Early evaluation of circulating tumor DNA as marker of therapeutic efficacy and prognosis in breast cancer patients during primary systemic therapy.

BACKGROUND: We assessed the potential role of serial circulating tumor DNA (ctDNA) as a biomarker to monitor treatment response to primary systemic therapy (PST) in breast cancer and evaluated the predictive value of ctDNA to further identify patients with residual disease. METHODS: We prospectively enrolled 208 plasma samples collected at three time points (before PST, after 2 cycles of treatment, before surgery) of 72 patients with stage Ⅱ-III breast cancer. Somatic mutations in plasma samples were identified using a customized 128-gene capture panel with next-generation sequencing. The correlation between early change in ctDNA levels and treatment response or long-term clinical outcomes was assessed. RESULTS: 37 of 72 (51.4%) patients harbored detectable ctDNA alterations at baseline. Patients with complete response showed a larger decrease in ctDNA levels during PST. The median relative change of variant allele fraction (VAF) was -97.4%, -46.7%, and +21.1% for patients who subsequently had a complete response (n = 11), partial response (n = 11), and no response (n = 15) (p = 0.0012), respectively. In addition, the relative change of VAF between the pretreatment and first on-treatment blood draw exhibited the optimal predictive value to tumor response after PST (area under the curve, AUC = 0.7448, p = 0.02). More importantly, early change of ctDNA levels during treatment have significant prognostic value for patients with BC, there was a significant correlation between early decrease of VAF and longer recurrence-free survival compared to those with an VAF increase (HR = 12.54; 95% CI, 2.084 to 75.42, p = 0.0063). CONCLUSION: Early changes of ctDNA are strongly correlated with therapeutic efficacy to PST and clinical outcomes in BC patients. The integration of preoperative ctDNA evaluation could help improving the perioperative management for BC patients receiving PST.

PROX1 promotes breast cancer invasion and metastasis through WNT/ß-catenin pathway via interacting with hnRNPK.

Background: The progressive, multifactorial and multistep dynamic process of metastasis is the primary cause of breast cancer (BC) lethality. PROX1 (Prospero-related homeobox 1), as a type of transcription factor that plays a key role in the formation of lymphatic vessels in animal embryonic development, has been proven to promote or suppress cancer in a variety of malignant tumors. However, molecular mechanisms behind PROX1 induced breast cancer metastases remain elusive. Methods: Changes of PROX1 expression and clinical significance of PROX1 in BC were evaluated by BC tissue, as well as public database. The functional role of PROX1 in metastases BC was analyzed by transwell assay in vitro, and by lung metastases model of nude mice in vivo via lentivirus mediated knockdown assays. Mechanism studies were performed by public database screening, western blot and PCR assay, immunoprecipitation, immunofluorescence staining and luciferase promoter assays. Results: In this study, we found that PROX1 was upregulated in breast cancer tissues; increased PROX1 expression in breast cancer was associated with tumor size, lymph node metastasis, ER and PR status. Meanwhile, PROX1 can promote breast cancer invasion and metastasis in vitro and in vivo. Furthermore, PROX1 can interact with hnRNPK to activate WNT/ß-catenin signaling in breast cancer cells. Moreover, the interaction of PROX1 and hnRNPK inhibits the ubiquitination of hnRNPK, and subsequently activates WNT pathway to promote the invasion and metastasis of breast cancer. Conclusions: In conclusion, our findings indicated PROX1 contributes to breast cancer EMT and metastasis and serves as a candidate diagnostic biomarker and promising therapeutic target for breast cancer.

Application Value of the Workshop Practice Teaching Method Based on Target-Oriented Study Modules on the Internet in Orthopedic Rehabilitation.

