Design of Temperature-Responsive Cell Culture Surfaces for Cell Sheet Engineering.

Temperature-responsive cell culture surfaces, which modulate cell attachment/detachment characteristics with temperature, have been used to fabricate cell sheets. Extensive study on fabrication of cell sheet with the temperature-responsive cell culture surface, manipulation, and transplantation of the cell sheet has established the interdisciplinary field of cell sheet engineering, in which engineering, biological, and medical fields closely collaborate. Such collaboration has pioneered cell sheet engineering, making it a promising and attractive technology in tissue engineering and regenerative medicine. This review introduces concepts of cell sheet engineering, followed by designs for the fabrication of various types of temperature-responsive cell culture surfaces and technologies for cell sheet manipulation. The development of various methods for the fabrication of temperature-responsive cell culture surfaces was also summarized. The availability of cell sheet engineering for the treatment and regeneration of damaged human tissue has also been described, providing examples of the clinical application of cell sheet transplantation in humans.

Nightlife clusters of coronavirus disease in Tokyo between March and April 2020.

We analysed associations between exposure to nightlife businesses and severe acute respiratory syndrome coronavirus 2 PCR test results at a tertiary hospital in Tokyo between March and April 2020. A nightlife group was defined as those who had worked at or visited the businesses. We included 1517 individuals; 196 (12.9%) were categorised as the nightlife group. After propensity score matching, the proportion of positive PCR tests in the nightlife group was significantly higher than that in the non-nightlife group (nightlife, 63.8%; non-nightlife, 23.0%; P < 0.001). An inclusive approach to mitigate risks related to the businesses needs to be identified.

The correlation of urinary podocytes and podocalyxin with histological features of lupus nephritis.

Objectives The objective of this study was to test the correlation of urinary podocyte number (U-Pod) and urinary podocalyxin levels (U-PCX) with histology of lupus nephritis. Methods This was an observational, cross-sectional study. Sixty-four patients were enrolled: 40 with lupus nephritis and 24 without lupus nephritis (12 lupus nephritis patients in complete remission and 12 systemic lupus erythematosus patients without lupus nephritis). Urine samples were collected before initiating treatment. U-Pod was determined by counting podocalyxin-positive cells, and U-PCX was measured by sandwich ELISA, normalized to urinary creatinine levels (U-Pod/Cr, U-PCX/Cr). Results Lupus nephritis patients showed significantly higher U-Pod/Cr and U-PCX/Cr compared with patients without lupus nephritis. U-Pod/Cr was high in proliferative lupus nephritis (class III±V/IV±V), especially in pure class IV (4.57 (2.02-16.75)), but low in pure class V (0.30 (0.00-0.71)). U-Pod/Cr showed a positive correlation with activity index ( r=0.50, P=0.0012) and was independently associated with cellular crescent formation. In contrast, U-PCX/Cr was high in both proliferative and membranous lupus nephritis. Receiver operating characteristic analysis revealed significant correlation of U-Pod/Cr with pure class IV, class IV±V and cellular crescent formation, and the combined values of U-Pod/Cr and U-PCX/Cr were shown to be associated with pure class V. Conclusions U-Pod/Cr and U-PCX/Cr correlate with histological features of lupus nephritis.

Stability of cervical esophagogastrostomy via hand-sewn anastomosis after esophagectomy for esophageal cancer.

The aim of the present study is to evaluate the outcome of hand-sewn esophagogastric anastomosis during radical esophagectomy for esophageal cancer. The outcomes of 467 consecutive esophageal cancer patients who underwent cervical esophagogastric anastomosis using interrupted and double-layered sutures after radical esophagectomy via right thoracotomy or thoracoscopic surgery were retrospectively reviewed. Anastomotic leakage, including conduit necrosis, occurred in 11 of 467 patients (2.4%); 7 of 11 (63.6%) cases experienced only minor leakage, whereas the other four (36.4%) patients had major leakage that required surgical or radiologic intervention, including two patients of conduit necrosis. Anastomotic leakages were more frequently observed after retrosternal reconstruction compared with the posterior mediastinal route (P < 0.0001). The median time to healing of leakage was 40 days (range: 14-97 days). Two patients (2/467, 0.4%) died in the hospital due to sepsis caused by the leakage and conduit necrosis. Twelve patients (2.6%) developed anastomotic stenosis, which was improved by dilatation in all patients. Hand-sewn cervical esophagogastric anastomosis is a stable and highly safe method of radical esophagectomy for esophageal cancer.

