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Immunoglobulin A (IgA) is a candidate antibody for oral passive immunization against mucosal pathogens like Shiga toxin-producing Escherichia coli (STEC). We previously established a mouse IgG monoclonal antibody (mAb) neutralizing Shiga toxin 1 (Stx1), a bacterial toxin secreted by STEC. We designed cDNA encoding an anti-Stx1 antibody, in which variable regions were from the IgG mAb and all domains of the heavy chain constant region from a mouse IgA mAb. Considering oral administration, we expressed the cDNA in a plant expression system aiming at the production of enough IgA at low cost. The recombinant-IgA expressed in Arabidopsis thaliana formed the dimeric IgA, bound to the B subunit of Stx1, and neutralized Stx1 toxicity to Vero cells. Colon injury was examined by exposing BALB/c mice to Stx1 via the intrarectal route. Epithelial cell death, loss of crypt and goblet cells from the distal colon were observed by electron microscopy. A loss of secretory granules containing MUC2 mucin and activation of caspase-3 were observed by immunohistochemical methods. Pretreatment of Stx1 with the plant-based recombinant IgA completely suppressed caspase-3 activation and loss of secretory granules. The results indicate that a plant-based recombinant IgA prevented colon damage caused by Stx1 in vivo., (© 2022. The Author(s).)

Quality of life of patients who undergo allogeneic hematopoietic stem cell transplantation (HSCT) temporally deteriorates and recovers over several years. We retrospectively evaluate the impact of chronic graft-versus-host disease (GVHD) and glucocorticoid on physical recovery. We included 162 patients who underwent their first allogeneic HSCT between October 2010 and December 2015 in a single hospital. All patients are planned to undergo physical function tests before and 1, 3, 12 months after allogeneic HSCT. Scores of knee extension strength and distance covered in the 6-min walk test (6MWT) recovered at the 12-month assessment. Both chronic GVHD and high dose glucocorticoid were associated with delayed recovery of body mass index (BMI), hand grip strength, knee extension strength, and duration of standing on one foot. Lung GVHD and high dose glucocorticoid had negative impact on the distance covered in the 6MWT. A multivariate analysis revealed that chronic GVHD and glucocorticoid was an independent risk factor for decreased BMI and delayed recovery of muscle strength, respectively. Our results suggest that high-risk patients who have chronic GVHD or who receive glucocorticoid therapy may require reduced dose of glucocorticoid and long-term physical support to recover physical function after transplantation.

Infection and inflammation can induce acute graft-vs.-host disease (aGVHD). We hypothesized that febrile neutropenia early after allogeneic hematopoietic cell transplantation (HCT) would increase the risk of aGVHD and non-relapse mortality (NRM). We retrospectively evaluated the impact of fever, C-reactive protein (CRP) concentration and blood stream infection (BSI) early after HCT on the incidence of grade II-IV aGVHD and NRM in 227 patients. Within 7 days after HCT, 91 (40.1%) patients experienced fever for at least 2 days (early-FN group). BSI occurred in 27 (11.9%) patients and the maximum CRP concentration was 2.57 mg/dl in the median. In a multivariate analysis, early-FN (hazard ratio (HR) 1.81, P = 0.007) and older recipient age (HR 1.68, P = 0.019) were significantly associated with the incidence of grade II-IV aGVHD. High-CRP and BSI were not significant risk factors for grade II-IV aGVHD. On the other hand, high-CRP was significantly associated with the incidence of NRM (HR 2.67, P = 0.004) in a multivariate analysis. In conclusion, although fever, CRP elevation and BSI are considered to be closely related events, they had different effects on the incidence of aGVHD and NRM. The development of early-FN after HCT may predict the risk of aGVHD.

Thermal management is a key technology to desterilize unused energy sources for building sustainable societies. However, conventional temperature measurement methods such as infrared thermography can detect only the surface temperature of objects because they use infrared light. We thus present a novel three-dimensional X-ray thermography using a phase-contrast X-ray imaging technique, which enables non-destructive observations of the inner thermal distribution of samples. The sensitivity of phase-contrast X-ray imaging is about 1000 times higher than that of conventional X-ray imaging. Therefore, temperature changes can be detected by using density changes caused by thermal expansion. We applied X-ray interferometric imaging (XI) that detects phase-shift by using a crystal X-ray interferometer. The highest sensitivity of XI was utilized to successfully obtain the first three-dimensional image that visualizes the thermal distribution in heated water nondestructively. Additionally, projection images visualizing the dynamic thermal flow in heated water were also obtained, and their distribution and diffusion velocity agreed well with those of the calculated images obtained by computational fluid dynamics analysis. These results show that the novel thermography enables nondestructive observations of inner temperature and thermal flow and can provide solutions for optimum thermal design of electrical devices, motors, and engines.