OBJECTIVE: To explore the application value of the workshop practice teaching method based on target-oriented study modules on the internet in orthopedic rehabilitation. METHODS: Thirty interns (2019-2020) in the rehabilitation department of our hospital were selected as the control group, another thirty interns (2021) in the rehabilitation department of our hospital were selected as the experimental group, and their materials were retrospectively reviewed. Both groups were given the three-month practice teaching. Besides, the conventional practice teaching method was applied to the control group, and the workshop practice teaching method based on target-oriented study modules on the internet was applied to the experimental group. After the practice teaching, the united examination paper and assessment table of the rehabilitation operation process were used to evaluate the interns' scores of theoretical knowledge about orthopedic rehabilitation, scores of practical skills, and comprehensive scores of clinical practice in the two groups. The evaluation team consisting of 5 guiding experts in the scientific research office assessed the teaching quality of the two methods. RESULTS: Compared with the control group, the experimental group had a notably higher score of theoretical knowledge about orthopedic rehabilitation, higher score of practical skills, and higher comprehensive score of clinical practice (92.47 ± 4.81 vs. 86.43 ± 5.12, 91.30 ± 5.68 vs. 81.53 ± 7.21, and 91.88 ± 2.45 vs. 83.98 ± 4.42, P < 0.001). According to the evaluation team, the teaching quality of the experimental group was observably higher than that in the control group (P < 0.05), and there was no remarkable difference in the scores of teachers' performance between the two groups (P > 0.05). CONCLUSION: The workshop practice teaching method based on target-oriented study modules on the internet, as a high-quality "Internet+" practice teaching mode, can improve the orthopedic interns' scores of theoretical knowledge and practical operation ability and enhance their comprehensive qualities of orthopedic rehabilitation in all respects.

Metastatic Pattern Discriminates Survival Benefit of Type of Surgery in Patients With De Novo Stage IV Breast Cancer Based on SEER Database.

Background: The role of surgery and surgery type in de novo stage IV breast cancer (BC) is unclear. Methods: We carried out a retrospective cohort study that included the data of 4,108 individuals with de novo stage IV BC abstracted from SEER (Surveillance, Epidemiology, and End Results) data resource from 2010 to 2015. The patients were stratified into the non-surgery group, breast-conserving (BCS) surgery group, and mastectomy group. Inverse probability propensity score weighting (IPTW) was then used to balance clinicopathologic factors. Overall survival (OS), as well as the breast cancer-specific survival (BCSS), was assessed in the three groups using Kaplan-Meier analysis and COX model. Subgroups were stratified by metastatic sites for analysis. Results: Of the 4,108 patients, 48.5% received surgery and were stratified into the BCS group (574 cases) and mastectomy group (1,419 cases). After IPTW balance demographic and clinicopathologic factors, BCS and mastectomy groups had better OS (BCS group: HR, 0.61; 95% CI: 0.49-0.75; mastectomy group: HR, 0.7; 95% CI: 0.63-0.79) and BCSS (BCS group: HR, 0.6; 95% CI, 0.47-0.75; mastectomy group: HR, 0.71; 95% CI, 0.63-0.81) than the non-therapy group. Subgroup analyses revealed that BCS, rather than mastectomy, was linked to better OS (HR, 0.66; 95% CI: 0.48-0.91) and BCSS (HR, 0.63; 95% CI: 0.45-0.89) for patients with bone-only metastasis. For patients with viscera metastasis or bone+viscera metastases, BCS achieved similar OS (viscera metastasis: HR, 1.05; 95% CI: 0.74-1.48; bone+viscera metastases: HR, 1.01; 95% CI: 0.64-1.61) and BCSS (viscera metastasis: HR, 0.94; 95% CI: 0.64-1.38; bone+viscera metastases: HR, 1.06; 95% CI: 0.66-1.73) in contrast with mastectomy. Conclusions: Local surgery for patients with distant metastasis (DS) exhibited a remarkable survival advantage in contrast with non-operative management. BCS may have more survival benefits for patients with de novo stage IV BC with bone-only metastasis than other metastatic sites. Decisions on de novo stage IV BC primary surgery should be tailored to the metastatic pattern.

Loss of HOXB3 correlates with the development of hormone receptor negative breast cancer.

