ß-arrestin2 is indispensable for the antidepressant effects of fluoxetine via inhibiting astrocytic pyroptosis in chronic mild stress mouse model for depression.

ß-arrestin2 is a versatile protein for signaling transduction in brain physiology and pathology. Herein, we investigated the involvement of ß-arrestin2 in pharmacological effects of fluoxetine for depression. A chronic mild stress (CMS) model was established using wild-type (WT) and ß-arrestin2-/- mice. Behavioral results demonstrated that CMS mice showed increased immobility time in the tail suspension test and forced swimming test, elevated concentrations of pro-inflammatory factors in peripheral blood, increased expression of pyroptosis-related proteins, and increased co-labeling of glial fibrillary acidic protein and Caspase1 p10 in the hippocampus compared to the CON group. Treatment with fluoxetine (FLX) ameliorated these conditions. However, compared with the ß-arrestin2-/- CMS group, these results of the ß-arrestin2-/- CMS + FLX group showed no significant changes. These results suggested that the above effects of FLX could be eliminated by knocking out ß-arrestin2. Mass spectrometry implying that FLX promoted the binding of ß-arrestin2 to the NLRP2 inflammasome of depressed mice. Subsequently, the results of the cellular experiments suggested that the 5HT2B receptor antagonist may attenuate L-kynurenine + ATP-induced cell pyroptosis by attenuating NLRP2 binding to ß-arrestin2. We further found that the lack of ß-arrestin2 eliminated the anti-pyroptosis effect of fluoxetine. In conclusion, ß-arrestin2 is an essential protein for fluoxetine to alleviate pyroptosis in the hippocampal astrocytes of CMS mice. Mechanistically, we found that the 5-HT2BR-ß-arrestin2-NLRP2 axis is vital for maintaining the antidepressant effects of fluoxetine.

Diagnostic Value of OPNI in Hepatocellular Carcinoma.

INTRODUCTION: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The overall prognosis of patients with HCC is not very ideal. Early detection of HCC has become one of the important means to reduce mortality. More and more studies have pointed out that inflammatory markers based on hematology, such as Onodera's prognostic nutritional index (OPNI), have important clinical significance. However, there is a lack of relevant research on the diagnosis of HCC by inflammatory markers. METHODS: We retrospectively enrolled 633 patients and prospectively recruited 121 consecutive patients, to explore the correlation between inflammatory markers and diagnosis of HCC. Based on the prognostic indicators, different diagnostic models were constructed and the diagnostic performance of different models was further compared. RESULTS: The best cutoff value of OPNI in the diagnosis of HCC is 43.925. Area under the receiver operating characteristics curve (AUC) of gender + age + AFP + OPNI diagnosis of HCC is 0.837 (95% CI: 0.702-0.868) in the retrospective cohort. Compared with gender + age + AFP, the Nagelkerke R2 of gender + age + AFP + OPNI increased from 0.234 to 0.426, and AUC increased by 0.0973 (95% CI: 0.0659-0.1290). DeLong test, net reclassification improvement (NRI) test, and integrated discrimination improvement (IDI) test are all statistically significant. In the prospective cohort, AUC of gender + age + AFP + OPNI diagnosis of HCC is 0.782 (95% CI: 0.696-0.869). Compared with gender + age + AFP, NRI test is statistically significant (categorical 0.0909 [95% CI: 0.0060-0.1759], p = 0.0359). CONCLUSION: In the process of monitoring the occurrence of HCC in patients with risk factors, OPNI can be included as appropriate to improve the accuracy of HCC diagnosis.

Prognostic Significance of Hemoglobin, Albumin, Lymphocyte and Platelet (HALP) Score in Hepatocellular Carcinoma.

