C-terminal binding protein 2 promotes high-glucose-triggered cell proliferation, angiogenesis and cellular adhesion of human retinal endothelial cell line.

PURPOSE: The proliferation and angiogenesis of human retinal endothelial cells (HRECs) are critical for the pathophysiology of diabetic retinopathy (DR). C-terminal binding protein 2 (CtBP2) has multiple biologic functions, but its effect on HRECs under high-glucose (HG) conditions is unclear. METHODS: The cell viability, angiogenesis, cellular adhesion and CtBP2 expression levels of HRECs were measured following treatment with different concentrations of glucose. Small interfering CtBP2-targeting RNA, wide-type and function mutant plasmid of CtBP2 were constructed and then were transfected into HRECs to evaluate the effects of CtBP2 on cell functions of HRECs. RESULTS: The expression of CtBP2 in HRECs was increased after HG treatment. HG treatment significantly increased cell proliferation, angiogenesis, and decreased relative gene expressions in gap junctions, tight junctions and adherens junctions. After CtBP2 was inhibited via siRNA, the changes induced by HG were partially restored. Conversely, only wild-type CtBP2 could increase cell proliferation and angiogenesis under HG condition. Mechanistically, we also found that CtBP2 exerted its functions to effect HG-induced changes via Akt signaling pathway. CONCLUSION: This study implicates that CtBP2 promotes HG-induced cell proliferation, angiogenesis and cellular adhesion, and CtBP2 might be a potential target in the prevention of DR.

The exosomes derived from CAR-T cell efficiently target mesothelin and reduce triple-negative breast cancer growth.

Genetically engineered T cells expressing a chimeric antigen receptor (CAR) have rapidly developed into a powerful and innovative therapeutic modality for cancer patients. However, the problem of dose-dependent systemic toxicity cannot be ignored. In this study, exosomes derived from mesothelin (MSLN)-targeted CAR-T cells were isolated, and we found that they maintain most characteristics of the parental T cells, including surface expression of the CARs and CD3. Furthermore, CAR-carrying exosomes significantly inhibited the growth of both endogenous and exogenous MSLN-positive triple-negative breast cancer (TNBC) cells. The expression of the effector molecules perforin and granzyme B may be a mechanism of tumor killing. More importantly, a highly effective tumor inhibition rate without obvious side effects was observed with the administration of CAR-T cell exosomes in vivo. Thus, the use of CAR-T cell exosomes has great therapeutic potential against MSLN-expressing TNBC.

Upregulation of microRNA-155 Enhanced Migration and Function of Dendritic Cells in Three-dimensional Breast Cancer Microenvironment.

Background: Dendritic cells (DCs) play an essential role in the induction and regulation of immune responses, including the activation of effector T lymphocytes for the eradication of cancers. However, the tumor microenvironment (TME) often leads to DCs dysfunction due to their immature state. MicroRNA-155 (miR-155) has emerged as a typical multifunctional gene regulator associated with immune system development and immune cell activation and differentiation.Methods: In this study, a three-dimensional TME model that closely mimics the microenvironment of breast cancer was prepared. MiR-155 overexpression and control vectors were constructed using lentivirus. The relative expression of miR-155 was determined by qRT-PCR. Cell viability, antigen uptake and cell surface marker expression were analyzed by live-dead staining and flow cytometry. The migration ability of bone marrow-derived DCs (BMDCs) was qualified by transwell assay. A mixed lymphocyte culture assay was used to assess T cell-specific proliferation. Cytokine levels were determined by ELISA.Results: We found that the expression of miR-155 in DCs was inhibited by the TME. Furthermore, upregulation of miR-155 enhanced the migration ability, uptake of antigen and elevated the expression of the mature DCs markers CD80 and MHCII. More importantly, overexpression of miR-155 in DCs significantly induced T cell proliferation and IFN-γ and IL-2 secretion.Conclusion: MiR-155 is a potential molecular regulator that may improve the efficacy of DCs-based tumor immunotherapy.

