NR2F2 alleviates pulmonary fibrosis by inhibition of epithelial cell senescence.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fatal, and aging-associated interstitial lung disease with a poor prognosis and limited treatment options, while the pathogenesis remains elusive. In this study, we found that the expression of nuclear receptor subfamily 2 group F member 2 (NR2F2), a member of the steroid thyroid hormone superfamily of nuclear receptors, was reduced in both IPF and bleomycin-induced fibrotic lungs, markedly in bleomycin-induced senescent epithelial cells. Inhibition of NR2F2 expression increased the expression of senescence markers such as p21 and p16 in lung epithelial cells, and activated fibroblasts through epithelial-mesenchymal crosstalk, inversely overexpression of NR2F2 alleviated bleomycin-induced epithelial cell senescence and inhibited fibroblast activation. Subsequent mechanistic studies revealed that overexpression of NR2F2 alleviated DNA damage in lung epithelial cells and inhibited cell senescence. Adenovirus-mediated Nr2f2 overexpression attenuated bleomycin-induced lung fibrosis and cell senescence in mice. In summary, these data demonstrate that NR2F2 is involved in lung epithelial cell senescence, and targeting NR2F2 may be a promising therapeutic approach against lung cell senescence and fibrosis.

Relationship between Platelet Index and Lipid Metabolism, Coagulation Function, and Inflammation in Diabetic Patients.

BACKGROUND: Platelets are overactive in type 2 diabetes mellitus (T2DM). This study analyzed the relationship between platelet index and lipid metabolism, coagulation function, and inflammation in T2DM patients. METHODS: This study enrolled 60 T2DM patients and 60 healthy subjects (age- and gender-matched). Mean platelet volume (MPV) and platelet distribution width (PDW) were evaluated, and their associations with lipid metabolism (TG and HDL-C), coagulation function (vWF and PAI-1), and inflammation (IL-6 and TNF-α) were analyzed. RESULTS: T2DM patients had increased MPV and PDW. Furthermore, the platelet index was correlated with the levels of TG, HDL-C, vWF, PAI-1, IL-6, and TNF-α. CONCLUSIONS: MPV and PDW are increased in T2DM patients. Moreover, platelet index was associated with lipid metabolism disorder, coagulation dysfunction, and inflammatory response in T2DM patients.

Dynamic evaluation of blood immune cells predictive of response to immune checkpoint inhibitors in NSCLC by multicolor spectrum flow cytometry.

Introduction: Immune checkpoint inhibitors (ICIs) only benefit a subset of cancer patients, underlining the need for predictive biomarkers for patient selection. Given the limitations of tumor tissue availability, flow cytometry of peripheral blood mononuclear cells (PBMCs) is considered a noninvasive method for immune monitoring. This study explores the use of spectrum flow cytometry, which allows a more comprehensive analysis of a greater number of markers using fewer immune cells, to identify potential blood immune biomarkers and monitor ICI treatment in non-small-cell lung cancer (NSCLC) patients. Methods: PBMCs were collected from 14 non-small-cell lung cancer (NSCLC) patients before and after ICI treatment and 4 healthy human donors. Using spectrum flow cytometry, 24 immune cell markers were simultaneously monitored using only 1 million PBMCs. The results were also compared with those from clinical flow cytometry and bulk RNA sequencing analysis. Results: Our findings showed that the measurement of CD4+ and CD8+ T cells by spectrum flow cytometry matched well with those by clinical flow cytometry (Pearson R ranging from 0.75 to 0.95) and bulk RNA sequencing analysis (R=0.80, P=1.3 x 10-4). A lower frequency of CD4+ central memory cells before treatment was associated with a longer median progression-free survival (PFS) [Not reached (NR) vs. 5 months; hazard ratio (HR)=8.1, 95% confidence interval (CI) 1.5-42, P=0.01]. A higher frequency of CD4-CD8- double-negative (DN) T cells was associated with a longer PFS (NR vs. 4.45 months; HR=11.1, 95% CI 2.2-55.0, P=0.003). ICIs significantly changed the frequency of cytotoxic CD8+PD1+ T cells, DN T cells, CD16+CD56dim and CD16+CD56- natural killer (NK) cells, and CD14+HLDRhigh and CD11c+HLADR + monocytes. Of these immune cell subtypes, an increase in the frequency of CD16+CD56dim NK cells and CD14+HLADRhigh monocytes after treatment compared to before treatment were associated with a longer PFS (NR vs. 5 months, HR=5.4, 95% CI 1.1-25.7, P=0.03; 7.8 vs. 3.8 months, HR=5.7, 95% CI 169 1.0-31.7, P=0.04), respectively. Conclusion: Our preliminary findings suggest that the use of multicolor spectrum flow cytometry helps identify potential blood immune biomarkers for ICI treatment, which warrants further validation.