Purpose: HALP score consisting of hemoglobin content, albumin content, lymphocyte count, and platelet count can comprehensively evaluate the inflammatory response and nutritional status. Many researchers have indicated that the HALP score is an effective predictor of the overall prognosis of various tumors. However, there is no relevant research to suggest whether the HALP score can predict the prognosis of patients with hepatocellular carcinoma (HCC). Patients and Methods: We retrospectively analyzed 273 HCC patients who underwent surgical resection. Hemoglobin content, albumin content, lymphocyte count, and platelet count in peripheral blood were measured for each patient. The relationship between the HALP score and overall survival (OS) was investigated. Results: With a mean of 56.69 ± 1.25 months follow-up, the 1-, 3-, and 5-year OS was 98.9%, 76.9%, and 55.3% for all patients, respectively. HALP scores (HR=1.708, 95% CI=1.192-2.448, P=0.004) were significant independent risk factors of OS. The 1-, 3-, and 5-year OS were 99.3%, 84.3%, and 63.4% for patients with high HALP scores; and 98.6%, 69.8%, and 47.5% for patients with low HALP scores, respectively (P=0.018). In TNM I-II stage patients, compared with high HALP scores, low HALP scores have worse OS (P=0.039). In AFP positive patients, compared with high HALP scores, low HALP scores have worse OS (P=0.042). Conclusion: Our research showed the preoperative HALP score is an independent predictive factor of overall prognosis, and a low HALP score indicates a worse prognosis in HCC patients who underwent surgical resection.

Changes in Inflammatory Markers Predict the Prognosis of Resected Hepatocellular Carcinoma with Child-Pugh A.

(1) Background: The reasons for changes in the inflammatory markers of patients with surgically resected hepatocellular carcinoma are unclear. We aimed to investigate the association of an inflammatory status with the prognosis of patients with hepatocellular carcinoma, who underwent surgical resection. (2) Methods: We retrospectively enrolled 91 patients with Child A hepatocellular carcinoma, who had received surgical resection, to explore the influence of preoperative inflammatory markers and postoperative changes on the prognosis. (3) Results: The platelet-to-lymphocyte ratio (PLR) and its alteration were independent prognostic factors. Patients with a low PLR had a significantly better recurrence-free survival (RFS) than those with a high PLR (1-year RFS of 88.5% versus 50.0%; 3-year RFS of 62.1% versus 25.0%, p = 0.038). The patients with a low PLR showed a significantly better overall survival (OS) than those with a high PLR (1-year OS of 98.9% versus 75.0%; 3-year OS of 78.2% versus 25.0%, p = 0.005). The patients whose PLR had increased at 6 months after operation showed a worse OS than patients whose PLR had decreased (1-year OS of 96.3% versus 98.4%; 3-year OS of 63.0% versus 79.7%, p = 0.048). However, neither the neutrophil-to-lymphocyte ratio nor Onodera's prognostic nutritional index had any prognostic significance. (4) Conclusions: The PLR and its alteration are significant prognostic factors for the RFS and OS of patients with Child A hepatocellular carcinoma who had received curative surgery.

Risk factors and antimicrobial resistance profiles of Pseudomonas putida infection in Central China, 2010-2017.

The aim of this study was to analyze the risk factors, clinical features, and antimicrobial resistance of Pseudomonas putida (P putida) isolated from Tongji Hospital in Wuhan, China.The data of 44 patients with P putida infections were retrospectively reviewed in this study. All cases of P putida strains were detected by the clinical laboratory of Tongji Hospital in the period of January 2010 to December 2017. Antimicrobial susceptibility testing was conducted using Kirby-Bauer method.Forty-four effective strains of P putida were isolated, including 32 inpatients and 12 outpatients. The 32 inpatients cases were obtained from various departments, which were urosurgery wards (n = 5, 15.6%), pediatrics wards (n = 4, 12.5%), hepatic surgery wards (n = 4, 12.5%), among others. The isolates had been discovered from urine specimens (28.2%), blood specimens (21.9%), sputum specimens (12.5%), and so on. Twenty-five patients had histories of catheterization before the isolation of P putida. Twenty-four patients were in immunocompromised states, 5 patients had undergone surgery, catheterization and were taking immunosuppressive therapy simultaneously. Polymicrobial infections were found in some P putida cases, especially Stenotrophomonas maltophilia, Pseudomonas aeruginosa, and Escherichia coli. All the patients had treated by antimicrobial before culture. Multi-drug-resistant strains were detected in 75% of P putida isolates. The P putida strains were resistant to trimethoprim/sulfamethoxazole (97.7%), aztreonam (88.6%), minocyline (74.3%), ticarcillin/clavulanic acid (72.7%), and sensitive to amikacin (86.4%), imipenem (62.8%), gentamicin (56.8%).Catheterization or other invasive procedures, immunocompromised states, and underlying diseases increased the risks of P putida infections. Moreover, the P putida strains were highly resistant to trimethoprim/sulfamethoxazole, aztreonam, minocyline, ticarcillin/clavulanic acid.

