Effects of Prone Positioning for Patients with Acute Respiratory Distress Syndrome Caused by Pulmonary Contusion: A Single-Center Retrospective Study.

Background: The effects of prone positioning (PP) on patients with acute respiratory distress syndrome (ARDS) caused by pulmonary contusion (PC) are unclear. We sought to determine the efficacy of PP among patients whose ARDS was caused by PC. Methods: A retrospective observational study was performed at an intensive care unit (ICU) from January 2017 to June 2021. ARDS patients with PaO2/FiO2 (P/F) < 150 mmHg were enrolled. During the study period, we enrolled 121 patients in the PP group and 117 in the control group. The changes in vital signs, laboratory tests, and compliance of the respiratory system (Crs) were recorded for 3 consecutive days. The mechanical ventilation time, duration of ICU stay, complications, extubation rate, 28-day ventilator-free days, and mortality were also recorded. Results: In the PP group, the P/F and Crs increased over time. Compared to the control group, the P/F and Crs improved in the PP group over 3 consecutive days (P < 0.05). Furthermore, the PP group also had shorter total mechanical ventilation time (5.1 ± 1.4 vs. 9.3 ± 3.1 days, P < 0.05) and invasive ventilation time (4.9 ± 1.2 vs. 8.7 ± 2.7 days, P < 0.05), shorter ICU stay (7.4 ± 1.8 vs. 11.5 ± 3.6days, P < 0.05), higher extubation rate (95.6% vs. 84.4%, P < 0.05), less atelectasis (15 vs. 74, P < 0.05) and pneumothorax (17 vs. 24, P > 0.05), more 28-day ventilator-free days (21.6 ± 5.2 vs. 16.2 ± 7.2 days, P < 0.05), and lower mortality (4.4% vs. 13.3%, P < 0.05). Conclusions: Among PC cases with moderate to severe ARDS, PP can correct hypoxemia more quickly, improve Crs, reduce atelectasis, increase the extubation rate, shorten mechanical ventilation time and length of ICU stay, and reduce mortality.

Oxytocin signalling in dendritic cells regulates immune tolerance in the intestine and alleviates DSS-induced colitis.

BACKGROUND: Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD) that is associated with immune dysfunction. Recent studies have indicated that the neurosecretory hormone oxytocin (OXT) has been proven to alleviate experimental colitis. METHODS: We investigated the role of OXT/OXT receptor (OXTR) signalling in dendritic cells (DCs) using mice with specific OXTR deletion in CD11c+ cells (OXTRflox/flox×CD11c-cre mice) and a dextran sulfate sodium (DSS)-induced colitis model. RESULTS: The level of OXT was abnormal in the serum or colon tissue of DSS-induced colitis mice or the plasma of UC patients. Both bone marrow-derived DCs (BMDCs) and lamina propria DCs (LPDCs) express OXTR. Knocking out OXTR in DCs exacerbated DSS-induced acute and chronic colitis in mice. In contrast, the injection of OXT-pretreated DCs significantly ameliorated colitis. Mechanistically, OXT prevented DC maturation through the phosphatidylinositol 4,5-bisphosphate 3-kinase (Pi3K)/AKT pathway and promoted phagocytosis, adhesion and cytokine modulation in DCs. Furthermore, OXT pre-treated DCs prevent CD4+ T cells differentiation to T helper 1 (Th1) and Th17. CONCLUSIONS: Our results suggest that OXT-induced tolerogenic DCs efficiently protect against experimental colitis via Pi3K/AKT pathway. Our work provides evidence that the nervous system participates in the immune regulation of colitis by modulating DCs. Our findings suggest that generating ex vivo DCs pretreated with OXT opens new therapeutic perspectives for the treatment of UC in humans.

Proteomics-based screening of the target proteins associated with antidepressant-like effect and mechanism of Saikosaponin A.

Depression is a commonly occurring neuropsychiatric disease with an increasing incidence rate. Saikosaponin A (SA), a major bioactive component extracted from Radix Bupleuri, possesses anti-malignant cell proliferation, anti-inflammation, anti-oxidation and liver protective effects. However, few studies have investigated SA's antidepressant effects and pharmacological mechanisms of action. Our study aimed to explore the anti-depression effect of SA and screen the target proteins regulated by SA in a rat model of chronic unpredictable mild stress (CUMS)-induced depression. Results showed that 8-week CUMS combined with separation could successfully produce depressive-like behaviours and cause a decrease of dopamine (DA) in rat hippocampus, and 4-week administration of SA could relieve CUMS rats' depressive symptoms and up-regulated DA content. There were 15 kinds of significant differentially expressed proteins that were detected not only between the control and CUMS groups, but also between the CUMS and SA treatment groups. Proline-rich transmembrane protein 2 (PRRT2) was down-regulated by CUMS while up-regulated by SA. These findings reveal that SA may exert antidepressant effects by up-regulating the expression level of PRRT2 and increasing DA content in hippocampus. The identification of these 15 differentially expressed proteins, including PRRT2, provides further insight into the treatment mechanism of SA for depression.

Overexpression of GSN could decrease inflammation and apoptosis in EAE and may enhance vitamin D therapy on EAE/MS.

