Enhancement of gap junctional intercellular communication of normal human dermal fibroblasts cultured on polystyrene dishes grafted with poly-N-isopropylacrylamide

Technology developed to allow recovery of cells without enzyme treatment, involving a dish grafted with a thermoreactive polymer gel of poly-N-isopropylacrylamide (PIPAAm), was found to significantly enhance gap junctional intercellular communication (GJIC) in normal human dermal fibroblasts (NHDF cells). NHDF cells were cultured for 4 days on PIPAAm-grafted dishes irradiated with various doses of electron beams, and GJIC was assayed by the scrape-loading dye transfer method. The area of dye transfer was greater in the PIPAAm-grafted dishes than in the control culture dishes, indicating that the PIPAAm-grafted dishes enhanced the GJIC of NHDF cells. Connexin-43 (Cx43) expression was analyzed because Cx43 is considered to be a main component of the gap junctional channel. PIPAAm-grafted dishes irradiated with 100, 250, or 500 kGy of electron beams showed significantly enhanced expression of Cx43-NP, Cx43-P1, and especially Cx43-P2. Enhanced expression of Cx43-P2, a functional transmembrane protein, may be related to the promotion of GJIC. These results suggest that the PIPAAm-grafted dish not only enables the enzyme-free recovery of a cell monolayer for use in the construction of a three-dimensional artificial tissue, but also significantly contributes to the enhancement of GJIC, which may partly promote tissue strength on the surface of the PIPAAm-grafted dish.     

Ex vivo rapamycin generates Th1/Tc1 or Th2/Tc2 Effector T cells with enhanced in vivo function and differential sensitivity to post-transplant rapamycin therapy.

Rapamycin prevention of murine graft-versus-host disease (GVHD) is associated with a shift toward Th2- and Tc2-type cytokines. Recently, we found that use of rapamycin during ex vivo donor Th2 cell generation enhances the ability of adoptively transferred Th2 cells to prevent murine GVHD. In this study, using a method, without antigen-presenting cells, of T-cell expansion based on CD3,CD28 costimulation, we evaluated whether (1) rapamycin preferentially promotes the generation of Th2/Tc2 cells relative to Th1/Tc1 cells, (2) rapamycin-generated T-cell subsets induce cytokine skewing after allogeneic bone marrow transplantation (BMT), and (3) such in vivo cytokine skewing is sensitive to post-BMT rapamycin therapy. Contrary to our hypothesis, rapamycin did not preferentially promote Th2/Tc2 cell polarity, because rapamycin-generated Th1/Tc1 cells secreted type I cytokines (interleukin [IL]-2 and interferon-gamma) did not secrete type II cytokines (IL-4, IL-5, IL-10, or IL-13) and mediated fasL-based cytolysis. Rapamycin influenced T-cell differentiation, because each of the Th1, Th2, Tc1, and Tc2 subsets generated in rapamycin had increased expression of the central-memory T-cell marker, L-selectin (CD62L). Rapamycin-generated Th1/Tc1 and Th2/Tc2 cells were not anergic but instead had increased expansion after costimulation in vitro, increased expansion in vivo after BMT, and maintained full capacity to skew toward type I or II cytokines after BMT, respectively; further, rapamycin-generated Th1/Tc1 cells mediated increased lethal GVHD relative to control Th1/Tc1 cells. Rapamycin therapy after BMT in recipients of rapamycin-generated Th1/Tc1 cells greatly reduced Th1/Tc1 cell number, greatly reduced type I cytokines, and reduced lethal GVHD; in marked contrast, rapamycin therapy in recipients of rapamycin-generated Th2/Tc2 cells nominally influenced the number of Th2/Tc2 cells in vivo and did not abrogate post-BMT type II cytokine skewing. In conclusion, ex vivo and in vivo usage of rapamycin may be used to modulate the post-BMT balance of Th1/Tc1 and Th2/Tc2 cell subsets.     