Lymphocyte cell population as a potential hematological index for early diagnosis of COVID-19.

The pandemic diseases caused by SARS-CoV-2 are now threatening human health and survival. Early diagnosis and isolation of mild or asymptomatic COVID-19 patients is important to control the spread of SARS-CoV-2. In this study, we investigate the potential clinical utility of lymphocyte CPD for early diagnosis of COVID-19. To investigate the potential of lymphocyte cell population data (lymphocyte CPD) for use in early diagnosis of coronavirus disease 2019 (COVID-19). Lymphocyte CPD of healthy control (n = 51), common cold patients (n = 49) and mild COVID-19 patients (n = 126) were generated using hematology analyzer. The parameters were subjected to sensitivity and specificity analysis to determine their suitability as biomarkers for early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Normality analysis showed that lymphocyte CPD followed a normal distribution. There were no significant differences in white blood cells (WBC) and lymphocyte (LY#) counts as well as the neutrophil-to-lymphocyte ratio (NLR) among the groups (p > 0.05). Lymphocyte volume standard deviation (LV-SD), lymphocyte conductivity standard deviation (LC-SD) and lymphocyte light scatter standard deviation (LS-SD) were significantly higher in the COVID-19 group than in common cold and control groups (p < 0.05). The corresponding mean lymphocyte light scattering  (MLS) was significantly reduced in the COVID-19 group, relative to the common cold group, but was significantly increased, when compared with the control group (p < 0.05). Moreover, there was no significant difference in mean lymphocyte volume (MLV) between the COVID-19 group and the common cold or control group (p > 0.05), but it was significantly higher in the common cold group than in the control group (p < 0.05). At a cutoff value ≥ 16.38, LS-SD was more sensitive and specific than other lymphocyte CPD parameters. At a cutoff value ≥ 11.89, LC-SD achieved 84.4 % sensitivity, 87.5 % specificity, and an area under the curve (AUC) of 0.888. However, at a cutoff value ≥ 15.95, LS-SD reached 81.3 % sensitivity, 75 % specificity and an AUC of 0.876. These results suggest that lymphocyte CPD parameters have great diagnostic potential for SARS-CoV-2 infection and can be used for early diagnosis of the disease.

Lupus-associated atypical memory B cells are mTORC1-hyperactivated and functionally dysregulated.

OBJECTIVES: A population of atypical memory B cells (AtMs) are greatly expanded in patients with active lupus, but their generation and pathophysiological roles are poorly defined. The aim of this study was to comprehensively characterise lupus AtMs with a purpose to identify therapeutic clues to target this B cell population in lupus. METHODS: Peripheral B cell subsets were measured by flow cytometry. Sorting-purified B cell subsets were subject to RNA sequencing and functional studies. Plasma cytokines and secreted immunoglobulins were detected by Luminex or ELISA. In situ renal B cells were detected by multiplexed immunohistochemistry. RESULTS: CD24-CD20hi AtMs were strongly increased in two Chinese cohorts of patients with treatment-naïve lupus. Gene expression profile indicated that B cell signalling and activation, lipid/saccharide metabolism and endocytosis pathways were abnormally upregulated in lupus AtMs. In addition, the mammalian target of rapamycin complex 1 (mTORC1) pathway was remarkably activated in lupus AtMs, and blocking mTORC1 signalling by rapamycin abolished the generation of T-bet+ B cells and terminal differentiation of lupus AtMs. Furthermore, lupus AtMs displayed a dysfunctional phenotype, underwent accelerated apoptosis, poorly co-stimulated T cells and produced proinflammatory cytokines. Interestingly, lupus AtMs were in a paradoxically differentiated status with markers pro and against terminal differentiation and enriched with antinucleosome reactivity. Finally, AtMs were accumulated in the kidneys of patients with lupus nephritis and associated with disease severity. CONCLUSIONS: These findings demonstrated that mTORC1-overactivated lupus AtMs are abnormally differentiated with metabolic and functional dysregulations. Inhibiting mTORC1 signalling might be an attractive option to target AtMs and to improve therapeutic effectiveness in patients with lupus.

