[The Observation of Liver and Kidney Injury and the Activation of Macrophages in the Overload Pressure Induced Cardiac Hypertrophy/Heart Failure Mouse Model].

OBJECTIVE: The purpose of this study is to investigate the injury of liver and kidney tissues in overload pressure induced cardiac hypertrophy/heart failure mice model and the changes of macrophage activation level. METHODS: 6-8 week-old C57BL/6 mice were subjected to transverse aortic constriction (TAC) surgery to establish the cardiac hypertrophy/heart failure mouse model induced by pressure overload, while the aortic was not ligated in the Sham group. At 4 weeks and 8 weeks after TAC, the mice of each group were subjected to echocardiography and blood collection. And mice were sacrificed to collect samples of the heart, liver, and kidney tissues. The contents of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil) and serum creatinine (Scr) in Sham group and two operation groups were determined. The histological changes of liver, heart and kidney tissues were observed by HE staining, and the expression of the marker of macrophage activation, F4/80 protein, was detected in the heart, liver and kidney tissue by immunohistochemical staining. RESULTS: Cardiac hypertrophy occurred at 4 weeks after TAC operation in C57BL/6 mice and developed into heart failure at 8 weeks after TAC. The echocardiography showed that, compared with the Sham group, the left ventricular end-diastolic posterior wall thickness (LVPWd) and the left ventricular internal diameter in diastole (LVIDd) were significantly increased, while the left ventricular ejection fraction (EF) and the left ventricular fractional shortening (FS) were significantly decreased ( P<0.05) in the 4-week-TAC group and 8-week-TAC group. The plasma content of ALT, AST, TBil and Scr in the 4-week-TAC group and 8-week-TAC group were significantly higher than those in the Sham group ( P<0.05). HE staining showed obvious liver pathological changes in TAC mice, such as vacuolation, mild hepatic sinusoid congestion and inflammatory infiltration in mice post 4 weeks after surgery, and such liver injury was worse in mice post 8 weeks after surgery. Besides, there was a slight damage in renal tissue shown by HE staining, such as slight glomerular injury and slight bleeding. F4/80 protein immunohistochemical staining results demonstrated that the activation of macrophages in the heart and liver in the 4-week-TAC group and 8-week-TAC group was significantly increased than that in the sham group ( P<0.05), but there was no significant difference in kidney tissues in groups. CONCLUSION: Macrophages are involved in the process of liver and kidney injury in cardiac hypertrophy/heart failure.

Circulating hematopoietic stem and progenitor cells are myeloid-biased in cancer patients.

Cancer is associated with a profound perturbation in myelopoiesis that results in the accumulation of myeloid-derived suppressor cells (MDSCs) to promote disease progression. Recent studies in mice suggest that tumor-derived factors could regulate the differentiation of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and subsequently contribute to dysregulation of hematopoiesis. However, the nature and role of HPSCs in patients with cancer remain unknown. Here we show, in detailed studies of the peripheral blood from 133 untreated patients with seven different types of tumors, that the composition of circulating HSPCs was significantly altered in patients with solid tumors. The frequencies of circulating granulocyte-monocyte progenitors (GMPs) were increased four to seven fold in all types of tumors examined, and the circulating hematopoietic precursors exhibited myeloid bias with a skew toward granulocytic differentiation in patients with solid tumors. These myeloid precursors are selectively enriched in tumor tissues, and the high levels of circulating GMPs were positively correlated with disease progression. By using cord blood-derived CD34(+) cells, we developed an in vitro short-term culture model to effectively induce the rapid generation of MDSCs. We found that, among the factors produced by various tumors, GM-CSF, granulocyte colony-stimulating factor, and IL-6 could not only promote the myeloid-biased differentiation, but also induce the differentiation of myeloid precursors into functional MDSCs. These findings suggest that the altered circulating HSPCs may serve as an important link between dysregulated bone marrow hematopoiesis and accumulated MDSCs in patients with cancer.

[Investigate the clonal origin of different phenotypic tumor-like cells in the epidermis and dermis of skin lesions in lymphomatoid papulosis].