The Role of Macrophage Phenotype in the Vascularization of Prevascularized Human Bone Marrow Mesenchymal Stem Cell Sheets.

With the development of tissue engineering and regenerative medicine, prevascularized bone marrow mesenchymal stem cell (BMSC) sheets have been regarded as a promising method for tissue regeneration. Furthermore, the inflammatory response is one of the main regulators of vascularization and the restoration of engineered tissue function; among them, macrophages and cytokines produced by them are considered to be the decisive factors of the downstream outcomes. This study investigated the effect of macrophages on the formation of microvascular-like structures of human umbilical vein endothelial cells (HUVECs) in BMSC sheets. First, a human monocytic leukemia cell line (THP-1 cells) was differentiated into derived macrophages (M0) with phorbol 12-myristate 13-acetate and further activated into proinflammatory macrophages (M1 macrophages) with interferon-γ and lipopolysaccharide or anti-inflammatory macrophages (M2 macrophages) with interleukin-4. Then, HUVECs and prevascularized sheets were treated with conditioned media (CM) from different macrophages, and the impact of macrophage phenotypes on vascularized network formation in prevascularized cell sheets was examined by hematoxylin and eosin staining, CD31 immunofluorescence staining and enzyme-linked immunosorbent assay. Our study showed that macrophages may guide the arrangement of endothelial cells through a paracrine pathway. Cell sheets that were cultured in the CM from M2 macrophages were thinner than those cultured in other media. At various time points, the levels of tumor necrosis factor alpha and vascular endothelial growth factor in prevascularized sheets cultured with CM(M1) was higher than that in sheets cultured with other media; however, the levels of platelet-derived growth factor in prevascularized sheets cultured with CM(M2) was higher than that in sheets cultured with other media. These findings suggest that the paracrine effect of macrophages can influence the formation of microvascular networks in prevascularized sheets by regulating the arrangement of cells, the thickness of the cell sheet and the secretion of cytokines related to angiogenesis. Macrophages with different phenotypes have unique effects on prevascularized sheets.

Serum Irisin: A Potential Diagnostic Marker for Insulin Resistance in Acne Vulgaris.

Background: Acne vulgaris (AV) is a chronic inflammatory disease of the pilosebaceous unit. Many factors are involved in the occurrence of acne. It has been confirmed that some adipokines play an important role in the development of AV. Irisin is a novel adipokine, which is highly expressed in skeletal muscle, liver, and fat. It improves insulin resistance (IR) by inducing the browning of white adipose tissue, increasing heat production and energy expenditure. Objective: The purpose of this study was to investigate the role of serum irisin as an adipokine to explore its function in the pathogenesis of AV and its correlation with IR, and whether it can be used as a potential biomarker of insulin sensitivity. Although the hyperinsulinemic-euglycemic clamp remains the gold standard for accurate determination of IR, it cannot be performed routinely. Various alternative simpler measures have been used, the most common being homeostasis model assessment. However, these metrics are limited by their accuracy, cost, and blood collection requirements.[1] Therefore, an effective and feasible serum biomarker is an attractive and relatively straightforward method, which may provide clinicians with a more accurate and simple method for the prediction and diagnosis of IR. IR can often be detected before other symptoms appear, so establishing an early diagnosis method will allow for the appropriate treatment of patients before the disease develops. Patients and Methods: The study included 171 subjects; 115 patients with newly diagnosed AV and 56 apparently healthy subjects. The contents of irisin and interleukin-1 alpha in serum were determined by enzyme-linked immunosorbent assay. The IR index was calculated by the homeostasis model. Results: Serum irisin levels in AV patients and control group were (24.0 ± 11.3) and (104.3 ± 27.0) ng/dl, respectively, which were significantly lower than those in control group (P < 0.001). Serum irisin was negatively correlated with IR (r = -0.711, P 0.001). The sensitivity of irisin was 100.0%, the specificity was 92.8%, and the cutoff point was 53.32. The decrease of serum irisin level could predict the patients with IR in acne. Conclusion: Serum irisin levels in AV patients were significantly decreased. Serum irisin showed acceptable performance criteria in the diagnosis of AV with IR. Serum irisin seems to be a good diagnostic and prognostic marker for IR. Further multi-center studies are needed to confirm this link, which could pave the way for new treatment options.