BACKGROUND: The homeobox gene family, encoding a specific nuclear protein, is essential for embryonic development, differentiation, and homeostasis. The role of the HOXB3 protein varies in different tumors. This study aims to explore the role of the HOXB3 gene in breast cancer. METHOD: Differentially expressed genes were screened by analyzing metastatic breast cancer gene chip data from TCGA and GEO databases. The function of the selected HOXB3 gene was also analyzed in different databases and through molecular biology methods, such as qRT-PCR, western blot and IF to verify bioinformatics findings. RESULTS: Both bioinformatics analyses and western blot showed that HOXB3 was lost in breast cancer compared to normal breast tissue. Survival analysis also showed that lower expression of HOXB3 was associated with poor prognosis. Bioinformatics analyses further showed that HOXB3 was positively correlated with hormone receptors. Metascape for GO analysis of GEO data provided possible mechanisms that HOXB3 could positively regulate cell adhesion, inhibit cell proliferation and activate immune response in breast cancer; moreover, GSEA included several cancer-associated pathways. CONCLUSION: In summary, HOXB3 expression was decreased in breast cancer, and it was associated with poor prognosis. It might become a new biomarker to predict prognosis of breast cancer.

Growth pattern can be used as a new characteristic to predict malignancy in breast cancer.

BACKGROUND: To date, anatomic tumor length is a key criterion for cancer staging and can be used to evaluate the effectiveness of therapies. This article describes growth pattern that can be used as a new characteristic to represent disease burden and tumor features and predict lymphatic metastasis. METHODS: Patients with breast cancer were included in this 10-year (1999-2008) hospital-based multicenter retrospective study. The pathologic length/height ratio was used to illustrate the correlation between tumor features, behaviors and treatments in breast malignancies. The most appropriate ratio was chosen based on the comprehensive evaluation of p value and changing trend of each characteristic. RESULTS: The sample consisted of 4211 women diagnosed with breast cancer. Among them, 2037 patients with complete pathologic length, width and height information were included in the final analysis. There were 2.34 ± 4.77 metastatic lymph nodes for spheroid tumors and 3.21 ± 5.82 for ellipsoid tumors when the cutoff point was 2. In addition, the proportion of ellipsoidal tumors gradually increased from 54.36 to 56.67% in the upper outer quadrant (UOQ) and from 6.7 to 9.03% in the central region with an increase in the cutoff point. The proportion of ER + PR + ellipsoid tumors significantly decreased from 50.1 to 45.35% and that of ER-PR ellipsoid tumors significantly increased from 32.73 to 36.24% with an increase in the cutoff point. Additionally, the best length/weight ratio to distinguish spheroid and ellipsoid tumors was 2. CONCLUSION: This study described for the first time how growth pattern is correlated with tumor malignancy and how it influences the selection of therapeutic strategies for patients.

SDF-1/CXCR4 axis promotes the growth and sphere formation of hypoxic breast cancer SP cells by c-Jun/ABCG2 pathway.

ATP-binding cassette sub-family G member 2 (ABCG2) confers to the major phenotypes of side population (SP) cells, the cancer stem-like cells. In this study, the SP cells displayed a distinctly higher ABCG2 expression level, sphere formation efficiency (SFE) and growth rate even under hypoxia condition. CXCR4 overexpression by pcDNA-CXCR4 transfection robustly increased ABCG2 expression, and promoted SFE and growth of hypoxic SP cells, while CXCR4 inhibitor AMD3100 could suppress the promotion. Additionally, we found that CXCR4 promoted the expression of c-Jun, a major gene in the oncogenic JNK/c-Jun pathway. Our data on electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays both showed that c-Jun directly bound with the ABCG2 promoter sequence. Moreover, overexpression of JNK/c-Jun promoted ABCG2 expression, SFE, and growth of hypoxic SP cells and the promotion could be rescued by c-Jun inhibitor SP600125. In conclusion, CXCR4 increases the growth and SFE of breast cancer SP cells under hypoxia through c-Jun-mediated transcriptional activation of ABCG2.

Ginsenoside Rh2 Inhibited Proliferation by Inducing ROS Mediated ER Stress Dependent Apoptosis in Lung Cancer Cells.