OBJECTIVE: To explore the correlation of the clone sources of MF-like cells in the epidermis and RS-like cells in the dermis of lymphomatoid papulosis (LyP), a compartive study of this two type cells in 8 LyP cases was carried out. METHODS: 8 cases of LyP (4 type A cases and 4 type C cases) were detected the immunophenotype first, and then comparative study between the MF-like cells and RS-like cells was conducted on T-cell receptor (TCR) gamma-chain gene rearrangement by combinating laser capture microdissectinon (LCM) and PCR. PCR products of one patient which had positive bands were selected randomly for sequencing and sequence alignment analysis. RESULTS: Monoclonal TCRgamma gene rearrangement was detected in 6 cases (75%) of the 8 cases. Detection rates of LyP type A and type C were 50% (2/4) and 100% (4/4), respectively. The rates of MF-like cells and RS-like cells were 75% (6/8) and 75% (6/8), respectively. Homology of the products with positive stripes in both MF-like cells and RS-like cells in one randomly selected case was compared and analyzed, and the results showed the homology of them was 106/108 (98%). CONCLUSION: The MF-like cells in the epidermis and the RS-like cells in the dermis of LyP may be from the same clone.

[Construction of shRNA expressing plasmid targeted rat NogoB and observation of the effection about NogoB on hepatic stellate cells].

OBJECTIVE: To construct shRNA expressing plasmid inhibiting rat NogoB and to observe its possible effect on rat primary hepatic stellate cells (HSCs) contraction. METHODS: Three pairs of shRNAs targeting different sequence of rat NogoB were designed and constructed into pSuper plasmid by DNA recombination technique. Culture-activated HSCs were transfected with NogoB-shRNA plasmids to scan the effective plasmid which could inhibit NogoB gene expression by Real-time PCR. And this depressant effect was also confirmed with Western blot. After NogoB was knocked-down effectively, ETA and ETB mRNA expression were assessed by Real-time PCR. RESULTS: Among the three pairs of recombinant plasmids, NogoB-shRNA2 plasmid could inhibit NogoB expression specifically. In HSCs, NogoB knockdown decreased the ratio of ETA and ETB. CONCLUSION: We constructed specific NogoB-shRNA expression plasmid successfully which might be involved in contraction of HSCs.

Advanced brain aging in Parkinson's disease with cognitive impairment.

Patients with Parkinson's disease and cognitive impairment (PD-CI) deteriorate faster than those without cognitive impairment (PD-NCI), suggesting an underlying difference in the neurodegeneration process. We aimed to verify brain age differences in PD-CI and PD-NCI and their clinical significance. A total of 94 participants (PD-CI, n = 27; PD-NCI, n = 34; controls, n = 33) were recruited. Predicted age difference (PAD) based on gray matter (GM) and white matter (WM) features were estimated to represent the degree of brain aging. Patients with PD-CI showed greater GM-PAD (7.08 ± 6.64 years) and WM-PAD (8.82 ± 7.69 years) than those with PD-NCI (GM: 1.97 ± 7.13, Padjusted = 0.011; WM: 4.87 ± 7.88, Padjusted = 0.049) and controls (GM: -0.58 ± 7.04, Padjusted = 0.004; WM: 0.88 ± 7.45, Padjusted = 0.002) after adjusting demographic factors. In patients with PD, GM-PAD was negatively correlated with MMSE (Padjusted = 0.011) and MoCA (Padjusted = 0.013) and positively correlated with UPDRS Part II (Padjusted = 0.036). WM-PAD was negatively correlated with logical memory of immediate and delayed recalls (Padjusted = 0.003 and Padjusted < 0.001). Also, altered brain regions in PD-CI were identified and significantly correlated with brain age measures, implicating the neuroanatomical underpinning of neurodegeneration in PD-CI. Moreover, the brain age metrics can improve the classification between PD-CI and PD-NCI. The findings suggest that patients with PD-CI had advanced brain aging that was associated with poor cognitive functions. The identified neuroimaging features and brain age measures can serve as potential biomarkers of PD-CI.