Correlation of catecholamine content and clinical influencing factors in depression among psoriasis patients: a case-control study.

OBJECTIVE: Our study sought to investigate the clinical influencing factors of psoriasis patients with depression, and analyze whether the content of monoamine neurotransmitters in plasma was correlated with depression incidence among psoriasis patients. METHODS: Ninety patients with psoriasis and 40 healthy volunteers (aged from18 to 60) were recruited and interviewed with a piloted questionnaire in both groups to obtain relevant information. The catecholamine in plasma from the two groups was analyzed by radioimmunoassay. The data were analyzed by SPSS statistical software. RESULTS: The mean Hamilton Depression Scale (HAMD) and mean Athens Insomnia Scale (AIS) scores of the psoriasis patients were higher than the control group. Dopamine content in the plasma was lower (comparing psoriasis patients without depression and the control group, and was negatively correlated with HAMD, AIS, and Psoriasis Area and Severity Index (PASI) scores in the psoriasis patients with depression. There was no significant difference in the epinephrine and norepinephrine contents in all groups. PASI scores were positively correlated with HAMD scores in psoriasis patients. The low dopamine content, Dermatology Life Quality Index, and high PASI scores were the risk factors for depression among the psoriasis patients. CONCLUSION: Psoriasis patients have a significantly higher risk of depression than healthy people, and higher PASI scores were linked to a higher incidence of depression. The dopamine levels of patients were influenced by both psoriasis and depression. The risk factors for depression in psoriasis patients are low dopamine levels in the plasma, severe skin lesions, and lower quality of life.

Psychological Impact of COVID-19 Cases on Medical Staff of Beijing Xiaotangshan Hospital.

PURPOSE: To investigate the psychological impact of cases of coronavirus disease 19 (COVID-19) on medical staff of Beijing Xiaotangshan Hospital. METHODS: The 287 online questionnaires were distributed to medical staff working at Beijing Xiaotangshan Hospital, comprising three main sections and 17 questions: basic information, current departmental position, and the 12-item General Health Questionnaire (GHQ-12). The threshold for emotional distress was defined to be a total score of 4 on the GHQ-12 and above. RESULTS: A total of 255 members of medical staff participating in this study presented an emotional distress rate of 17%. Members who were male, aged 50-59, married with children, positioned as doctors, and in administration were the population with the highest rate of emotional distress. Furthermore, the severity of emotional distress among those under 30 was significantly lower than those aged 30-39 and 50-59. Doctors and other occupations shared a lower level of satisfaction on routine activities compared with nurses, so did staff in the administration compared with those who were working in screening or logistic departments. Besides, males and staff of the confirmation department had more difficulty in concentrating than females and those of the screening department, respectively. CONCLUSION: Medical staff working at Xiaotangshan Hospital underwent relatively low levels of emotional distress thanks to sufficient medical and psychological preparations. However, special attention should be paid to those who were male, married with children, senior, doctors, in administration, and in the confirmation department.