Ginsenoside Rh2 (G-Rh2), a component extracted from roots of ginseng, exhibited anti-cancer pharmacological activities by inhibiting proliferation and inducing apoptosis in lung cancer cells. However, the mechanisms of G-Rh2 suppressing lung cancer development remained elusive. This study tried to investigate the possible mechanism involved in anti-proliferative effect of G-Rh2 in lung cancer cells. As results, G-Rh2 inhibited the proliferation of H1299 cells in a dose-dependent manner by inducing cell apoptosis. Activating transcription factor 4 (ATF4), CCAAT/enhancer-binding protein homologous protein (CHOP), and caspase-4 were involved in G-Rh2-induced apoptosis of H1299 cells. It was also found that G-Rh2 could up-regulate expressions of ATF4, CHOP and caspase-4 in H1299 cells in a dose-dependent manner. In addition, NAC (N-acetylcysteine, a reactive oxygen species (ROS) scavenger) treatment dramatically decreased ROS generation in H1299 cells; both of NAC and 4-PBA (4-phenylbutyrate, a specific endoplasmic reticulum (ER) stress inhibitor) administration impaired apoptosis and expression levels of ATF4, CHOP and caspase-4 in G-Rh2 incubated H1299 cells. In vivo assays extended the significance of these results, showing that G-Rh2 inhibited lung cancer growth and the inhibition effects of G-Rh2 in tumor growth were significantly reduced by inhibition of ER stress. In conclusion, G-Rh2 inhibited proliferation of H1299 cells by inducing ROS mediated ER stress dependent cell apoptosis.

The Function of SDF-1-CXCR4 Axis in SP Cells-Mediated Protective Role for Renal Ischemia/Reperfusion Injury by SHH/GLI1-ABCG2 Pathway.

Renal ischemia-reperfusion (I/R) injury ranks as the primary cause of acute renal injury with severe morbidity and mortality. Side population (SP) cells have recently drawn increasing attention due to their critical role in injury repair and regeneration. Unfortunately, the underlying mechanism involved in renal I/R remains poorly elucidated. Here, pronounced increases of stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) were substantiated in I/R kidneys from C57BL/6 mice subjected to clamp the bilateral renal pedicles to mimic renal ischemia. Similar up-regulation of them was also determined in SP cells upon simulated ischemia/reperfusion (SI/R). In contrast to non-SP cells, SP cells exhibited higher viability, apoptosis resistance, chemotaxis, and paracrine actions following SI/R treatment, and these were further enhanced after SDF-1 stimulation. Interestingly, blocking CXCR4 signaling with AMD3100 notably ameliorated the above effects. Mechanism analysis corroborated that SDF-1/CXCR4 further induced the expression of ATP-binding cassette transporter ABCG2, an essential element for SP-mediated kidney regeneration after renal I/R injury. Moreover, AMD3100 pretreatment strikingly attenuated ABCG2 elevation in SP cells. Additionally, sonic hedgehog (SHH)-Gli 1 signaling was involved in SDF-1/CXCR4-mediated ABCG2 expression. When SP cells pretreated with AMD3100 were intravenously injected into I/R mice, SP cell-mediated decreases in blood urea nitrogen, serum creatinine, and histological score of kidney were noticeably attenuated, indicating that blocking CXCR4 pathway mitigated the therapeutic function of SP cells in renal I/R injury. Together, this research suggests that SDF-1/CXCR4 axis might act, via Shh-Gli1-ABCG2 signaling, as a positive regulator of SP cell-based therapies for renal I/R by Shh-Gli 1-ABCG2 signaling.

PRSS8 is Downregulated and Suppresses Tumour Growth and Metastases in Hepatocellular Carcinoma.