[Preliminary research on the differentiation of human amniotic epithelial cells into female germ cells].

OBJECTIVE: To establish the method for isolation and culturation of human amniotic epithelial cells (hAECs) in vitro, and to investigate the differentiation potential of hAECs towards germ cells. METHODS: hAECs were isolated from human term placenta and identified by immunocytochemistry. hAECs were sequential induced to form germ cells in medium supplemented with 5% human follicular fluid. Morphological changes were observed by inverted microscope. The oestradiol levels in the spent media were assayed. The expression of germ cell special genes and protein were examined by Real-time PCR and Western blot separately. RESULTS: Most of the isolated hAECs were polygon and of typical alabstone-like appearance, could express cytokeratin 19, the specific marker of epithelial cells, as well as octamer-binding protein 4 (Oct-4), a specific marker of embryonic stem cells (ESCs). After induced in medium supplemented with 5% human follicular fluid for 14 days, these stem cells grew as aggregates morphologically. Aliquots of supernatant of culture were collected and were assayed for oestradiol using chemiluminescence immunoassay (CLIA), although oestradiol was absent in the control group, the levels of the induction group were increased by day 8 and reached the maxima by day 10. Our study showed that hAECs treated with human follicular fluid were able to express germ cell genes, including growth differentiation factor 9 (GDF9) and deleted in azoospermia-like (DAZL) (P < 0.05), but without synaptonemal complex protein 3 (SCP3) (P > 0.05). The DAZL protein were also examined (P < 0.05). CONCLUSION: Germ cells could be induced in vitro from hAECs by human follicular fluid.

TP63 fusions drive multicomplex enhancer rewiring, lymphomagenesis, and EZH2 dependence.

Gene fusions involving tumor protein p63 gene (TP63) occur in multiple T and B cell lymphomas and portend a dismal prognosis for patients. The function and mechanisms of TP63 fusions remain unclear, and there is no target therapy for patients with lymphoma harboring TP63 fusions. Here, we show that TP63 fusions act as bona fide oncogenes and are essential for fusion-positive lymphomas. Transgenic mice expressing TBL1XR1::TP63, the most common TP63 fusion, develop diverse lymphomas that recapitulate multiple human T and B cell lymphomas. Here, we identify that TP63 fusions coordinate the recruitment of two epigenetic modifying complexes, the nuclear receptor corepressor (NCoR)-histone deacetylase 3 (HDAC3) by the N-terminal TP63 fusion partner and the lysine methyltransferase 2D (KMT2D) by the C-terminal TP63 component, which are both required for fusion-dependent survival. TBL1XR1::TP63 localization at enhancers drives a unique cell state that involves up-regulation of MYC and the polycomb repressor complex 2 (PRC2) components EED and EZH2. Inhibiting EZH2 with the therapeutic agent valemetostat is highly effective at treating transgenic lymphoma murine models, xenografts, and patient-derived xenografts harboring TP63 fusions. One patient with TP63-rearranged lymphoma showed a rapid response to valemetostat treatment. In summary, TP63 fusions link partner components that, together, coordinate multiple epigenetic complexes, resulting in therapeutic vulnerability to EZH2 inhibition.

An in situ molecular signature to predict early recurrence in hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND & AIMS: To develop an in situ molecular signature to predict postsurgical recurrence in hepatocellular carcinoma (HCC) patients. METHODS: Immunohistochemistry was performed using tissue microarrays containing both tumoral and peri-tumoral regions of the advancing tumor edge from 336 HCC patients (289 were positive for hepatitis B virus) who underwent curative resection. Forty-nine variables were analyzed in the training set (n=151) using support vector machine and stepwise algorithms to develop a classifier to predict recurrence within 1 year, which was mainly caused by invasion or metastasis from the primary tumors. The classifier was further validated in an independent cohort of 185 patients (71 internal and 114 external). RESULTS: The final signature was composed of eight IHC features: CD80(T), B7-DC(T), HLA-DR(P), FasL(P), Bcl-2(T), Ki-67(T), cyclin D1(T), and CK19(T). In the independent test set, this classifier reliably predicted recurrence within 1 year (sensitivity, 69.1%; specificity, 65.0%) with an odds ratio of 4.149 (95% CI, 2.189-7.864). Based on a multivariate logistic model, the in situ molecular signature provided significant predictive power independent of tumor number, tumor size, vascular invasion and BCLC classification (p=0.001). The highest potential clinical impact of the classifier was observed in early-stage (BCLC classification 0-A) patients (p<0.0001), and the classifier was also predictive of the time-to-recurrence and overall survival (both p<0.0001). CONCLUSIONS: This in situ molecular classifier could provide a novel approach to identify patients who are at greatest risk for postsurgical recurrence of HCC and may benefit from intensive clinical follow-up or chemopreventive strategies.