Effects of FOXJ2 on TGF-ß1-induced epithelial-mesenchymal transition through Notch signaling pathway in non-small lung cancer.

As one member of Forkhead box transcription factors, Forkhead box J2 (FOXJ2) has been found to be involved in epithelial-mesenchymal transition (EMT) process. However, the role and mechanism of FOXJ2 in non-small cell lung cancer (NSCLC) and EMT regulation have not been fully revealed. In this paper, it was revealed that the expression of FOXJ2 was lower in NSCLC samples compared with matched peritumoral lung tissue. We demonstrated that FOXJ2 expression was down-regulated by transforming growth factor-ß1 (TGF-ß1) treated, and overexpression of FOXJ2 inhibited TGF-ß1-induced EMT. Mechanistically, knocking out the expression of FOXJ2 promoted EMT by increasing the expression of Notch1 and NICD. This study implicates the potential value of FOXJ2 as a molecular marker for NSCLC.

Methylation of OPCML promoter in ovarian cancer tissues predicts poor patient survival.

BACKGROUND: Ovarian cancer is a lethal gynecological malignancy largely due to the lack of biomarkers for early detection and treatment options. Opioid binding protein/cell adhesion molecule-like gene (OPCML) has a tumor-suppressor function in ovarian cancer, and epigenetic inactivation of OPCML induces oncogenic transformation of human ovarian surface epithelial cells. METHODS: This study investigated OPCML promoter methylation levels in ovarian cancer tissues. A total of 30 normal ovarian, 85 benign ovarian tumor, and 102 ovarian cancer tissues were subjected to quantitative methylation-specific PCR analysis of OPCML methylation. Four ovarian cancer cell lines were cultured and treated with the DNA demethylation agent 5-aza-2'-deoxycytidine (5-AZA) for restoring OPCML expression. RESULTS: The data showed that 80 of 102 (78.4%) ovarian cancer tissues and 28 of 85 (32.9%) benign ovarian tumors had a methylated OPCML gene promoter. In contrast, there was no OPCML gene promoter methylation in any of the 30 normal ovarian samples. OPCML promoter methylation was significantly associated with an older age of the patients (p=0.022), an advanced pathological stage of ovarian cancer (p=0.023), and poor overall survival of ovarian cancer patients (p<0.001). Multivariate analysis data showed that pathological stage, age, and OPCML promoter methylation were all independent factors to predict overall survival of patients. Furthermore, 5-AZA was able to restore expression of OPCML mRNA and protein in ovarian cancer cell lines. CONCLUSIONS: These data indicate that detection of OPCML gene promoter methylation could be a useful biomarker for predicting the prognosis of ovarian cancer patients and disease progression.

Detection of circulating methylated opioid binding protein/cell adhesion molecule-like gene as a biomarker for ovarian carcinoma.

BACKGROUND: Hypermethylation of the opioid binding protein/cell adhesion molecule-like (OPCML) gene is frequently observed in ovarian carcinoma. We evaluated the detection of circulating hypermethylated OPCML for detecting ovarian carcinoma and assessing its prognosis. METHODS: We studied 85 tissue samples including 45 ovarian cancer tissues and 40 normal ovarian tissues and blood samples from 45 ovarian cancer patients and 20 healthy individuals. Bisulfite sequencing and methylation-sensitive restriction enzyme-PCR (MSRE-PCR) were used to detect the frequency of OPCML hypermethylation. RESULTS: We detected that the frequency of OPCML hypermethylation for tissue and serum samples in ovarian carcinoma were 86.7% (39/45) and 80.0% (36/45), respectively, but none was detected in ovarian tissue and serum of healthy individuals. The frequency of OPCML hypermethylation in endometrioid carcinoma, serous cystadenocarcinoma, mucinous cystadenocarcinoma, clear cell carcinoma, and undifferentiated carcinoma were 80.0%, 85.5%, 50.0%, 80.0%, and 100%, respectively (p > 0.05). The frequencies of OPCML hypermethylation in patients were also different in terms of tumor differentiation degree. We detected hypermethylated OPCML in the sera of 50% of well differentiated, 62.5% of moderately differentiated, 93.1% of poorly differentiated tumors (p < 0.05). The frequency of OPCML hypermethylation at FIGO stage I was 42.9%, stage II was 66.7%, stage III was 85.7%, stage IV was 100% (p < 0.05). CONCLUSIONS: Detection OPCML methylation in the serum is useful for ovarian carcinoma diagnosis.