BACKGROUND: Protease serine 8 (PRSS8), a trypsin-like serine peptidase, has been shown to function as a tumour suppressor in various malignancies. The present study aimed to investigate the expression pattern, prognostic value and the biological role of PRSS8 in human hepatocellular carcinoma (HCC). METHODS: PRSS8 expression in 106 HCC surgical specimens was examined by Real-time polymerase chain reaction (PCR) and immunohistochemistry, and its clinical significance was analysed. The role of PRSS8 in cell proliferation, apoptosis and invasion were examined in vitro and in vivo. RESULTS: PRSS8 mRNA and protein expression were decreased in most HCC tumours from that in matched adjacent non-tumour tissues. Low intratumoral PRSS8 expression was significantly correlated with poor overall survival (OS) in patients with HCC (P = 0.001). PRSS8 expression was an independent prognostic factor for OS (hazard ratio [HR] = 1.704, P = 0.009). Furthermore, restoring PRSS8 expression in high metastatic HCCLM3 cells significantly inhibited cell proliferation and invasion. In contrast, silencing PRSS8 expression in non-metastatic HepG2 cells significantly enhanced cell growth and invasion. Moreover, our in vivo data revealed that attenuated PRSS8 expression in HepG2 cells greatly promoted tumour growth, while overexpression of PRSS8 remarkably inhibited tumour growth in an HCCLM3 xenograft model. Enhanced cell growth and invasion ability mediated by the loss of PRSS8 expression was associated with downregulation of PTEN, Bax and E-cadherin and an upregulation in Bcl-2, MMP9 and N-cadherin. CONCLUSIONS: Our data demonstrate that PRSS8 may serve as a tumour suppressor in HCC progression, and represent a valuable prognostic marker and potential therapeutic target for HCC.

WITHDRAWN: CXCR4 promotes growth and sphere formation of hypoxic breast cancer side 7 population cells via activation of c-Jun/ABCG2 pathway.

Ahead of Print article withdrawn by publisher.

Gypenoside Attenuates ß Amyloid-Induced Inflammation in N9 Microglial Cells via SOCS1 Signaling.

Reducing ß amyloid- (Aß-) induced microglial activation is believed to be effective in treating Alzheimer's disease (AD). Microglia can be activated into classic activated state (M1 state) or alternative activated state (M2 state), and the former is harmful; in contrast, the latter is beneficial. Gypenoside (GP) is the major bioactive constituent of Gynostemma pentaphyllum, a traditional Chinese herb medicine. In this study, we hypothesized that GP attenuates Aß-induced microglial activation by ameliorating microglial M1/M2 states, and the process may be mediated by suppressor of cell signaling protein 1 (SOCS1). In this study, we found that Aß exposure increased the levels of microglial M1 markers, including iNOS expression, tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and IL-6 releases, and coadministration of GP reversed the increase of M1 markers and enhanced the levels of M2 markers, including arginase-1 (Arg-1) expression, IL-10, brain-derived neurotrophic factor (BDNF), and glial cell-derived neurotrophic factor (GDNF) releases in the Aß-treated microglial cells. SOCS1-siRNA, however, significantly abolished the GP-induced effects on the levels of microglial M1 and M2 markers. These findings indicated that GP attenuates Aß-induced microglial activation by ameliorating M1/M2 states, and the process may be mediated by SOCS1.

Interleukin-37 suppresses tumor growth through inhibition of angiogenesis in non-small cell lung cancer.

BACKGROUND: Interleukin-37 (IL-37), a newly identified member of the IL-1 family, has been known to play an immunosuppressive role in a variety of inflammatory disorders, but whether it participates in the regulation of pathogenesis of non-small cell lung cancer (NSCLC) has not been investigated. METHODS: Real-time polymerase chain reaction (PCR), western blotting, and immunohistochemical staining were employed to detect IL-37 expression in NSCLC tissues and corresponding adjacent tissues. The correlations between IL-37 expression and clinicopathological characteristics, prognosis were analyzed. Stable clone with overexpression of IL-37 was generated in H1299 cell lines. Cell growth, cell cycle and cell apoptosis assays were carried out for detecting proliferation and apoptosis of H1299 cells. The effects of IL-37 on NSCLC progression in vivo was performed in a xenografted lung tumor model in nude mice. The concentrations of IL-37 and VEGF in the s growth medium supernatants were quantified by ELISA. The antiangiogenic effect of IL-37 on HUVEC was measured by tube formation assay. RESULTS: IL-37 mRNA and protein expressions were significantly decreased in NSCLC tissues, and decreased intratumoral IL-37 expression was significantly associated with tumor state, TNM stage and poor prognosis in NSCLC patients. In addition, intratumoral IL-37 expression was an independent prognostic factors for Overall survival (hazard ratio = 2.047; P = 0.011). Overexpression of IL-37 exerted no direct effect on cell proliferation and apoptosis of H1299 lung cancer cells in vitro, but significantly inhibited tumor growth in a H1299 xenograft model in vivo. Furthermore, there was no significant change in immune cell infiltration in IL-37 over-expressing tumors; instead, we found decreased microvessel density (MVD) and VEGF levels in IL-37-expressing tumors. Additional studies showed IL-37 could directly inhibit HUVEC cells growth and capillary structure formation. Finally, we found that decreased IL-37 expression was associated with high MVD in NSCLC patients. CONCLUSIONS: Our findings demonstrate a protective role for IL-37 in lung cancer development, possibly through inhibiting tumor angiogenesis. IL-37 could serve as a promising therapeutic target for NSCLC.