Elevated Hepatic CD1d Levels Coincide with Invariant NKT Cell Defects in Chronic Hepatitis B Virus Infection.

Activation of invariant NKT (iNKT) cells manifests antiviral immune responses in vivo. However, clinical trials have failed to show consistent hepatitis B virus (HBV) DNA reduction postadministration of iNKT cell-specific agonist α-galactosylceramide (α-GalCer). In this study, we aimed to investigate HBV infection-related iNKT cell defects and explore iNKT cell-based therapeutic potential for chronic hepatitis B (CHB). Liver specimens from 30 HBV-infected hepatocellular carcinoma patients were collected for CD1d/hepatitis B surface Ag (HBsAg) staining and/or intrahepatic iNKT cell assay. Two hundred and six chronic HBV-infected patients (including 130 CHB patients) were enrolled in the study of circulating iNKT cell frequency and function. We found that liver and hepatoma tissue that positively stained for HBsAg had higher CD1d expression as compared with HBsAg negatively stained counterparts. The elevated CD1d expression in infected tissue is supposed to facilitate the iNKT cell-based antiviral effects locally. However, iNKT cell defects that related with disease progression suggested iNKT cells attenuated their effects during chronic HBV infection. The residual iNKT cells in CHB patients showed aberrant activation and hyporesponsiveness to α-GalCer. Exogenous IL-2 fully rescued α-GalCer-induced expansion of iNKT cells from CHB patients, and synergistic effects of IL-2 and IL-15 helped to recover the CD1d-dependent IFN-γ production. In conclusion, our results highlight the increased CD1d expression in HBV-infected liver and differential iNKT cell defects associated with disease progression during chronic HBV infection. The reversibility of iNKT cell defects suggests protective immune responses could be partially recovered in CHB.

Roles of TGF-ß signaling pathway in endoplasmic reticulum stress in endothelial cells stimulated with cigarette smoke extract.

To investigate the role of signaling pathway in the effect of endoplasmic reticulum stress (ER stress) in endothelial cells stimulated with cigarette smoke extract (CSE). Human umbilical vein endothelial cells (HUVECs) were cultured and divided into 3 groups: CSE-stimulated group, CSE-stimulated with 4-PBA group, and negative control group. HUVECs were cultured and stimulated with CSE at concentrations of 5%, 10% and 20%, respectively, mRNA of CXCL-8 and GRP78 was detected by real-time PCR. ELISA was performed to test the expression of CXCL-8 protein, and neutrophils migration was detected by Transwell board test. The NF-κB, ERK, p38MAPK and transforming growth factor beta (TGF-ß) were detected by flow cytometry. The mRNA of CXCL-8 and GRP78 increased in CSE-stimulated HUVECs (P<0.05). Furthermore, it was concentration-dependent. 4-PBA significantly reduced the expression of CXCL-8 protein (P<0.05) and neutrophil migration (P<0.05). The TGF-ß, rather than the NF-κB, ERK and P38MAPK pathway might be involved in ER stress stimulated by CSE. CSE induced neutrophils migration by increasing the expression of CXCL-8 in endothelial cells. ER stress might play a role in the effect of neutrophils migration stimulated with CSE, and TGF-ß pathway may contribute to the ER stress in HUVECs.

Roles of TGF-β Signaling Pathway in Endoplasmic Reticulum Stress in Endothelial Cells Stimulated with Cigarette Smoke Extract / 华中科技大学学报（医学）（英德文版）