[Construction of three-dimensional dermoid tissue based on cell sheets technology in vitro].

OBJECTIVE: To explore a new strategy for constructing three-dimensional dermoid tissue in vitro by using cell sheets technology. METHODS: Rabbit bone marrow mesenchymal stem cells (rBMSCs) were isolated from bone marrow of New Zealand white rabbits and cultured by whole bone marrow adherent method. Human dermal fibroblasts (HDFs) were cultured and passaged in vitro. The 2nd generation rBMSCs and the 3rd generation HDFs were cultured in a culture dish for 2 weeks with cell sheets conditioned medium respectively to obtain a monolayer cell sheets. Human umbilical vein endothelial cells (HUVECs) were inoculated on rBMSCs sheet to construct pre-vascularized cell sheet. During the culture period, the morphological changes of the cell sheet were observed under an inverted phase contrast microscope. At 1, 3, 7, and 14 days, HE staining and CD31 immunofluorescence staining were performed to observe the cell distribution and microvascular network formation. The rBMSCs sheet was used as control. The pre-vascularized cell sheet (experimental group) and rBMSCs sheet (control group) cultured for 7 days were placed in the middle of two HDFs sheets, respectively, to prepare three-dimensional dermoid tissues. After 24 hours of culture, CD31 immunofluorescence staining and collagen type Ⅰ and collagen type Ⅲ immunohistochemical stainings were performed to evaluate cell distribution and collagen expression. RESULTS: HDFs and rBMSCs sheets were successfully prepared after 2 weeks of cell culture. After inoculation of HUVECs on rBMSCs sheet for 3 days, HUVECs could be seen to rearrange on rBMSCs sheet and forming vacuoles. The reticular structure was visible at 7 days and more obvious at 14 days. The formation of vacuoles between the cell sheets was observed by HE staining, and the vacuoles became more and more obvious, the thickness of the membranes increased significantly with time. CD31 immunofluorescence staining showed the microvascular lumen formation. However, only the thickness of rBMSCs sheet increasing was observed, with no changes in cell morphology or cavitation structure. The three-dimensional dermoid tissue observation showed that the endothelial cells in the experimental group were positive expressions, and the rBMSCs, HDFs, and HUVECs cells were arranged neatly. The endothelial cells were negative expressions and randomly arranged in the control group. The collagen type Ⅰ and collagen type Ⅲ were positive expression in the experimental group and the control group. But compared with control group, experimental group presented a "honeycomb" network connection, where the matrix was distributed regularly, and cells were arranged tightly. The difference in the expression of collagen type Ⅰ and collagen type Ⅲ between the experimental group and the control group was not significant ( P>0.05). CONCLUSION: Three-dimensional dermoid tissue is successfully constructed by using cell sheet technology. The cell matrix distribution of the pre-vascularized cell sheet constructed by HUVECs and rBMSCs sheet is relatively regular, which has the potential to form tissue engineered dermis.

Controlling cell viability and bacterial attachment through fabricating extracellular matrix-like micro/nanostructured surface on titanium implant.

Good osseointegration and gingival epithelial sealing play a key role in preventing peri-implantitis of dental implants. In addition to antibacterial qualities, the transmucosal surface of the implant is beneficial to the growth of fibroblasts and epithelial cells, while its body surface is suitable for the growth of osteoblasts and is resistant to epithelial cells and fibroblasts. In this study, both microgrooves mimicking the extracellular matrix (ECM) and titanium (Ti) dioxide nanotubes with different parameter settings were produced on Ti surfaces. The behavior of MG63 osteoblasts, L929 fibroblasts, SCC epithelial cells and Porphyromonas gingivalis on these decorated Ti surfaces was detected to quantify their performances in terms of osseointegration, biological sealing and antimicrobial ability. Via a scoring method based on these results, we concluded that 100-50-20-10-5 µm width grooves arranged in the horizontal direction at 2 µm depth were the priority for the design of the implant's transmucosal surface. By changing the depth to 3.6 µm and further decorating with 55 nm nanotubes, a best surface design for the implant body was acquired. Hierarchical ECM-like micro/nano patterns could provide novel designs for dental implants to achieve excellent gingival epithelial sealing and osseointegration, which would facilitate the clinical application of dental implants.