Enhanced SLC34A2 in breast cancer stem cell-like cells induces chemotherapeutic resistance to doxorubicin via SLC34A2-Bmi1-ABCC5 signaling.

Even though early detection methods and treatment options are greatly improved, chemoresistance is still a tremendous challenge for breast cancer therapy. Breast cancer stem cells (BCSCs) represent a subpopulation that is central to chemoresistance. We aim to investigate the relationship between SLC34A2 and chemoresistance in BCSCs and identify the underlying mechanisms by which SLC34A2 regulates chemoresistance in BCSCs. Fluorescence Activated Cell Sorting (FACS) analysis showed the presence of a variable fraction of CD44(+)CD24(-) cells in 25 out of 25 breast cancer samples. We cultured primary breast cancer sample cells and breast cancer cell line cells to induce sphere formation in serum-free medium. Following sorting of CD44(+)CD24(-) cells from spheres, we showed that CD44(+)CD24(-) cells displayed stem cell-like features and were resistant to chemotherapy drug doxorubicin. Significantly, enhanced SLC34A2 expression correlated with chemoresponse and survival of breast cancer patients. We subsequently indicated that increased SLC34A2 expression in BCSCs directly contributed to their chemoresistance by a series of in vitro and in vivo experiments. Furthermore, we demonstrated that SLC34A2 induced chemoresistance in BCSCs via SLC34A2-Bmi1-ABCC5 signaling. Finally, we showed that ABCC5 was a direct transcriptional target of Bmi1 by chromatin immunoprecipitation (ChIP). In conclusion, our work indicated that decreased SLC34A2 expression sensitized BCSCs to doxorubicin via SLC34A2-Bmi1-ABCC5 signaling and shed new light on understanding the mechanism of chemoresistance in BCSCs. This study not only bridges the missing link between stem cell-related transcription factor (Bmi1) and ABC transporter (ABCC5) but also contributes to development of potential therapeutics against breast cancer.

miR-215 functions as a tumor suppressor in epithelial ovarian cancer through regulation of the X-chromosome-linked inhibitor of apoptosis.

Epithelial ovarian cancer (EOC) accounts for 90% of all ovarian cancer, which is the third most common gynaecological malignancy worldwide. Dysregulation of miRNAs is involved in the development of different types of EOC. The present study was designed to investigate the role of abnormal expression of miR-215 in the development of EOC and to elucidate the possible molecular mechanisms. mRNA expression of miR-215 was significantly decreased in EOC tissues and cell lines. Upregulation of miR-215 inhibited cell proliferation, promoted apoptosis and increased sensitivity to chemotherapy drugs in EOC cells. In contrast, downregulation of miR-215 increased cell proliferation, inhibited apoptosis and decreased sensitivity to chemotherapy drugs in EOC cells. In addition, the X-chromosome-linked inhibitor of apoptosis (XIAP) expression was significantly increased in EOC tissues and cell lines. Downregulation of XIAP inhibited cell proliferation, promoted apoptosis and increased sensitivity to chemotherapy drugs in EOC cells. Upregulation of miR-215 notably inhibited the expression of XIAP. Moreover, overexpression of XIAP significantly inhibited miR-215-exerted decrease of proliferation, increase of apoptosis and increase of sensitivity to chemotherapy drugs. In conclusion, we identified miR-215 as a potential tumor suppressor in patients with EOC downregulating expression of the oncogenic regulator XIAP. The data demonstrate that miR-215/XIAP pathway may serve as novel therapeutic targets and prognostic markers in patients with EOC.

ABCG2 protects kidney side population cells from hypoxia/reoxygenation injury through activation of the MEK/ERK pathway.