To investigate the role of signaling pathway in the effect of endoplasmic reticulum stress (ER stress) in endothelial cells stimulated with cigarette smoke extract (CSE).Human umbilical vein endothelial cells (HUVECs) were cultured and divided into 3 groups:CSE-stimulated group,CSE-stimulated with 4-PBA group,and negative control group.HUVECs were cultured and stimulated with CSE at concentrations of 5％,10％ and 20％,respectively,mRNA of CXCL-8 and GRP78 was detected by real-time PCR.ELISA was performed to test the expression of CXCL-8 protein,and neutrophils migration was detected by Transwell board test.The NF-κB,ERK,p38MAPK and transforming growth factor beta (TGF-β) were detected by flow cytometry.The mRNA of CXCL-8 and GRP78 increased in CSE-stimulated HUVECs (P＜0.05).Furthermore,it was concentration-dependent.4-PBA significantly reduced the expression of CXCL-8 protein (P＜0.05) and neutrophil migration (P＜0.05).The TGF-β,rather than the NF-κB,ERK and P38MAPK pathway might be involved in ER stress stimulated by CSE.CSE induced neutrophils migration by increasing the expression of CXCL-8 in endothelial cells.ER stress might play a role in the effect of neutrophils migration stimulated with CSE,and TGF-β pathway may contribute to the ER stress in HUVECs.

Nucleoside analogs prevent disease progression in HBV-related acute-on-chronic liver failure: validation of the TPPM model.

PURPOSE: This study aimed to evaluate the efficacy and safety of entecavir, lamivudine and telbivudine for treating patients with HBV-ACLF and to validate the Tongji prognostic predictor model (TPPM) in these patients. METHODS: In this retrospective study, we enrolled 283 patients with HBV-ACLF (100 treated with entecavir, 98 treated with lamivudine and 85 treated with telbivudine). There were no significant differences in baseline clinical and virological characteristics among patients treated with entecavir, telbivudine or lamivudine. RESULTS: There were no significant differences in the 4- and 12-week survival rates of entecavir-, telbivudine- and lamivudine-treated patients (79.00, 81.18 and 86.73 %, respectively, at 4 weeks; 67.00, 65.88 and 73.47 %, respectively, at 12 weeks). Patients in all three groups achieved an improvement in the model for end-stage liver disease (MELD) score. Using the Hosmer-Lemeshow test, the validation of the TPPM score for HBV-ACLF demonstrated a good degree of fit with disease prognosis. Based on this unique group of patients, the TPPM score with an AUC of 0.787 was superior to the MELD score, which had an AUC of 0.736 in the prediction of 12-week mortality. The TPPM had an AUC of 0.733, and the MELD score had an AUC of 0.672 in the prediction of 4-week mortality. Using a cutoff value of 0.22 for 12-week mortality prediction by the TPPM, the positive predictive value was 49.66 %, with a negative predictive value of 89.55 %. CONCLUSION: Treatment with nucleoside analogs including entecavir, lamivudine and telbivudine prevented disease progression and increased the survival of patients with HBV-ACLF. Validation of the established TPPM scoring system in this study confirmed its superior predictive value for HBV-ACLF patients when compared with the MELD system.

CD8low T-cell subpopulation is increased in patients with chronic hepatitis B virus infection.

Recent studies suggest that CD8(+) T cells with down-regulated CD8 expression (CD3(+)CD8(low) T cells) represented as a distinct phenotype of CD8(+) T cells are increased and linked to disease severity in some chronically persistent infection, such as chronic HIV and parasite infection. However, the role of CD3(+)CD8(low) T cells in the context of chronic HBV infection is poorly understood. In this study, peripheral blood samples of 47 chronic hepatitis B patients and 19 healthy controls were collected and tested for the frequency and phenotype of CD8(low) T cells. The circulating CD8(low) T cells were significantly more frequent in the patients compared to those in healthy controls, and the CD8(low) T cells in the patients expressed less IFN-γ and more mTGF-ß1 than those in the controls, suggesting their type-2 polarized and suppressive properties. Meanwhile, the concentrations of plasma soluble HLA class I molecules were found elevated in the patients, and positively associated with the frequencies of CD8(low) T cells. Furthermore, the CD8(low) T-cell frequency in the HLA-A2-positive patients (n=21) was found negatively correlated with the T-cell responsiveness against the HBc18₋27 peptide, the latter was impaired as revealed by IFN-γ Elispot assay. Our findings suggested that a better understanding of the involvement of CD8(low) T cells in chronically persistent HBV infection would add to our knowledge of the impaired T-cell response in the patients.

Performance evaluation of the Maxwell 16 System for extraction of influenza virus RNA from diverse samples.