[Effects of omeprazole on the proliferation and apoptosis of hepatoma cell line HepG2].

OBJECTIVE: To investigate the effects of omeprazole (OME), a proton pump inhibitor, on the proliferation and apoptosis of human hepatoma cell line HepG2. METHODS: HepG2 cells were cultured to the logarithmic phase, and then treated with OME of different concentrations (10, 20, 40, 80, 160 mg/L) for 24 h or 48 h. Cell proliferation was evaluated by MTT assay, DNA synthesis was measured with 5-ethynyl-2'-deoxyuridine (Edu) fluorescent assay and the apoptosis of cells was measured by the Hoechst33342 assay. RESULTS: MTT assay showed that OME (40, 80 and 160 mg/L concentrations) could inhibit the proliferation of HepG2 cells for 24 h or 48 h treatment (P < 0.05) and 80 mg/L group has strongest effect. Compared with that of 24 h treatment, the same concentration of OME could inhibit HepG2 more significantly with 48 h treatment. After different concentrations of OME treatment for 24 h and then incubation with Edu for 2 h, compared with the control group, the proportion of Cells in S phase in 20, 40, 80, 160 mg/L groups decreased. Hoechst33342 staining demonstrated that treatment with OME (40, 80,160 mg/L) for 24 h could significantly promote the cell apoptosis. CONCLUSION: Omeprazole could inhibit human hepatoma cell line HepG2 cell proliferation and promote apoptosis.

[Expression of ACTN2, alpha-actin and TNNT2 in rat bone marrow-derived mesenchymal stem cells induced by low frequency pulsed electromagnetic fields].

OBJECTIVE: To study the expression of Actinin, alpha 2 (ACTN2), alpha-actin and Troponin T type 2 (TNNT2) in rat bone marrow-derived mesenchymal stem cells (rBMSCs) induced by low frequency pulsed electromagnetic fields (PEMFs). METHODS: The rBMSCs were isolated by adherence method and the third-generation of the rBMSCs were randomly divided into PEMFs groups, 5-Azacytidine groups (5-Aza), PEMFs +5-Aza group and control groups. PEMFs groups with complete medium were exposed to 50 Hz, 1 mT PEMFs for 30 min every day, lasting for 10 d, 15 d and 20 d, respectively. 5-Aza groups were induced by 10 micromol/L 5-Aza for 1 day, then the medium was changed to complete medium without 5-Aza. PEMFs +5-Aza group were firstly induced by 10 micromol/L 5-Aza for 1 day, and then were exposed to PEMFs according to the way of PEMFs group. And control groups were only cultured with complete medium, rBMSCs' growth status and morphological features were observed by inverted phase microscope. The mRNA expressions of ACTN2 and TNNT2 were determined by real-time fluorescent quantitation PCR. And the protein expression of the ACTN2, alpha-actin and TNNT2 were detected with Western blot method. RESULTS: The result showed that the mRNA expression level of the ACTN2 and TNNT2 and the protein expression level of the ACTN2, alpha-actin and TNNT2 were significantly higher in the PEMFs group, 5-Aza group and PEMFs+ 5-Aza group when compared with the control group. CONCLUSION: PEMFs may induce rBMSCs differentiation into cardiomyocyte-like cells in vitro.

Endothelium-coated tumor clusters are associated with poor prognosis and micrometastasis of hepatocellular carcinoma after resection.