Nanometre-scale magnetic field distributions in materials such as those at oxide interfaces, in thin layers of spintronics devices, and at boundaries in magnets have become important research targets in materials science and applied physics. Electron holography has advantages in nanometric magnetic field observations, and the realization of aberration correctors has improved its spatial resolution. Here we show the subnanometre magnetic field observations inside a sample at 0.67-nm resolution achieved by an aberration-corrected 1.2-MV holography electron microscope with a pulse magnetization system. A magnetization reduction due to intermixing in a CoFeB/Ta multilayer is analyzed by observing magnetic field and electrostatic potential distributions simultaneously. Our results demonstrate that high-voltage electron holography can be widely applied to pin-point magnetization analysis with structural and composition information in physics, chemistry, and materials science.

Cytokines are required for γ-retroviral transduction of human CD34+ cells. However, cytokines may reduce engraftment of CD34+ cells and may not be necessary for their lentiviral transduction. We sought to optimize transduction and engraftment of human CD34+ cells using lentiviral vectors. Single 24 h transduction of human CD34+ cells with human immunodeficiency virus type 1 (HIV1)-based lentiviral vectors in media containing stem cell factor (SCF), FMS-like tyrosine kinase 3 (FLT3) ligand, thrombopoietin (each 100 ng ml⁻¹) and 10% fetal bovine serum was compared with various cytokine conditions during ex vivo culture and assayed using humanized xenograft mice for 6 months after transplantation. Serum-free media improved transduction efficiency of human CD34+ cells. Interleukin-3 (20 ng ml⁻¹) had little effect on transduction efficiency or engraftment. Threefold higher cytokine mixture (each 300 ng ml⁻¹) reduced engraftment of CD34+ cells. SCF alone (100 ng ml⁻¹) proved insufficient for maintaining engraftment ability and reduced transduction efficiency. Short-term prestimulation had little effect on transduction efficiency or engraftment, yet 24 h prestimulation showed higher transduction efficiency, higher gene expression levels and lower engraftment. In summary, 24 h prestimulation followed by single 24-h lentiviral transduction in serum-free media with SCF, FLT3 ligand and thrombopoietin yields high transduction efficiency to engrafting human CD34+ cells, and is applicable in human clinical gene therapy trials.

Although there is growing evidence that estrogens promote tumor progression in epithelial ovarian cancer, the molecular mechanisms accounting for this are still unclear. Selective estrogen receptor modulators (SERMs) mimic estrogen action in certain tissues while opposing it in others. The molecular mechanisms of the effects of SERMs such as raloxifene on the tumor progression of epithelial ovarian cancer are also still unclear. Here, we show that various genomic actions of estrogen differ from those of raloxifene in human ovarian cancer cell lines expressing estrogen receptor alpha (ERalpha). 17beta-Estradiol (E2) induced the gene expression of c-Myc and IGF-1 and increased the binding of ERalpha to the AP1 site of the promoters of c-Myc and IGF-1. ERalpha silencing abolished the E2-stimulated c-Myc expression. E2 induced the recruitment of co-activators such as SRC-1, SRC-3 and CBP to the promoters of c-Myc and IGF-1, and SRC-1 silencing abolished both the E2-stimulated c-Myc expression and cell-cycle progression. In contrast, although raloxifene increased the binding of ERalpha to the AP1 site of the promoters of c-Myc and IGF-1, raloxifene had no effect on the gene expression of c-Myc or IGF-1. Raloxifene induced the recruitment of co-repressors such as HDAC2, N-CoR and SMRT to the promoter of IGF-1. Thus, the difference between the genomic actions exerted by estrogen and raloxifene in human ovarian cancer cell lines expressing ERalpha appear to be dependent on the recruitment of co-regulators.

Recent studies have shown bone marrow (BM) cells to differentiate into a variety of cell types and to thereby participate in the reconstitution of damaged organs. In the present study, we examined the extent to which BM-derived cells are incorporated into glomeruli during recovery from experimentally induced nephritis. To investigate the localization of BM cells in glomeruli, chimeric mice were prepared by transplanting BM cells from green fluorescent protein (GFP) transgenic mice into wild-type mice. Five weeks later, glomerulonephritis was induced by intravenous injection of Habu snake venom. Groups of mice were then killed every few days for 42 d, and harvested kidney samples were subjected to immunohistochemical and immunoelectron microscopic analyses with the aim of detecting the presence of GFP(+) cells within glomeruli. Chimeric animals injected with Habu venom developed proliferative glomerulonephritis within 1-3 d. The lesion gradually subsided and the glomerular structure returned to normal within 42 d. Consistent with the disease course, large numbers of GFP(+) cells were present within glomeruli on d 1-3, but most had disappeared by d 7. Nevertheless, some GFP(+) cells did remain within glomeruli showing mesangial proliferative changes, and were found to express thrombomodulin (TM), a specific endothelial cell marker. These GFP-TM-double-positive cells accounted for a mean of 1.31-2.24% of the total glomerular nuclei from d 7 through d 42, levels that remained stable for at least 12 mo. It thus appears that BM cells can give rise to endothelial cells that participate in the remodeling of glomeruli.