This study evaluated the performance of the Maxwell 16 System (Promega) for extraction of influenza virus (flu-v) RNA from diverse samples compared to a classical manual method (QIAamp Kit, QIAGEN). Following extraction by the two methods, all samples were analyzed by Real-time RT-PCR. Results revealed that the use of the standard Maxwell 16 protocol (Maxwell 16-S) resulted in good linearity and precision across a wide concentration range and higher sensitivity of detection from flu-v stock suspensions than the manual method. Compared with the latter method, Maxwell 16-S extracted RNA more efficiently (higher RNA yield and/or fewer PCR inhibitors) from throat swabs and bronchoalveolar lavage fluids, while both methods performed comparably on fecal samples from human and poultry in terms of overall threshold cycle values and detection rates although the Maxwell 16-S co-purified more inhibitors from fecal samples. The capacity of this system to remove inhibitors from fecal matrix was improved by using a modified Maxwell 16 protocol with a reduced sample input, which eliminated all false-negatives produced by the Maxwell 16-S. These findings suggest that the Maxwell 16 System is suitable for RNA extraction from multiple-source samples for diagnosis of influenza and viral load determination and that a proper reduction in starting sample volume may improve the detection of flu-v from complex matrices such as feces. Additionally, this system allows flexible sample throughput and labor-saving sample processing with little or no risk of cross-contamination.

Inhibitory function of Tregs via soluble FGL2 in chronic hepatitis B.

CD4(+)CD25(+)CD127(dim/-) regulatory T cells (Tregs) have been implicated in suppressing T cell immune responses to hepatitis B virus (HBV), but the inhibition mechanism has not being clear yet. This study investigated the effects of soluble FGL2 (sFGL2) secreted by Tregs on immune suppression in chronic HBV-infected patients. We verified that sFGL2 protein and mRNA were highly expressed in Tregs. The separated Tregs by using magnetic beads from peripheral blood mononuclear cells (PBMCs) in 20 patients with chronic hepatitis B were co-cultured with PBMCs at a ratio of 1:3 with anti-CD3 stimulating antibody or FGL2 blocking antibody. The proliferation index of CD8(+)T cells after blocking FGL2 was higher than that in blank group (3.58±0.18 vs. 3.28±0.17, P=0.034) in 18 of 20 samples, and lower than that in CD3 stimulation group (3.82±0.19, P=0.026) in 16 of 20 samples. The IFN-γ secreted in the mixed culture in the absence of Tregs was higher than that in the culture in the presence of Tregs, but it could be abolished by FGL2 blocking antibody. These results suggest that sFGL2 protein secreted by Tregs suppresses the proliferation and function of CD8(+) T cells in chronic hepatitis B.

Downregulation of FGL2/prothrombinase delays HCCLM6 xenograft tumour growth and decreases tumour angiogenesis.

BACKGROUND: Fibrinogen-like protein 2 (FGL2), which directly generates thrombin from prothrombin without activation of the conventional coagulation cascade, was shown to be overexpressed in various human malignant tumours. AIMS: Herein, we aimed to investigate its expression pattern, biological function and mechanism of action in hepatocellular carcinoma (HCC). METHODS: FGL2 expression and colocalization with fibrin was examined in 15 HCC tissues. FGL2 downregulation was performed by targeting microRNA in a HCCLM6 cell line in which FGL2 was highly expressed in xenografts of nude mice. The effects of FGL2 knockdown on tumour growth and angiogenesis were evaluated in vitro and in vivo. Cytometric bead arrays were employed to identify FGL2-regulated signalling pathways. RESULTS: FGL2 was overexpressed in HCC tissues and colocalized with fibrin deposition. Knockdown of FGL2 expression in HCCLM6 cells (hFGL2(low) HCCLM6) resulted in delayed xenografts tumour growth within an observation period of 42 days and decreased vascularization, which was accompanied by decreased phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). In vitro hFGL2(low) HCCLM6 cells exhibited decreased proliferation without significant induction of apoptosis. Overexpression of FGL2 in HCCLM6 cells or addition of recombinant hFGL2 protein induced phosphorylation of p38-MAPK and ERK1/2 involving protease-activated receptors (PARs).activation. CONCLUSIONS: FGL2 contributes to HCC tumour growth and angiogenesis in a thrombin-dependent manner, and downregulation of its expression might be of therapeutic significance in HCC.