Breast cancer resistance protein 1 (BCRP1/ABCG2) is used to identify the side population (SP) within a population of cells, which is enriched for stem and progenitor cells in different tissues. Here, we investigated the role of extracellular signal-regulated kinase (ERK) 1/2 in the signaling mechanisms underlying ischemic/hypoxic conditions in kidney SP cells. Kidney SP cells were isolated using Hoechst 33342 dye-mediated fluorescein-activated cell sorting and then incubated under hypoxia/reoxygenation (H/R) with or without verapamil, a selective BCRP1/ABCG2 inhibitor. ABCG2 expression, ERK activity, cell viability, metabolic activity, and membrane damage were tested after H/R treatment. To evaluate the role of ERK 1/2 on the expression and function of ABCG2, the expression of mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), which preferentially activates ERK, was upregulated by transfection with the recombinant sense expression vector pcDNA3.1-MEK and downregulated by pretreatment with U0126, a specific MEK inhibitor. We found that hypoxia activated ERK activity in the kidney SP cells but not in non-SP cells both in vitro and in vivo. Overexpression of MEK mimicked hypoxia-induced ABCG2 expression. Contrarily, U0126 inhibited hypoxia- and MEK-upregulated ABCG2 expression. Furthermore, H/R induced significant increases in nuclear, metabolic, and membrane damage in both SP cells and non-SP cells; however, this H/R-induced cytotoxicity was much more severe in non-SP cells than in SP cells. Notably, the viability of kidney SP cells was enhanced by MEK overexpression and inhibited by U0126. Verapamil treatment reversed MEK-induced viability of kidney SP cells. When administered systemically into animals with renal ischemia/reperfusion injury, the SP cells significantly improved renal function, accelerated mitogenic response, and reduced cell apoptosis. However, this improved therapeutic potential of SP cells was significantly reduced by pretreatment with verapamil. Collectively, these findings provide evidence for a crucial role for the MEK/ERK-ABCG2 pathway in protecting kidney SP cells from ischemic/hypoxic injury.

Impact of HLA-G 14-bp polymorphism on acute rejection and cytomegalovirus infection in kidney transplant recipients from northwestern China.

Human leukocyte antigen (HLA)-G plays an important role in promoting transplant tolerance and helping human cytomegalovirus (CMV) to subvert host defenses. Strong evidence suggests that HLA-G 14-bp insertion/deletion polymorphism influences the stability of HLA-G mRNAs and levels of protein expression. We hypothesized that HLA-G 14-bp polymorphism of recipients has an influence on the risk of acute rejection (AR) and CMV infection. We investigated the impact of HLA-G 14-bp polymorphism on a total of 363 unrelated Chinese Han individuals who included 42 kidney transplant recipients with AR, 43 recipients with CMV infection, 102 recipients with stable allograft function (STA), and 176 healthy controls (HC). No statistically significant difference was found between all kidney transplant patients and HC (P=0.149). But, our data showed an increased frequency of homozygous genotype +14/+14 bp (P(c)=0.004) and allele +14 bp (P(c)=0.002) in patients with AR when compared with STA, with the odds ratio of 3.17 and 2.28, respectively. Moreover, we found that the frequency of the -14/-14 bp genotype (P(c)=0.008) and the -14 bp allele (P(c)=0.016) was increased in patients with CMV infection when compared with STA, with the OR of 2.66 and 1.96, respectively. Multivariate analysis further demonstrated that HLA-G homozygous +14 bp and -14 bp genotypes were an independent risk factor for allograft rejection and CMV infection, respectively. In conclusion, this study identified an important genetic risk factor for acute allograft rejection, and it was the first to show a significant correlation between HLA-G 14-bp polymorphism and CMV infection after kidney transplantation from northwestern China.

The role of SDF-1-CXCR4/CXCR7 axis in the therapeutic effects of hypoxia-preconditioned mesenchymal stem cells for renal ischemia/reperfusion injury.