BACKGROUND: Distinct morphologic features of microvascular endothelium exist in tumor tissues. The objective of this study was to investigate the prognostic value of endothelium-coated tumor clusters (ECTCs) in hepatocellular carcinoma (HCC). METHODS: ECTCs were evaluated by immunohistochemical staining for cluster of differentiation 34 (CD34) (a cell surface glycoprotein which is expressed specifically on tumor microvascular endothelium in HCC) in 239 specimens from patients with primary HCC. Overall survival (OS) and time to recurrence (TTR) were determined using Kaplan-Meier analysis and a Cox proportional hazards regression model. Levels of terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining, and Ki-67 expression, and E-cadherin expression were assessed to determine tumor apoptosis, proliferation, and invasiveness, respectively. RESULTS: The presence of ECTCs was associated with a poor prognosis in all patients and in patient subgroups stratified by tumor size, TNM classification, and Barcelona Clinic Liver Cancer stage and tumor invasiveness. In a multivariate Cox proportional hazards analysis, the presence of ECTCs emerged as an independent prognostic indicator of both poor OS (P = .001; hazard ratio, 1.949) and shorter TTR (P < .001; hazard ratio, 2.085). Furthermore, the presence of ECTCs was associated with micrometastatic endothelium-coated emboli (P < .001; chi-square test) and early relapse after resection (P < .001; chi-square test). In addition, patients who had endothelium-coated emboli, in which tumor cells displayed high proliferation and low apoptosis, had poor OS and shorter TTR. CONCLUSIONS: The current results suggested that the presence of ECTCs was an efficient, simple, and convenient predictor of a poor prognosis in patients with HCC that potentially may serve as a novel target for the prevention and treatment of HCC metastasis.

[Effects of verapamil on the proliferation and migration of cervical cancer cells].

OBJECTIVE: To investigate the effects of Verapamil, a L-type calcium channel blocker, on the proliferation and migration of human cervical cancer cells. METHODS: Human cervical cancer cell lines (HeLa cells) were incubated with serum-free media for 24 hours. These cells were then treated with Verapamil for different time points. Cell proliferation was evaluated by MTT assay and EdU fluorescent assay. The migration of cells was measured by the wound healing assay. Results MTT assay demonstrated that, Verapamil (1, 5, 10 and 20 microg/mL concentrations) could inhibit the proliferation of HeLa cells after 48 or 72 hours treatment. The inhibition rate for 48 h-treatment was 20.87% +/- 1.71%, 23.55% +/- 4.46%, 28.65% +/- 1.32%, 27.37% +/- 3.05% respectively (P < 0.05). After 5, 10 and 20 microg/mL concentrations of verapamil treatment for 24h, the proportion of HeLa cells in the stage of DNA synthesis was 15.7% +/- 1.2%, 11.7% +/- 0.3%, 2.8% +/- 0.5% as evaluated with EdU fluorescent assay, which was significantly lower than that of control (27.34% +/- 2.1%, P < 0.05). With the increase of drug concentrations, Verapamil could significantly suppressed the DNA synthesis in HeLa cell. Cell migration assay revealed that, treatment with Verapamil (5, 10 and 20 microg/mL) for 24 hours significantly inhibited cells migration distance (P < 0.05). CONCLUSION: Calcium channel blocker Verapamil could inhibit proliferation and migration of the cervical cancer HeLa cells.

[Effects of verapamil on the proliferation, DNA synthesis and migration of human hepatoma cell line HepG2].