Biochemical Characterization of Function and Structure of RseP, an Escherichia coli S2P Protease.

Intramembrane-cleaving proteases (I-CLiPs) are a group of membrane-associated proteases with a unique feature: they are believed to cleave their substrate within the hydrophobic lipid bilayer, even though peptide bond hydrolysis requires a water molecule. Escherichia coli RseP, which belongs to the S2P zinc metalloprotease family of I-CLiPs, plays an essential role in activation of a cell envelope stress response through cleavage of anti-σE protein RseA, a single-span transmembrane protein. A recent study showed that it also cleaves remnant signal peptides generated upon membrane translocation of secretory proteins. Here, we describe several methods for characterization of the proteolytic functions and structure of RseP mainly in vivo, including a proteolytic activity assay using model substrates, an in vitro analysis of cleavage of signal peptides in a detergent solution and in the membrane vesicles, structural analysis of membrane-embedded RseP based on the thiol modifiability of introduced cysteine residues, and the protein interaction analysis by in vivo cross-linking protocols.

GABA-producing Bifidobacterium dentium modulates visceral sensitivity in the intestine.

BACKGROUND: Recurrent abdominal pain is a common and costly health-care problem attributed, in part, to visceral hypersensitivity. Increasing evidence suggests that gut bacteria contribute to abdominal pain perception by modulating the microbiome-gut-brain axis. However, specific microbial signals remain poorly defined. γ-aminobutyric acid (GABA) is a principal inhibitory neurotransmitter and a key regulator of abdominal and central pain perception from peripheral afferent neurons. Although gut bacteria are reported to produce GABA, it is not known whether the microbial-derived neurotransmitter modulates abdominal pain. METHODS: To investigate the potential analgesic effects of microbial GABA, we performed daily oral administration of a specific Bifidobacterium strain (B. dentiumATCC 27678) in a rat fecal retention model of visceral hypersensitivity, and subsequently evaluated pain responses. KEY RESULTS: We demonstrate that commensal Bifidobacterium dentium produces GABA via enzymatic decarboxylation of glutamate by GadB. Daily oral administration of this specific Bifidobacterium (but not a gadB deficient) strain modulated sensory neuron activity in a rat fecal retention model of visceral hypersensitivity. CONCLUSIONS & INFERENCES: The functional significance of microbial-derived GABA was demonstrated by gadB-dependent desensitization of colonic afferents in a murine model of visceral hypersensitivity. Visceral pain modulation represents another potential health benefit attributed to bifidobacteria and other GABA-producing species of the intestinal microbiome. Targeting GABAergic signals along this microbiome-gut-brain axis represents a new approach for the treatment of abdominal pain.

Orbital Fat Volumetry and Water Fraction Measurements Using T2-Weighted FSE-IDEAL Imaging in Patients with Thyroid-Associated Orbitopathy.

BACKGROUND AND PURPOSE: The quantitative evaluation of orbital fat proliferation and edema and the assessment of extraocular muscles are useful for diagnosing and monitoring thyroid-associated orbitopathy. To evaluate therapy-induced quantitative changes in the orbital fat of patients with thyroid-associated orbitopathy, we performed volumetric and water fraction measurements by using T2-weighted FSE iterative decomposition of water and fat with echo asymmetry and least-squares estimation (FSE-IDEAL) imaging. MATERIALS AND METHODS: Orbital FSE-IDEAL images of 30 volunteers were acquired twice within 1 week. Nine patients with thyroid-associated orbitopathy underwent FSE-IDEAL imaging before and after methylprednisolone pulse therapy, and the treatment results were assessed by using their pre- and post-methylprednisolone pulse therapy clinical activity scores. We performed volumetric and water fraction measurements of orbital fat by using FSE-IDEAL imaging and evaluated interscan differences in the volunteers. In patients with thyroid-associated orbitopathy, we compared pre- and posttherapy orbital fat measurements and assessed the correlation between the pretherapy values and clinical activity score improvement. RESULTS: The reproducibility of results obtained by the quantitative evaluation of orbital fat in volunteers was acceptable. After methylprednisolone pulse therapy, the water fraction in the orbital fat of patients with thyroid-associated orbitopathy was significantly decreased (P < .001). There was a significant positive correlation between the pretherapy water fraction and clinical activity score improvement (right, r = 0.82; left, r = 0.79) and a significant negative correlation between the pretherapy volume and clinical activity score improvement (bilateral, r = -0.84). CONCLUSIONS: Volumetric and water fraction measurements of orbital fat by using FSE-IDEAL imaging are feasible and useful for monitoring the effects of therapy and for predicting the response of patients with thyroid-associated orbitopathy to methylprednisolone pulse therapy.