Delta mean neutrophil volume (ΔMNV) is comparable to procalcitonin for predicting postsurgical bacterial infection.

BACKGROUND: The Coulter LH750 (Beckman Coulter, Brea, CA) analyzer can determine intrinsic biophysical properties of white blood cell (WBC), known as cell population data. Previous studies have shown that mean neutrophil volume (MNV) was significantly increased in postsurgical patients with bacterial infection. To further validate its potential clinical usefulness, we investigate the changes in MNV before and after surgery, called ΔMNV. We also compare the ΔMNV with procalcitonin (PCT) and C-reactive protein (CRP) in terms of diagnostic sensitivity and specificity for postsurgical bacterial infection. METHODS: Blood samples from 300 healthy controls, 219 cardiac surgical patients without postsurgical infection, and 31 cardiac surgical patients complicated with postsurgical bacterial infection were studied. RESULTS: There are no statistically significant differences for WBC count and neutrophil percentage prior to or after surgery between postsurgical noninfected and infected patients. However, the ΔMNV is significantly increased in postsurgical infected patients when compared with noninfected patients (P < 0.05). The receiver-operating characteristics analysis reveals the ΔMNV and PCT have largest areas under curves (0.92, 0.93 on the second day and 0.94, 0.99 on the third day postsurgery, respectively) compared to other parameters. CONCLUSION: ΔMNV shows comparable sensitivity and specificity to PCT and superior sensitivity and specificity to WBC or CRP for predicting postsurgical bacterial infection. The potential clinical application of this parameter merits further exploration in a larger prospective study.

A study of the methylation status of opioid binding protein/cell adhesion molecule-like gene in ovarian cancer using nested methylation-specific polymerase chain reaction detection.

BACKGROUND: The goal was to improve the methylation-specific PCR (MSP) method and investigate the methylation status of the OPCML gene in carcinoma tissues from ovarian cancer patients. METHODS: MSP and nested MSP methods were used to examine the methylation status of the OPCML gene promoter in ovarian cancer, borderline tumor, and normal ovary tissues. RESULTS: Methylation of the OPCML gene was detected in 58.3% (14/24) and 83.3% (20/24) of the specimens from ovarian cancer patients using MSP and nested MSP methods, respectively. No methylation was observed in normal ovarian tissues using either method. CONCLUSIONS: The modified nested MSP method showed better sensitivity. The methylation of the OPCML gene was significantly higher in ovarian cancer than in normal tissue. The detection of OPCML gene methylation could serve as one of the molecular markers for the diagnosis of ovarian cancer.

Immune Cell-Derived Extracellular Vesicles - New Strategies in Cancer Immunotherapy.