OBJECTIVE: To study the effects of verapamil (VR) on proliferation, DNA synthesis and migration of human hepatoma cell line HepG2. METHODS: HepG2 cells were treated with different concentration of VR (0,1, 5, 10, 15, 20 microg/mL) for different time point. Cell proliferation was evaluated by MTT assay and DNA synthesis was determined by 5-ethynyl-2'-deoxyuridine (Edu) fluorescent assay. The migration of cells was measured by Scratch wound migration assay. RESULTS: MTT assay demonstrated that the different concentration of VR (1, 5, 10, 15, 20 microg/mL) could inhibit the proliferation of HepG2 cells after 24 h and 48 h treatment (P < 0.05) except 1, 5 microg/mL group processing for 24 h (P > 0.05). Edu fluorescent assay showed that, the proportion of HepG2 cells in the stage of DNA synthesis was (51.5 +/- 3.78)%, (52.4 +/- 3.26)%, (53.1 +/- 1.94)%, (39.6 +/- 4.25)%, (40.2 +/- 2.67)%, (42.6 +/- 3.13)% respectively for the treatment of 0, 1, 5, 10, 15, 20 microg/mL concentration of VR for 24 h. DNA synthesis in the cells treated with VR (10, 15 and 20 microg/mL concentration) was lower than that in the cells treated with 0, 1, 5 microg/mL concentrations of VR (P < 0.05). Scratch wound migration assay indicated that treatment with VR (10, 15 and 20 microg/mL) for 24 h significantly inhibited cells migration distance (P < 0.05). CONCLUSION: Calcium channel blocker VR could inhibit the cell proliferation and migration of human hepatoma cell line HepG2.

Biological Select Agents and Toxins Management in Taiwan: From Past to Present.

Introduction: Before 2016, there were no specific regulations or guidelines for the management of biological select agents and toxins (BSATs) in Taiwan. The Taiwan Centers for Disease Control responded to the global health security agenda in 2016 and made use of the Joint External Evaluation tool: International Health Regulations to evaluate Taiwan's epidemic prevention system capacities, including BSAT management. For technical areas that did not meet the highest requirements, the regulations and guidelines are now in place to strengthen the management of BSATs. Methods: In 2017, a survey on the BSAT entities management status in Taiwan was conducted to understand the gap between BSAT practice and international policies, and to improve BSAT management based on the findings. Results and Discussion: After 3 years of promotion, relevant management regulations and supervision mechanisms have been established. In 2021, the evaluation will be conducted again and it is expected that Taiwan's BSAT management capacity will reach the level of international biosafety and biosecurity.

Reprogramming immunosuppressive myeloid cells by activated T cells promotes the response to anti-PD-1 therapy in colorectal cancer.

Overcoming local immunosuppression is critical for immunotherapy to produce robust anti-tumor responses. Myeloid-derived suppressor cells (MDSCs) are key regulators of immunosuppressive networks and promote tumor progression. However, it remains unclear whether and how tumor-infiltrating MDSCs are shaped in response to anti-PD-1 treatment and what their impact on therapeutic efficacy is in colorectal cancer (CRC). In this study, the levels of infiltrating MDSCs were significantly higher in the non-responding organoids and were selectively reduced in the responding group, with MDSCs showing increased apoptosis and attenuated functional activity after anti-PD-1 treatment. A negative correlation between T-cell activation and MDSC function was also observed in fresh human CRC tissues. Mechanistic studies revealed that autocrine IFN-α/ß upregulated TRAIL expression on activated T cells to elicit MDSC apoptosis via the TRAIL-DR5 interaction and acted synergistically with TNF-α to inhibit MDSC function of suppressing the T-cell response through the JNK-NMDAR-ARG-1 pathway. Moreover, blockade of IFN-α/ß and TNF-α abolished the therapeutic efficacy of anti-PD-1 treatment by preserving the frequency and suppressive activity of infiltrating MDSCs in a CRC mouse model. This result suggested that reprogramming MDSCs by IFN-α/ß and TNF-α from activated T cells was necessary for successful anti-PD-1 treatment and might serve as a novel strategy to improve the response and efficacy of anticancer therapy.

Retinoic Acid Synthesis Deficiency Fosters the Generation of Polymorphonuclear Myeloid-Derived Suppressor Cells in Colorectal Cancer.