Vascular Wall Imaging of Unruptured Cerebral Aneurysms with a Hybrid of Opposite-Contrast MR Angiography.

BACKGROUND AND PURPOSE: Inflammation and degeneration of the intracranial saccular aneurysm wall play a major role in aneurysm formation, development and subsequent rupture. The aim of this study was to characterize the walls of unruptured intracranial aneurysms by using a hybrid of opposite-contrast MRA at 3T. MATERIALS AND METHODS: Fourteen consecutive patients with 17 unruptured intracranial aneurysms who initially underwent clipping surgery were prospectively evaluated. All aneurysms were scanned preoperatively by using a hybrid of opposite-contrast MRA in 3T high-resolution MR imaging. We classified intraoperative findings of atherosclerotic plaques in the aneurysms into 3 grades: grade A (major plaques), grade B (minor plaques), and grade C (no plaques). The contrast ratio of the high-intensity area was also measured relative to the background low-intensity area inside the carotid artery. RESULTS: Findings from preoperative plaque imaging of the aneurysm corresponded to the intraoperative findings in 15 of 16 aneurysms (excluding 1 that was impossible to visualize in its entirety due to anatomic reasons). Overall sensitivity and specificity of the hybrid of opposite-contrast MRA were 88.9% and 100%, respectively. During the operation, 4 aneurysms were classified as grade A; 5, as grade B; and 7, as grade C. The means of the contrast ratio for grades A, B, and C were 0.72 ± 0.03, 0.34 ± 0.30, and -0.02 ± 0.09, respectively. CONCLUSIONS: The hybrid of opposite-contrast MRA can detect visible atherosclerotic plaques in the unruptured aneurysm wall, and the contrast ratio in intracranial aneurysms correlated with their presence and extent. A study including a larger series is needed to validate the diagnostic potential of this imaging technique.

Characterization of mesenchymal progenitor cells in crown and root pulp from human mesiodentes.

OBJECTIVE: Mesiodentes are usually found in the central position of the upper or lower jaw as supernumerary teeth. Here, we obtained 10 mesiodentes and three permanent teeth (PT) and separated the dental pulp (DP) from these into crown and root portions. We then characterized and compared the isolated crown portion-derived cells (crown cells) with root portion-derived cells (root cells) using a range of in vitro assays. MATERIALS AND METHODS: Crown cells and root cells were examined for cell surface marker expression, colony-forming unit-fibroblast (CFU-F), cell proliferation, cell cycle characteristics and markers, and osteogenic and adipogenic differentiation. RESULTS: The proportion of CD105-positive cells (CD105(+) cells) in the crown cells vs the root cells varied among the mesiodentes, but not among the PT. When there were more CD105(+) cells in the root cells than in the crown cells, the root cells showed higher CFU-F, proliferation capacity, and osteogenic differentiation capacity. In contrast, when the crown cells contained more CD105(+) cells than the root cells, the crown cells showed the higher CFU-F, proliferation capacity, and osteogenic differentiation capacity. In addition, the sorted CD105(+) cells showed higher CFU-F and proliferation capacity than the sorted CD105(-) cells. CONCLUSION: These results indicated that proportion of CD105(+) cells is an effective means of characterizing DP-derived cells in mesiodentes.

An empirical model for gas phase acidity and basicity estimation.