Increased interleukin-32 expression in chronic hepatitis B virus-infected liver.

OBJECTIVES: The role of interleukin-32 (IL-32) in chronic hepatitis B (CHB) remains unclear. In order to identify the role of IL-32 in CHB, we detected the expression levels of IL-32 in liver samples of CHB patients and analyzed the correlation between IL-32 and liver inflammation/fibrosis. METHODS: Real-time PCR and immunohistochemistry were used to detect the expression levels of IL-32 in liver tissues of patients with CHB. The correlation between hepatic IL-32 expression and the severity of liver inflammation/fibrosis was assessed using Spearman's correlation. RESULTS: Hepatic IL-32 expression was increased in CHB patients and increased with the severity of liver inflammation/fibrosis. Moreover, hepatic IL-32 expression was significantly positively correlated with serum ALT level and negatively related with serum ALB level. CONCLUSIONS: Our results suggest that IL-32 could be implicated in HBV-related liver inflammation/fibrosis. We believe that IL-32 might play important roles in the pathogenesis of CHB.

Entecavir treatment prevents disease progression in hepatitis B virus-related acute-on-chronic liver failure: establishment of a novel logistical regression model.

BACKGROUND: Hepatitis B-related acute-on-chronic liver failure (ACLF) has a poor prognosis with very high mortality. Unfortunately, most prognostic predictive models of liver failure are complicated and offer suboptimal sensitivity. Experience in entecavir (ETV)-treated patients with hepatitis B virus (HBV)-ACLF is limited. AIMS: This study was designed to evaluate the efficacy and safety of ETV in patients with HBV-ACLF and to develop a novel model (Tongji prognostic predictor model, TPPM) for prognostic prediction of HBV-ACLF patients. METHOD: In this retrospective study, 248 patients with HBV-ACLF were enrolled. There were no significant differences in baseline clinical and virologic characteristics between patients treated with and without ETV. RESULTS: The 1- and 3-month survival rates of patients in the ETV-treated group (n = 124) were 72.58 and 61.29%, respectively, significantly higher than that in NA-free group (n = 124), which were 53.23 and 45.97%, respectively. By Hosmor and Lemeshow test, TPPM for HBV-ACLF had a very good degree of fit with disease prognosis. Based on this unique group of patients, the TPPM scoring offered a better prediction value in both specificity and sensitivity for 3-month mortality of patients with HBV-ACLF compared with MELD scoring system with statistically significant difference. In the patients with HBV-ACLF, using a cutoff of 0.22 for 3-month predicted mortality by TPPM, the positive predictive value was 93.6% and negative predictive value 91.3%. CONCLUSION: ETV treatment prevented disease progression and increased the survival of patients with HBV-ACLF. The established TPPM scoring system offers superior predictor value in both specificity and sensitivity for HBV-ACLF patients when compared with MELD.

Interleukin(IL)-4 promotion of CXCL-8 gene transcription is mediated by ERK1/2 pathway in human pulmonary artery endothelial cells.

Interleukin-4 is central to allergic pulmonary inflammatory responses, but its contribution to airway neutrophilia remains controversial. The endothelium plays a critical role in regulating leukocyte recruitment and migration during inflammation. However, its response to IL-4 is reported to either increase or decrease the production of neutrophil chemotactic factors. We hypothesized that these conflicting findings may be due to the tissue and the size of the vessels from which endothelial cells have been derived. The expression of CXCL-8 by human primary culture umbilical veins endothelial cells (HUVECs), human pulmonary artery endothelial cells (HPAECs), and human pulmonary microvascular endothelial cells (HPMECs) when stimulated with recombinant human IL-4 (rhIL-4) was studied. The chemoattractant property of the cells' supernatants for neutrophils was evaluated using Boyden chambers. The role of the nuclear factor-κB (NF-κB), and mitogen-activated protein kinases (MAPK) in IL-4-induced HPAECs was studied using Western blotting and electrophoretic mobility shift assay (EMSA). We demonstrated that IL-4 increased the mRNA expression and the protein production of CXCL-8 in HPAECs, but not in HUVECs and HPMECs. The supernatants of HAPECs stimulated by IL-4 significantly promoted neutrophils migration in a dose-dependent manner, and was significantly attenuated by an inhibitor of CXCL-8. We also found that extracellular-regulated protein kinase1/2 (ERK1/2) is activated by IL-4 in HPAECs, but not JUN-N-terminal protein kinase (JNK) or p38 MAPK pathway. Furthermore, NF-κB-DNA binding activity, phosphorylation of IκBα and p65 levels were not affected by rhIL-4 in HAPECs. These findings indicate marked functional differences in the response of micro and macro-ECs to IL-4. ERK1/2, rather than NF-κB, JNK and p38 MAPK signaling, plays a role in IL-4 induced chemokine activation. Our results suggest that inhibition of ERK1/2 may be a possible target for airway neutrophilia in allergic lung diseases.