Metabolism is reprogrammed in cancer to fulfill the demands of malignant cells for cancer initiation and progression. Apart from its effects within cancer cells, little is known about whether and how reprogramed metabolism regulates the surrounding tumor microenvironment (TME). Myeloid-derived suppressor cells (MDSC) are key regulators of the TME and greatly affect tumor progression and therapeutic responses. In this study, our results revealed that retinol metabolism-related genes and enzymes were significantly downregulated in human colorectal cancer compared with adjacent colonic tissues, and tumors exhibited a defect in retinoic acid (RA) synthesis. Reduced ADH1-mediated retinol metabolism was associated with attenuated RA signaling and accumulated MDSCs in colorectal cancer tumors. Using an in vitro model, generating MDSCs from CD34+ myeloid precursors, we found that exogenous RA could abrogate the generation of polymorphonuclear MDSCs (PMN-MDSC) with negligible impact on myeloid differentiation. Mechanistically, RA could restrain the glycolytic capacity of myeloid cells, which in turn activated the AMP-activated protein kinase (AMPK) pathway, further impairing the suppressive capacity of myeloid cells. Supplementation with RA could significantly delay tumor growth, with reduced arginase-1-expressing myeloid cells and increased CD8+ and granzyme B+ T cells in both colitis-associated and implanted MC38 mouse colorectal cancer models. Our results indicated that the defect in ADH1-mediated RA synthesis could provide a possible mechanism that fosters the generation of PMN-MDSCs in colorectal cancer and that restoring RA signaling in the TME could serve as a promising therapeutic strategy to abrogate the generation of PMN-MDSCs.

Glutamine Deprivation Promotes the Generation and Mobilization of MDSCs by Enhancing Expression of G-CSF and GM-CSF.

Solid tumors are often challenged by hypoxic and nutrient-deprived tumor microenvironments (TME) as tumors progress, due to limited perfusion and rapid nutrient consumption. While cancer cells can demonstrate the ability to survive in nutrient-deprived conditions through multiple intrinsic alterations, it is poorly understood how nutrient-deprived cancer cells co-opt the TME to promote cancer cell survival and tumor progression. In the present study, we found that glutamine deprivation markedly potentiated the expression of G-CSF and GM-CSF in mouse mammary cancer cells. The IRE1α-JNK pathway, which is activated by glutamine starvation, was found to be important for the upregulation of these cytokines. G-CSF and GM-CSF are well-known facilitators of myelopoiesis and mobilization of hematopoietic progenitor cells (HPC). Consistently, as tumors progressed, we found that several myeloid HPC compartments were gradually decreased in the bone marrow but were significantly increased in the spleen. Mechanistically, the HPC-maintaining capacity of the bone marrow was significantly impaired in tumor-bearing mice, with lower expression of HPC maintaining genes (i.e., CXCL12, SCF, ANGPT1, and VCAM1), and reduced levels of mesenchymal stem cells and CXCL12-producing cells. Furthermore, the mobilized HPCs that displayed the capacity for myelopoiesis were also found to accumulate in tumor tissue. Tumor-infiltrating HPCs were highly proliferative and served as important sources of immunosuppressive myeloid-derived suppressor cells (MDSCs) in the TME. Our work has identified an important role for glutamine starvation in regulating the expression of G-CSF and GM-CSF, and in facilitating the generation of immunosuppressive MDSCs in breast cancer.

[Effects of placental growth factor on the secretion of proinflammatory cytochemokines in vascular endothelial cells].

OBJECTIVE: Placental growth factor (PIGF) mediates angiogenesis and inflammation, and is up-regulated in early and advanced atherosclerotic lesions. But its effects on the function of vascular endothelial cells is still largely unknown. This study was designed to test the effects of PIGF on the secretion of proinflammatory cytochemokines in vascular endothelial cells. METHODS: Cultured human umibilical vein endotheial cells (HUVECs) of passages 3-5 were used. Growth-arrested HUVECs were treated with 5, 10, 25 or 50 ng/mL recombinant human PIGF (rhPLGF) for 4, 8, 24 or 48 hours. Monocyte chemotactic protein-1 (MCP-1), soluble vascular cell adhesion molecule-1 (sVCAM-1) and P-selectin secreted from HUVECs were measured by ELISA. RESULTS: Treated with rhPIGF at 10-50 ng/mL for 48 h, the production of MCP-1 from the HUVECs increased significantly. Treatment with rhPIGF at 5-50 ng/mL for 4 h, the release of P-selectin from the HUVECs increased significantly. But rhPIGF (5-50 ng/mL) had no effect on sVCAM secretion. CONCLUSION: Exogenous PIGF could modulate the secretion function of vascular. endothelial cells, stimulating MCP-1 and P-selectin release in HUVECs.