Gas phase acidity and basicity estimation models have been developed for acidic and basic functional groups of amino acid side-chains and also for a number of small organic molecules. The acidic functional groups include aliphatic and aromatic alcohol, and aliphatic and aromatic carboxylic acid, and the basic functional groups include aliphatic, aromatic and hetero-aromatic amines, and also pyridino-, pyrazolo- and imidazolo-groupings. The models are described in terms of a linear combination of descriptors that highly influence reactivity at the reaction centres of the functional groups. In order to describe the chemical environments of the deprotonating and protonating sites, atomic descriptors such as the effective atomic electronegativity and effective atomic polarizability of the atoms in the reaction field and the electrostatic potentials at the reaction sites have been introduced. The coefficient of determination (r(2)) of each model is above 0.8, apart from the imidazole model. The models are readily applicable, ranging from simple organic molecules to proteins.

Jumonji domain-containing protein 2B silencing induces DNA damage response via STAT3 pathway in colorectal cancer.

BACKGROUND: Jumonji domain-containing protein 2B (JMJD2B), directly targeted by hypoxia-inducible factor 1α, maintains the histone methylation balance important for the transcriptional activation of many oncogenes. Jumonji domain-containing protein 2B has been implicated in colorectal cancer (CRC) progression; however, the mechanism remains unclear. METHODS: Immunofluorescence and western blotting detected phosphorylated histone H2AX, characteristic of double-strand breaks, and comet assay was used to investigate DNA damage, in CRC cells after JMJD2B small interfering RNA (siRNA) transfection. We assessed the resulting in vitro responses, that is, cell cycle progression, apoptosis, and senescence coupled with JMJD2B silencing-induced DNA damage, studying the regulatory role of signal transducers and activators of transcription 3 (STAT3). The JMJD2B silencing anti-cancer effect was determined using an in vivo CRC xenograft model. RESULTS: Jumonji domain-containing protein 2B knockdown induced DNA damage via ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related pathway activation, resulting in cell cycle arrest, apoptosis, and senescence in both normoxia and hypoxia. Signal transducers and activators of transcription 3 suppression by JMJD2B silencing enhanced DNA damage. Intratumoural injection of JMJD2B siRNA suppressed tumour growth in vivo and activated the DNA damage response (DDR). CONCLUSIONS: Jumonji domain-containing protein 2B has an essential role in cancer cell survival and tumour growth via DDR mediation, which STAT3 partially regulates, suggesting that JMJD2B is a potential anti-cancer target.

Circulating microRNAs as biomarkers for early detection of diffuse-type gastric cancer using a mouse model.

BACKGROUND: Diffuse-type gastric cancer (DGC) exhibits rapid disease progression and a poor prognosis. There are no effective serum biomarkers for early detection of DGC. We have established an E-cadherin/p53 double conditional knockout (DCKO) mouse line that recapitulates human DGC morphologically and molecularly. In this study we tried to identify circulating microRNAs (miRNAs) as non-invasive biomarkers for DGC diagnosis using DCKO mice. METHODS: We performed miRNA microarray and quantitative reverse transcription-PCR analyses of tissue and serum samples from DCKO mice with DGC and age-matched littermate controls. RESULTS: Comparative analyses showed that mouse and human primary gastric cancers have similar miRNA expression patterns. Next, we selected some candidate miRNAs highly expressed in sera and cancer tissues of DCKO mice for further evaluation. TaqMan quantitative RT-PCR analyses indicated that four of them, miR-103, miR-107, miR-194 and miR-210, were significantly upregulated in sera of both early and advanced-stage DGC-bearing mice compared with in corresponding controls. Receiver-operating characteristic curve analyses demonstrated that these four miRNAs can discriminate DGC-positive cases from normal ones with high sensitivity and specificity. CONCLUSION: These observations suggest that this mouse model of DGC is useful for identifying serum biomarkers, and we found circulating miRNAs that can accurately detect DGC at an early stage.

Ocular volumetry using fast high-resolution MRI during visual fixation.