Fabrication of vascularized and scaffold-free bone tissue using endothelial and osteogenic cells differentiated from bone marrow derived mesenchymal stem cells.

Over-dependence on existing synthetic scaffolds and insufficient vascularization limit the development of tissue engineered bone (TEB). The purpose of this study is to fabricate vascularized and scaffold-free bone tissue using cell sheet technology and to assess its feasibility to repair critical-sized calvarial defects in rats. Firstly, the pre-vascularized cell sheet was formed by seeding BMSC-derived endothelial cells (ECs) on an undifferentiated BMSCs cell sheet layer in vitro. After 3 days of co-culture, ECs migrated and rearranged to form lumens on the BMSC sheet. Secondly, osteogenic cell sheet was formed by inducing osteogenic differentiation of high density BMSCs. Then, the pre-vascularized cell sheet was stacked on BMSC-derived osteogenic cell sheet to fabricate a scaffold-free construct for bone regeneration. Finally, the scaffold-free construct with both angiogenic and osteogenic potential was implanted into critical-sized calvarial defects in adult Wistar rats. Results showed that more functional perfused blood vessels and new bone tissue formed in the pre-vascularized group than that in the controls (both empty and non-pre-vascularized cell sheet group). This study indicates a new promising strategy for bone tissue regeneration.

High frequency of circulating follicular helper T cells is correlated with B cell subtypes in patients with ankylosing spondylitis.

T follicular helper (Tfh) cells are known to support effector B cells and enhance autoimmunity; however, the association between the Tfh cells and B cells in ankylosing spondylitis (AS) is unclear. The aim of the present study was to measure the frequency of circulating cluster of differentiation (CD)4+ C-X-C chemokine receptor type 5 (CXCR5)+ Tfh cells and B cell subtypes in peripheral blood from patients with AS, and evaluate the correlation of these factors. Percentages of peripheral blood circulating CD4+CXCR5+ Tfh cells and B cell subtypes were measured via flow cytometry and the disease activity of individual patients was measured using the Bath AS Disease Activity Index (BASDAI). The potential association among these measures was analyzed via Spearman's or Pearson's correlations. In comparison with those in healthy controls (HC), significantly increased percentages of CD4+CXCR5+ cTfh, CD4+CXCR5+ programmed death 1+, CD4+CXCR5+ inducible T cell costimulator (ICOS)+, CD3+CD8-CXCR5+ interleukin (IL)-21+ T cells, CD19+CD27high plasmablast and CD19+CD38+ antibody-secreting B cells were detected in patients with AS, whereas there was no significant difference in CD19+CD27- naïve B cells and CD19+CD27+ memory B cells. When Patients with AS were divided into high and low activity groups, significantly higher percentages of CD4+CXCR5+, CD3+CD8-CXCR5+IL-21+ T cells, CD19+CD27- naïve B cells and CD19+CD38+ antibody-secreting B cells, and lower CD19+CD27+ memory B cells were detected in high activity AS group compared with the low activity AS group. In addition, percentages of CD4+CXCR5+ circulating (c)Tfh, CD3+CD8-CXCR5+IL-21+ T and CD19+CD38+ antibody-secreting B cells were positively correlated with BASDAI values. Furthermore, the percentage of CD4+CXCR5+ cTfh cells was positively correlated with CD19+CD38+ antibody-secreting B cells and the percentage of CD3+CD8-CXCR5+IL-21+ T cells was positively correlated with CD19+CD27- naïve B cells in patients with AS. These findings suggest that CD4+CXCR5+ cTfh, CD3+CD8-CXCR5+IL-21+ T and CD19+CD38+ antibody-secreting B cells may participate in the pathogenesis of AS because of their distinct functions. As such, levels of cTfh and B cell subtypes may be a useful biomarker for the evaluation of disease activity in patients with AS.