Immune cell-derived extracellular vesicles (EVs) have increasingly become the focus of research due to their unique characteristics and bioinspired applications. They are lipid bilayer membrane nanosized vesicles harboring a range of immune cell-derived surface receptors and effector molecules from parental cells. Immune cell-derived EVs are important mediators of intercellular communication that regulate specific mechanisms of adaptive and innate immune responses. However, the mechanisms underlying the antitumor effects of EVs are still being explored. Importantly, immune cell-derived EVs have some unique features, including accessibility, storage, ability to pass through blood-brain and blood-tumor barriers, and loading of various effector molecules. Immune cell-derived EVs have been directly applied or engineered as potent antitumor vaccines or for the diagnosis of clinical diseases. More research applications involving genetic engineering, membrane engineering, and cargo delivery strategies have improved the treatment efficacy of EVs. Immune cell-derived EV-based therapies are expected to become a separate technique or to complement immunotherapy, radiotherapy, chemotherapy and other therapeutic modalities. This review aims to provide a comprehensive overview of the characteristics and functions of immune cell-derived EVs derived from adaptive (CD4+ T, CD8+ T and B cells) and innate immune cells (macrophages, NK cells, DCs, and neutrophils) and discuss emerging therapeutic opportunities and prospects in cancer treatment.

The morphological changes of monocytes in peripheral blood as a potential indicator for predicting active pulmonary tuberculosis.

BACKGROUND: Monocytes play a crucial role in immune response against Mycobacterium tuberculosis infection. The purpose of this current study was to investigate the morphology present on monocytes in peripheral blood from patients with active pulmonary tuberculosis (APTB) and the laboratory performance of the changes for discriminating cases from normal healthy subjects (NHS). METHOD: A total of 71 peripheral blood samples from patients with APTB, and 65 samples from NHS were analyzed. The mean monocyte volume with its distribution width and mean monocyte conductivity as well as monocyte light scatter were detected by VCS technology used on the LH750 hematology analyzer. Correlations of these changes with the serum cytokine level in the immune alterations were further evaluated. The Receiver operating characteristic curve (ROC) analysis was used to highlight the clinical implication. RESULTS: In APTB patients, the mean monocyte volume showed significant difference associated with an evident elevation in the mean monocyte volume distribution width compared to those in NHS. Furthermore, the mean monocyte volume had positive relationship with the serum level of interleukine-1ß response to M. tuberculosis infection. Simultaneous measurement of the mean monocyte volume and its distribution width was able to distinguish active infection with an excellent sensitivity of 84.5% and specificity of 90.5% comparable to those obtained from pro-inflammatory cytokine interleukine-6 identifying APTB with great accuracy. CONCLUSION: The morphological changes of monocytes particular increased mean volume may be a potential indicator to predict active tuberculosis infection.

The VCS parameters: Potential hematological indicators for predicting antituberculosis drug-induced neutropenia.

BACKGROUND: The morphological changes in activated neutrophils associated with antituberculosis drugs can be measured by volume, conductivity, and scatter (VCS) technology on the Coulter LH750 hematology analyzer. We conducted the current study to further validate the clinical usefulness of the neutrophil VCS parameters in predicting drug-induced neutropenia. METHODS: Peripheral blood samples were collected from 52 patients with drug-induced neutropenia, 309 patients without any abnormal CBC, and 237 healthy controls. The mean neutrophil volume (MNV) with its distribution width (NDW) and the mean neutrophil scatter (MNS) were studied. RESULTS: We observed a significant increase in the MNV and NDW as well as a significant decrease in the MNS in neutropenia patients approximately one week prior to development of neutropenia compared to healthy controls as well as to case controls. In addition, the delta MNV and delta MNS were respectively correlated well with delta absolute neutrophil counts when neutropenia occurred. The ROC curve analyses showed that the MNV、NDW and MNS had larger areas under curves compared to conventional parameters. With a cutoff of 150.15 for the MNV, a sensitivity of 84.4% and specificity of 75.7% were achieved prior to neutropenia. CONCLUSION: The neutrophil VCS parameters may be clinically useful as potential hematological indicators for predicting antituberculosis drug-induced neutropenia.

Increased levels of exhaled sICAM1, sVCAM1, and sE-selectin in patients with non-small cell lung cancer.