BACKGROUND AND PURPOSE: Volumetry may be useful for evaluating treatment response and prognosis of intraocular lesions. Phantom, volunteer, and patient studies were performed to determine whether ocular MR volumetry is reproducible. MATERIALS AND METHODS: Half-Fourier single-shot RARE and FSPGR sequences at 1.5T with a 76-mm-diameter surface coil were optimized to obtain still ocular images. Volumetry accuracies of each sequence were compared with simulated subretinal phantom volumes. Ocular volumetry was performed in 15 volunteers twice in 1 week by using contiguous axial images of the globes while the subjects stared at a target, and images were acquired in 2 seconds before the subjects were instructed to blink, with this process repeated as necessary. Imaging, intraobserver, and interobserver reproducibility for volumes of the whole eyeball and anterior chamber were assessed. Ocular volumetry was also performed in 6 patients with intraocular tumors before and after treatment. RESULTS: The phantom study demonstrated that measurement error rates with RARE were significantly lower than with FSPGR (P<.01). The volunteer study demonstrated excellent imaging and intraobserver reproducibility of RARE volumetry for whole eyeballs and anterior chambers (P<.01). Although no interobserver differences were observed in anterior chamber volume measurement (P=.33), there was a significant difference between the 2 observers in eyeball volume measurement (P<.01). Follow-up volumetric data were useful for treatment decisions in all patients. CONCLUSIONS: Ocular volumetry from contiguous ultrafast RARE images obtained during visual fixation is feasible in volunteer and patient studies and is superior to FSPGR images.

Synergistic effect of PKC activation and actin filament disruption on carbonate apatite-facilitated lymphocyte transfection.

Leukemia and lymphoma cells are potential targets for genetic manipulation in cancer therapy. On the other hand, genetically modified autologous lymphocytes expressing a chimeric antigen against a receptor overexpressed in tumor cells or tumor vasculature are promising cell-based therapeutics for cancer.However, the lack of a smart device for efficient transgene delivery to the lymphocytes poses the major obstacle to the successful clinical applications of these attractive approaches. Recently, we developed a carbonate apatite-based nanocarrier system for effective intracellular delivery and release of DNA molecules, achieving very high level of transgene expression in both primary and cancer cells. Although its efficacy in human T leukemia cells is relatively poor, immobilization of fibronectin and/or chimeric E-cadherin-Fc on particle surface could enhance transgene delivery in presence of an actin filament disrupter. Here, we report for the first time that simultaneous stimulation of human T leukemia cells by a protein kinase C (PKC) activator, a Ca(2+) ionophore and an actin filament disrupter dramatically accelerated carbonate apatite-mediated transgene delivery in the cells, resulting in over 100-fold more efficacy than commcercially available lipofectamine.

Effect of a selective neutrophil elastase inhibitor on early recovery from body water imbalance after transthoracic esophagectomy.

The objective of the study was to evaluate the efficacy of sivelestat, a selective neutrophil elastase inhibitor, on body fluid balance after transthoracic esophagectomy. Esophagectomy with elective lymphadenectomy may induce excessive release of neutrophil elastase, which then promotes vascular permeability and an excessive water shift from the intravascular space to the peripheral compartment. Body fluid imbalance after esophagectomy often leads to circular instability, a decrease of urine output, and a delay in the shift to a diuretic state. The study was designed as a case-control study with a historical control group. A retrospective analysis was performed to examine our hypothesis that sivelestat improves abnormal body fluid retention and prevents subsequent pulmonary complications. To reveal the direct influence of sivelestat on the postoperative course, we avoided using steroids or other diuretic agents. Eighty-eight patients who underwent thoracic esophagectomy with extended lymphadenectomy from 2000 to 2008 were divided into two groups: those treated from 2003 to 2008, who all received postoperative administration of sivelestat (n=60); and those treated from 2000 to 2002, who did not receive sivelestat and were used as the control group (n=28). Both groups received fluid management using the same protocol. The time to reach a diuretic state, time until extubation of the tracheal tube, and development of delayed respiratory dysfunction were compared between the groups using univariate and multivariate analysis. The time until a shift to a diuretic state was significantly shorter after treatment with sivelestat (p<0.0001) and with a shorter operation time (p<0.0001). The tracheal tube was extubated significantly earlier in the sivelestat group (p<0.0001) and the incidence of delayed respiratory dysfunction was also significantly lower (p=0.0028) in this group. Multivariate logistic regression analysis showed that a delay in a shift to a diuretic state was a strong independent risk factor for the time to tracheal extubation (odds ratio 2.539, p=0.0056) and occurrence of delayed respiratory dysfunction (odds ratio 1.989, p=0.0104). Sivelestat treatment was not independently associated with reduced pulmonary complications, but the diuretic state was strongly regulated by sivelestat treatment (odds ratio 0.044, p=0.0003). Thus, administration of sivelestat has a beneficial influence on recovery from body water imbalance through a more rapid return to a diuretic state after esophagectomy, which contributes to prevention of subsequent pulmonary complications.