Transplantation of NIT-1 cells expressing pD-L1 for treatment of streptozotocin-induced diabetes.

BACKGROUND: Programmed death-1 ligand-1 (PD-L1, CD274, B7-H1) has been identified as the ligand for the immunoinhibitory receptor programmed death-1 and has been demonstrated to play a role in the regulation of immune responses and peripheral tolerance. In this study, we tested the effect of PD-L1-transfected pancreatic beta-cell line established from a transgenic NDD/Lt mouse (NIT) on the alloresponse and streptozotocin-induced diabetes. METHODS: The diabetes model was established by a low dose of streptozotocin in Balb/C mice. PD-L1 transfected NIT cell line was established, namely NIT-PD-L1. NIT-1, empty vector-transfected NIT-1, or NIT-PD-L1 cells were transplanted into diabetic mice by intraperitoneal injection, respectively. Proliferation and apoptosis of splenic lymphocytes were detected by labeling with carboxy fluorescein succinimidyl ester or AnnexinV-Cy5 and proliferation index (PI). Cytokines were determined by enzyme-linked immunosorbent assay and flow cytometry analysis. RESULTS: When compared with the controls, overexpression of PD-L1 on NIT-1 cells markedly prolonged allograft survival in diabetic mice. In mixed cells reaction, splenic lymphocytes from NIT-PD-L1-transplanted diabetic mice co-culture with mitomycin C-treated NIT-PD-L1 showed the lowest proliferative response but severe apoptosis. In addition, NIT-PD-L1 suppressed interferon-gamma but up-regulated interleukin-4 and -10 productions by those lymphocytes in vitro and in vivo. CONCLUSION: Our data demonstrated that overexpression of PD-L1 on pancreatic beta cells significantly can prolong allograft survival, and it is associated with inhibition of lymphocytes activation and proliferation, induction of lymphocytes apoptosis.

Preparation and identification of scFv and bsFv against transferrin receptor.

To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively. Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.

CTLA4-Ig-modified dendritic cells inhibit lymphocyte-mediated alloimmune responses and prolong the islet graft survival in mice.

The induction of antigen specific tolerance is critical for prevention and treatment of allograft rejection. In this study, we transfected CTLA4-Ig gene into dendritic cells (DCs), and investigated their effect on inhibition of lymphocyte activity in vitro and induction of immune tolerance on pancreatic islet allograft in mice. An IDDM C57BL/6 murine model induced by streptozotocin is as model mouse. The model mice were transplanted of the islet cells isolated from the BALB/c mice to their kidney capsules, and injected of CTLA4-Ig modified DCs (mDCs). The results showed that mDCs could significantly inhibit T lymphocyte proliferation and induce its apoptosis; whereas, unmodified DCs (umDCs) promoted the murine lymphocyte proliferation. Compared with injection of umDCs and IgG1 modified DCs, the injection of mDCs prolonged IDDM mice's allograft survival, and normalized their plasma glucose (PG) levels within 3 days and maintained over 2 weeks. The level of IFN-gamma was lower and the level of IL-4 was higher in mDCs treated recipient mice than that in control mice, it indicated that mDCs led to Th1/Th2 deviation. After 7 days of islet transplantation, HE stain of the renal specimens showed that the islets and kidneys were intact in structure, and islet cells numbers are increased in mDCs treated mice. Our studies suggest that DCs expressing CTLA4-Ig fusion protein can induce the immune tolerance to islet graft and prolong the allograft survival through the inhibition of T cell proliferation in allogeneic mice.