In vitro hypoxic preconditioning (HP) of mesenchymal stem cells (MSCs) could ameliorate their viability and tissue repair capabilities after transplantation into the injured tissue through yet undefined mechanisms. There is also experimental evidence that HP enhances the expression of both stromal-derived factor-1 (SDF-1) receptors, CXCR4 and CXCR7, which are involved in migration and survival of MSCs in vitro, but little is known about their role in the in vivo therapeutic effectiveness of MSCs in renal ischemia/reperfusion (I/R) injury. Here, we evaluated the role of SDF-1-CXCR4/CXCR7 pathway in regulating chemotaxis, viability and paracrine actions of HP-MSCs in vitro and in vivo. Compared with normoxic preconditioning (NP), HP not only improved MSC chemotaxis and viability but also stimulated secretion of proangiogenic and mitogenic factors. Importantly, both CXCR4 and CXCR7 were required for the production of paracrine factors by HP-MSCs though the former was only responsible for chemotaxis while the latter was for viability. SDF-1α expression was upregulated in postischemic kidneys. After 24 h systemical administration following I/R, HP-MSCs but not NP-MSCs were selectively recruited to ischemic kidneys and this improved recruitment was abolished by neutralization of CXCR4, but not CXCR7. Furthermore, the increased recruitment of HP-MSCs was associated with enhanced functional recovery, accelerated mitogenic response, and reduced apoptotic cell death. In addition, neutralization of either CXCR4 or CXCR7 impaired the improved therapeutic potential of HP-MSCs. These results advance our knowledge about SDF-1-CXCR4/CXCR7 axis as an attractive target pathway for improving the beneficial effects of MSC-based therapies for renal I/R.

Induction of CD4+ CD25+ Foxp3+ T regulatory cells by dendritic cells derived from ILT3 lentivirus-transduced human CD34+ cells.

Immunoglobulin-like transcript 3 (ILT3) belongs to a family of inhibitory receptors with cytoplasmic immunoreceptor tyrosine based inhibitory motifs (ITIMs). Numerous studies have reported that increased ILT3 expression is associated with the tolerogenic properties of antigen-presenting cells (APCs) including dendritic cells (DCs). In this study, human CD34(+) hematopoietic stem/progenitor cells (HPSCs) were transduced with self-inactivating lentiviral vector carrying the ILT3 gene, and then induced to differentiate into DCs. Long-term and sustained transgene expression were observed. Importantly, DCs differentiated from ILT3-transduced HPSCs expressed high levels of human ILT3 and acquired strong tolerogenic capacity. This effect was associated with markedly decreased expression of co-stimulatory molecules (CD80, CD86) and down-regulation of NF-κB. Functionally, ILT3(high) DCs showed a reduced capacity to stimulate allogeneic T cell proliferation and increased the production of CD4(+)CD25(+)Foxp3(+) T regulatory cells with immunosuppressive activity. These results demonstrate that DCs derived from ILT3-transduced human CD34(+)HPSCs display tolerogenic properties to induce T regulatory cells in vitro.

[The influence of rapamycin on BTLA expression of human peripheral blood T lymphocytes].

AIM: To explore the effect of rapamycin (RPM) on the expression of B and T lymphocyte attenuate (BTLA) on human peripheral blood T lymphocytes, providing a experimental basis for application of RPM to organ transplantation and autoimmune diseases. METHODS: Human peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and then the T cells were isolated from PBMCs with immunomagnetic beads. Concanavalin A (Con A) used to stimulate and activate the human peripheral blood T cells. The proliferation of T cells was detected by MTT colorimetry. The levels of IL-2 and IFN-γ in cell culture supernatant were detected by ELISA. The expression of BTLA on T cells was assayed by Flow cytometry. RESULTS: The expression of BTLA on T cells treated with various concentrations (10-1 000 ng/L) of RPM had no significant difference, while had significant difference (P<0.01) by compared with RPM non-treatment group. ELISA detection manifested that compared with the untreated group different concentrations of RPM could significantly inhibit the IL-2 and IFN-γ secretion and significantly inhibited T lymphocyte proliferation (P<0.01). CONCLUSION: RPM has little effect on expression BTLA, but has stronger inhibition on lymphocyte proliferation and inflammatory cytokines secretion. Suggesting that RPM is suitable for the treated of organ transplant rejection and autoimmune diseases.