Autoantibody against activating transcription factor-2 in patients with systemic sclerosis.

OBJECTIVES: To determine the prevalence and clinical correlation of autoantibody to activating transcription factor (ATF)-2, a transcription factor of ATF/CREB family, in patients with systemic sclerosis (SSc). METHODS: Anti-ATF-2 Ab was examined by ELISA and immunoblotting using human recombinant ATF-2. ATF-2 activity to bind target DNA was evaluated by ELISA using a plate coated with oligonucleotide containing the consensus binding site for ATF-2. RESULTS: IgG anti-ATF-2 Ab levels in SSc patients (n=69) were significantly higher than those in normal controls (n=26). SSc patients positive for IgG anti-ATF-2 Ab had significantly longer disease duration, more frequent presence of decreased %VC and %DLco, and elevated levels of serum IgG, serum IgA, and erythrocyte sedimentation rates than those negative. More-over, IgG anti-ATF-2 Ab levels correlated inversely with %VC or %DLco. The presence of anti-ATF-2 Ab in SSc patients was confirmed by immunoblotting analysis. IgG isolated from serum samples of SSc patients positive for IgG anti-ATF-2 Ab by ELISA slightly but significantly inhibited ATF-2 activity compared with normal controls. CONCLUSIONS: These results suggest that anti-ATF-2 Ab is a new autoantibody in SSc and that it serves as a novel serological marker for inflammation and lung involvement in SSc.

Disrupting actin filaments promotes efficient transfection of a leukemia cell line using cell adhesive protein-embedded carbonate apatite particles.

Tumor cells such as leukemia and lymphoma cells are obvious and attractive targets for gene therapy. Gene transfer and expression for cytokine and immunomodulatory molecules in various kinds of tumor cells have been shown to mediate tumor regression and antimetastatic effects. Moreover, genetically modified leukemia cells expressing costimulatory molecules or cytokines are likely to have significant therapeutic roles for patients with leukemia. One of the major hurdles to the successful implementation of these promising approaches is the lack of a suitable nanocarrier for transgene delivery and expression in a safe and effective manner. Recently, we reported on the development of a safe, efficient nanocarrier system of carbonate apatite that can assist both intracellular delivery and release of DNA, leading to very high level of transgene expression in cancer and primary cells. However, its efficiency in human lymphocytes is poor. We show here that nanocrystals of carbonate apatite, when electrostatically associated with fibronectin and/or E-cadherin-Fc, accelerated transgene delivery in a human T leukemia cell line (Jurkat). Moreover, transgene expression efficiency could be enhanced dramatically with the cell adhesive protein-embedded particles finally up to 150 times by selectively disrupting the actin filaments.

Pharmacokinetics of 5-fluorouracil in elderly Japanese patients with cancer treated with S-1 (a combination of tegafur and dihydropyrimidine dehydrogenase inhibitor 5-chloro-2,4-dihydroxypyridine).

S-1 is an oral anticancer agent that combines tegafur, a prodrug of 5-fluorouracil (5-FU), and 5-chloro-2,4-dihydroxypyridine (CDHP), an inhibitor of dihydropyrimidine dehydrogenase. We examined the effects of aging on the pharmacokinetics of the components of S-1. The median area under the concentration-time curve (AUC) of active 5-FU did not significantly differ between 10 patients 75 years or older and 53 patients younger than 75 years (P = 0.598, Mann-Whitney U test). It is interesting to note that the median oral clearance of tegafur in patients 75 years or older was significantly lower than that in patients younger than 75 years (P = 0.011). Furthermore, the median AUC of CDHP was significantly higher in patients 75 years or older than in those younger than 75 years (P = 0.004). This effect was caused by reduced renal function in the elderly, because CDHP is excreted in the urine by glomerular filtration. The opposing effects of aging on the oral clearance of tegafur and the AUC of CDHP may offset each other, leading to unchanged systemic exposure of 5-FU.