and neck squamous cell carcinoma and other poorly immunogenic neoplasms through activating NK cells and inducing tumor-specific immunoglobulin that may cooperatively elicit antibody-dependent cellular cytotoxicity activity.