[Study on the expression of activin-A in myocardial ischemia postconditioning].

OBJECTIVE: To analyze the relationship between the hydroxy free radical (* OH) and the activity of lactate dehydrogenase (LDH) and to study the potential role of activin-A in myocardial ischemia postconditioning for the protection of myocardium. METHODS: Forty healthy adult Sprague-Dawley rats (gender unconcerned) were randomly divided into four different groups (n = 10 in each group): sham-operation, ischemia (I), ischemia/reperfusion (I/R) and ischemia postconditioning (postcon). In anesthetized open-chest rats, the left anterior descending artery (LAD) was ligated. Sham operation: the surgical procedure was identical to other groups, but the LAD was ligated for only 30 min, and not occluded. Ischemia: the LAD was occluded for 30 min; Ischemia/reperfusion: the LAD was occluded for 30 min and reperfused for 1 h; Postconditioning: after LAD occlusion for 30 min, at the start of reperfusion, three cycles of 30 s reperfusion and 30 s LAD re-occlusion preceded the 1h of reperfusion. The expression of activin-A in myocardium was detected by ELISA in all groups. The ability of suppression on the hydroxy free radical (* OH) and the activity of lactate dehydrogenase (LDH) in myocardium taken from each group were measured by spectrophotometer. RESULTS: Compared with the sham-operated group, the expression of activin-A was significantly elevated in ischemia, ischemia/reperfusion and postconditiong groups, with the highest in the ischemia/reperfusion group. And postconditioning significantly decreased the expression of activin-A in the ischemia/ reperfusion injured myocardium [(343.05 +/- 25.8) microg/mg protein vs (470.6 +/- 33.1) microg/mg protein, P < 0.05]. The expression of activin-A was negatively correlated with the ability of suppression on the hydroxy free radical and LDH separately. CONCLUSION: The result indicated that activin-A was involved in myocardial ischemia/reperfusion injury, and down-regulation of activin-A might be one of the cardioprotective mechanisms of postconditioning.

Immunosuppressive Immature Myeloid Cell Generation Is Controlled by Glutamine Metabolism in Human Cancer.

Tumor-associated myeloid cells are one of the prominent components of solid tumors, serving as major immune regulators for the tumor microenvironment (TME) and an obstacle for immune-checkpoint blocking (ICB) therapy. However, it remains unclear how metabolic processes regulate the generation of suppressive myeloid cells in the TME. Here, we found that hematopoietic precursor cells are enriched in the tissues of several types of human cancer and can differentiate into immature myeloid cells (IMC). Tumor-infiltrating IMCs are highly immunosuppressive, glycolytic, and proliferative, as indicated by high levels of M-CSFR, Glut1, and Ki67. To elucidate the role of metabolism in regulating the generation of IMCs, we induced suppressive IMCs from hematopoietic precursor cells with GM-CSF and G-CSF in vitro We found that the generation of suppressive IMCs was accompanied by increased glycolysis, but not affected by glucose deprivation due to alternative catabolism. Generation of IMCs relied on glutaminolysis, regardless of glucose availability. Glutamine metabolism not only supported the expansion of IMCs with glutamine-derived α-ketoglutarate but also regulated the suppressive capacity through the glutamate-NMDA receptor axis. Moreover, inhibition of glutaminase GLS1 enhanced the therapeutic efficacy of anti-PD-L1 treatment, with reduced arginase 1+ myeloid cells, increased CD8+, IFNγ+ and granzyme B+ T cells, and delayed tumor growth in an ICB-resistant mouse model. Our work identified a novel regulatory mechanism of glutamine metabolism in controlling the generation of suppressive IMCs in the TME.