Functional analysis of PTPN11/SHP-2 mutants identified in Noonan syndrome and childhood leukemia

Noonan syndrome (NS) is characterized by short stature, characteristic facial features, and heart defects. Recently, missense mutations of PTPN11, the gene encoding protein tyrosine phosphatase (PTP) SHP-2, were identified in patients with NS. Further, somatic mutations in PTPN11 were detected in childhood leukemia. Recent studies showed that the phosphatase activities of five mutations identified in NS and juvenile myelomonocytic leukemia (JMML) were increased. However, the functional properties of the other mutations remain unidentified. In this study, in order to clarify the differences between the mutations identified in NS and leukemia, we examined the phosphatase activity of 14 mutants of SHP-2. We identified nine mutations, including a novel F71I mutation, in 16 of 41 NS patients and two mutations, including a novel G503V mutation, in three of 29 patients with leukemia. Immune complex phosphatase assays of individual mutants transfected in COS7 cells showed that ten mutants identified in NS and four mutants in leukemia showed 1.4-fold to 12.7-fold increased activation compared with wild-type SHP-2. These results suggest that the pathogenesis of NS and leukemia is associated with enhanced phosphatase activity of mutant SHP-2. A comparison of the phosphatase activity in each mutant and a review of previously reported cases showed that high phosphatase activity observed in mutations at codons 61, 71, 72, and 76 was significantly associated with leukemogenesis.     

Association between Vitamin D and Heart Failure Mortality in 10,974 Hospitalized Individuals

A broad range of chronic conditions, including heart failure (HF), have been associated with vitamin D deficiency. Existing clinical trials involving vitamin D supplementation in chronic HF patients have been inconclusive. We sought to evaluate the outcomes of patients with vitamin D supplementation, compared with a matched cohort using real-world big data of HF hospitalization. This study was based on the Diagnosis Procedure Combination database in the Japanese Registry of All Cardiac and Vascular Datasets (JROAD-DPC). After exclusion criteria, we identified 93,692 patients who were first hospitalized with HF between April 2012 and March 2017 (mean age was 79 ± 12 years, and 52.2% were male). Propensity score (PS) was estimated with logistic regression model, with vitamin D supplementation as the dependent variable and clinically relevant covariates. On PS-matched analysis with 10,974 patients, patients with vitamin D supplementation had lower total in-hospital mortality (6.5 vs. 9.4%, odds ratio: 0.67, p < 0.001) and in-hospital mortality within 7 days and 30 days (0.9 vs. 2.5%, OR, 0.34, and 3.8 vs. 6.5%, OR: 0.56, both p < 0.001). In the sub-group analysis, mortalities in patients with age < 75, diabetes, dyslipidemia, atrial arrhythmia, cancer, renin-angiotensin system blocker, and β-blocker were not affected by vitamin D supplementation. Patients with vitamin D supplementation had a lower in-hospital mortality for HF than patients without vitamin D supplementation in the propensity matched cohort. The identification of specific clinical characteristics in patients benefitting from vitamin D may be useful for determining targets of future randomized control trials.     

A digest of the Evidence-Based Clinical Practice Guideline for Nephrotic Syndrome 2020

Clinical and Experimental Nephrology -     

A multi-institutional survey of the quality of life after treatment for uterine cervical cancer: a comparison between radical radiotherapy and surgery in Japan

This study aimed to research the post-treatment quality of life (QOL) between radiotherapy (RT)- and operation (OP)-treated early cervical cancer survivors, using separate questionnaires for physicians and patients. We administered an observational questionnaire to patients aged 20–70 years old with Stages IB1–IIB cervical cancer who had undergone RT or OP and without recurrence as outpatients for ≥6 months after treatment. We divided 100 registered patients equally into two treatment groups (n = 50 each). The average age was 53 and 44 years in the RT and OP groups, respectively. The RT group included 34 and 66% Stage I and II patients, respectively, whereas the OP group included 66 and 34% Stage I and II patients, respectively. The OP group included 58% of patients with postoperative RT. Combination chemotherapy was performed in 84 and 48% of patients in the RT and OP groups, respectively. On the physicians’ questionnaire, we observed significant differences in bone marrow suppression (RT) and leg edema (OP). On the patients’ questionnaire, significantly more patients had dysuria and leg edema in the OP group than in the RT group, and severe (Score 4–5) leg edema was significantly higher in the post-operative RT group than in the OP only group. The frequency of sexual intercourse decreased after treatment in both groups. On the patients’ questionnaire, there were no significant differences between the two groups regarding sexual activity. These findings are useful to patients and physicians for shared decision-making in treatment choices. The guidance of everyday life and health information including sexual life after treatment is important.     

Development and characterization of a new monoclonal antibody against SARS-CoV-2 NSP12 (RdRp)

SARS-CoV-2 NSP12, the viral RNA-dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID-19. To facilitate research on SARS-CoV-2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N-terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID-19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club-like, vascular endothelial cells, and a range of immune cells, despite wide-spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady-state expression or extensive posttranslation modifications that limit antibody reactivity during SARS-CoV-2 replication.