We investigated the role of circulating bone marrow cells (BMC) in cardiomyocyte regeneration. BMC, isolated from transgenic mice expressing enhanced green fluorescent protein (GFP), were transplanted into lethally irradiated C57BL6 mice. Five weeks after bone marrow transplantation (BMT), flow cytometric analysis for GFP-positive cells confirmed reconstitution of transplanted bone marrow. Bone marrow transplant mice subsequently underwent left coronary artery ligation (myocardial infarction) or sham-operation, and were killed at 1 mo or 3 mo after operation. Infarct size was similar in bone marrow transplant mice at 1 mo (47.1 +/- 5.9%) and at 3 mo (45.3 +/- 7.8%), and echocardiography at 2 and 8 wk revealed decreasing left ventricular function. In infarcted heart, GFP-positive cells that expressed desmin and troponin T-C were identified by confocal microscopy. GFP and troponin T-C double-positive cells were predominantly in the peri-infarcted region (1 mo, 365 +/- 45 cells/50 sections; 3 mo: 458 +/- 100 cells/50 sections; p < 0.05 versus noninfarct, infarct, and sham-operated regions). Furthermore, BMC mobilization and differentiation into cardiomyocytes was found to be complete within 1 mo after myocardial infarction. These results demonstrate that circulating BMC undergo mobilization and differentiation in cardiac cells after myocardial infarction. Future studies are required to determine the molecular signaling mechanisms responsible for this phenomenon.

The optimal treatment for natural killer (NK) cell leukemia after chronic active Epstein-Barr virus (CAEBV) infection has not been determined. A 15-year-old boy presented with NK cell leukemia following CAEBV infection for 5 years. The peripheral blood and BM had an increased number of CD3(-)CD56(+) large granular lymphocytes and a monoclonal integration of the EBV genome was detected. Chemotherapy was not sufficiently effective to control the disease. Allogeneic BMT from an HLA-identical sister was performed using a conditioning regimen consisting of total body irradiation, cyclophosphamide and thiotepa. The patient is disease-free with a perfect performance status 24 months after BMT. This is the first report to show that allogeneic BMT is potentially able to cure NK cell leukemia after CAEBV infection.

Structural characterization of the colistin (CL) components were carried out using Frit-fast atom bombardment liquid chromatography/mass spectrometry (Frit-FAB LC/MS), tandem mass spectrometry (MS/MS) and the amino acid analysis proposed by MARFEY, and the total structures of 4 minor components including the absolute configuration of the constituent amino acids were proposed. The structures of the minor components were the same as those of the main component colistin A or B except that L-leucine is replaced by L-valine or L-isoleucine.

Total structures of 13 minor components of bacitracin (BC) were proposed, and their antimicrobial activities were investigated. The components of BC including bacitracins A (BC-A) and F (BC-F) were isolated by preparative HPLC and were hydrolyzed under acidic conditions. The resulting amino aids were derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanineamide and were separated by HPLC to determine their absolute configurations. It was found that the N-terminal amino acids of BC-A and its related components were epimerized during the hydrolysis to yield their enantiomers. The formation of these artifactual amino acids suggests that our previously proposed structures of the BC minor components are incorrect; therefore, the structures were corrected based on these results. The structures of the BC minor components were the same as that of BCs-A and -F except that one to three of the L-isoleucines, including the N-terminal one, were replaced by L-valines. These structures were confirmed by tandem mass spectrometry under fast atom bombardment (FAB) conditions and Frit-FAB liquid chromatography/mass spectrometry. Based on the UV spectra of the BC components determined by photodiode array detection-HPLC analysis, a new systematic nonmenclature was proposed for the minor components. The isolated components were also used for the determination of their minimal inhibition concentrations and it was found that BC-A is 2 approximately 8 times more potent than the other minor components against strains of Micrococcus luteus and Staphylococcus aureus.

The structural characterization of minor components of bacitracin (BC) complex was carried out using a technique of liquid chromatography/mass spectrometry (LC/MS). Satisfactory total ion current chromatogram of BC complex and excellent mass spectra of many components were given by Frit-fast atom bombardment (FAB) LC/MS analytical system, and the structures of 13 minor components could be proposed. The 13 minor components were classified into two groups, bacitracin A (BC-A) related components and bacitracin F (BC-F) related components depending on their common N-terminal moieties. The structures of BC-A related components and BC-F related components were the same as those of BC-A and BC-F, respectively, except that one to three of isoleucine and leucine residues are replaced by valines. The BC-F related components were degradation products of BC-A related components through the same degradation process as that of BC-A.