AIM: The mortality of lung cancer remains high and methods for early diagnosis are still lacking. Recently, exhaled breath condensate (EBC) has been considered a potential tool for obtaining biological information leading to a reliable diagnosis of non-small cell lung cancer (NSCLC). OBJECTIVE: This study assessed the potentials of exhaled and serum concentrations of soluble(s) forms of intercellular adhesion molecule 1 (sICAM1), vascular cell adhesion molecule 1 (sVCAM1), and E-selectin as biomarkers for diagnosis and predicting metastasis in NSCLC patients. METHODS: We enrolled 33 patients with NSCLC, 35 patients with chronic obstructive pulmonary disease (COPD) and 30 healthy controls. EBC and serum samples from subjects were collected at the time of diagnosis and, where applicable, 3 months after surgical treatment. Measurements of sICAM1, sVCAM1, and sE-selectin were determined by enzyme immunoassay. RESULTS: Concentrations of sICAM1, sVCAM1, and sE-selectin in the EBC and sera of NSCLC patients were significantly elevated compared to COPD patients and healthy controls. The exhaled and serum levels of sICAM1 and sVCAM1, but not sE-selectin, decreased significantly after tumor resection from pre-surgery levels. In addition, analyzed results showed a correlation between exhaled sICAM1 levels and disease progression of NSCLC patients. CONCLUSIONS: Our results suggest that the levels of sICAM1, sVCAM1, and sE-selectin in EBC and sera of NSCLC patients are higher than those of COPD patients or healthy controls. Moreover, exhaled sICAM1 may have prognostic value and potential as a biomarker for the diagnosis and prognosis of patients with lung cancer.

The role of C-terminal binding protein 2 in Schwann cell differentiation after sciatic nerve crush.

C-terminal binding protein 2 (CtBP2), as a transcriptional repressor, plays an essential role in development and tumorigenesis. However, its distribution and function in peripheral system lesion and repair are still unknown. Here, we investigated the spatiotemporal expression of CtBP2 in rat sciatic nerve crush model. Western blot analysis revealed that CtBP2 was expressed in normal sciatic nerve. It gradually decreased, reached minimal levels at 7 days after crush, and then returned to the normal level at 4 weeks. We observed that CtBP2 is mainly expressed in Schwann cells (SCs). In vitro, we induced SC differentiation via cyclic adenosine monophosphate (cAMP) and found that CtBP2 expression was downregulated during the process of differentiation. CtBP2-specific siRNA inhibited the cAMP-induced expression of the immature SC marker P75(NTR), and exogenous CtBP2 expression upregulated the expression of P75(NTR). Taken together, we hypothesized that peripheral nerve crush-induced downregulation of CtBP2 in the sciatic nerve was associated with SC differentiation, and CtBP2 likely played an important role in peripheral nerve injury and regeneration.

FOXJ2 expression in rat spinal cord after injury and its role in inflammation.

Foxj2 (forkhead box J2), a novel member of the forkhead/HNF3 family, binds DNA with a dual sequence specificity. It may play a role in maintenance and survival of developing and adult neurons. However, its expression and function in the central nervous system lesion are still unclear. In this study, we performed a spinal cord injury (SCI) model in adult Sprague-Dawley rats and investigated the dynamic changes of Foxj2 expression in the spinal cord. Western blot analysis revealed that Foxj2 was present in normal spinal cord. It gradually increased, reached a peak at day 5 after SCI, and then declined during the following days. Double immunofluorescence staining revealed wide expression of Foxj2, which is detected in neurons and astrocytes. After injury, Foxj2 expression was increased predominantly in astrocytes, which highly expressed proliferating cell nuclear antigen, a marker for proliferating cells. And knockdown of Foxj2 in cultured primary astrocytes by siRNA showed that Foxj2 played an important role in lipopolysaccharide-induced inflammatory responses. These results suggested that Foxj2 may be involved in the pathophysiology of SCI, and further research is needed to have a good understanding of its function and mechanism.