The decrease of gelsolin (GSN) in the blood has been reported in multiple sclerosis (MS) patients and experimental allergic encephalomyelitis (EAE) animals, but the protective effect of GSN on EAE/MS lacks of evidence. In our study, we increased the GSN level in EAE by injecting GSN-overexpress lentivirus (LV-GSN) into the lateral ventricle and caudal vein and found that GSN administration can delay the onset and decrease the severity of EAE. Vitamin D is proven to have a therapeutic effect on MS/EAE; however, we previously found that vitamin D caused a downregulation of GSN, which might limit vitamin D efficacy. In our current research, we obtained a better symptom and a slowing down progression in EAE after combining vitamin D treatment with a proper increase of GSN. Furthermore, we discovered that the mediation of vitamin D on GSN might occur through the vitamin D receptor (VDR) by using gene interruption and overexpression to regulate the level of VDR in PC12 cells (a rat sympathetic nerve cell line). We also confirmed the anti-apoptotic function of GSN by GSN RNA interference in PC12. Collectively, these results support the therapeutic effect of GSN in EAE, which might enhance Vitamin D therapy in EAE/MS.

Vitamin D-Binding Protein Acts in the Actin Scavenge System and Can Have Increased Expression During Aspirin Therapy.

While the clinical efficacy of aspirin in cerebral thrombosis prevention has been well established, its mechanism of action is still controversial. In an effort to better understand these mechanisms and to identify potential biomarkers, comparative proteomic analysis between 18 patients both pre-aspirin treatment at the time of cerebral thrombotic onset (control group) and post-aspirin treatment (experiment group) was carried out using two-dimensional gel electrophoresis (2-DE) in combination with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDITOF/ MS). Of the 228 2-DE identified differentially expressed protein spots, 11 proteins showed more than a 1.5-fold difference. Of these, vitamin D-binding protein (DBP) and actin were further examined via Western blot and showed consistent results, with DBP levels significantly increased post-aspirin treatment (114.04 ± 16.69) relative to pre-treatment (66.33 ± 5.61) while actin showed the opposite trend (p < 0.01 for both comparisons). Next, co-immunoprecipitation analysis of DBP and actin showed direct binding. Furthermore, a protein-protein interaction network of DBP and the other differentially expressed proteins was constructed using Ingenuity Pathway Analysis software. These results suggest that DBP acts in the actin scavenge system and consequently the increase in DBP levels correlated with aspirin therapy in cerebral thrombotic patients. These findings also suggest that aspirin may prevent platelet aggregation and thrombosis through the actions of DBP and other DBP related proteins.

Mmu-miR-125b overexpression suppresses NO production in activated macrophages by targeting eEF2K and CCNA2.

BACKGROUND: MicroRNAs have been shown to be important regulators of the immune response and the development of the immune system. It was reported that microRNA-125b (miR-125b) was down-regulated in macrophages challenged with endotoxin. However, little is known about the function and mechanism of action of miR-125b in macrophage activation. Macrophages use L-arginine to synthesize nitric oxide (NO) through inducible NO synthase (iNOS), and the released NO contributes to the tumoricidal activity of macrophages. METHODS: Luciferase reporter assays were employed to validate regulation of a putative target of miR-125b. The effect of miR-125b on endogenous levels of this target were subsequently confirmed via Western blot. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to determine the expression level of miR-125b in macrophage. MTS assays were conducted to explore the impact of miR-125b overexpression on the cell viability of 4T1 cells. RESULTS: Here, we demonstrate that mmu-miR-125b overexpression suppresses NO production in activated macrophages and that LPS-activated macrophages with overexpressed mmu-miR-125b promote 4T1 tumor cell proliferation in vitro and 4T1 tumor growth in vivo. CCNA2 and eEF2K are the direct and functional targets of mmu-miR-125b in macrophages; CCNA2 and eEF2K expression was knocked down, which mimicked the mmu-miR-125b overexpression phenotype. CONCLUSIONS: These data suggest that mmu-miR-125b decreases NO production in activated macrophages at least partially by suppressing eEF2K and CCNA2 expression.

The Clinical Impact of Ventilator-Associated Events: A Prospective Multi-Center Surveillance Study.

OBJECTIVE: The Centers for Disease Control and Prevention (CDC) has developed an approach to ventilator-associated events (VAE) surveillance. Using these methods, this study was performed to investigate VAE incidences and to test whether VAEs are associated with poorer outcomes in China. DESIGN: A 4-month, prospective multicenter surveillance study between April and July 2013. SETTING: Our study included 15 adult intensive care units (ICUs) of 15 hospitals in China. PATIENTS: Patients admitted to ICUs during the study period METHODS: Patients on mechanical ventilation (MV) were monitored for VAEs: ventilator-associated conditions (VACs), infection-related ventilator-associated complications (IVACs), and possible or probable ventilator-associated pneumonia (VAP). Patients with and without VACs were compared with regard to duration of MV, ICU length of stay (LOS), overall hospital LOS, and mortality rate. RESULTS: During the study period, 2,356 of the 5,256 patients admitted to ICUs received MV for 8,438 ventilator days. Of these patients, 636 were on MV >2 days. VACs were identified in 94 cases (4.0%; 11.1 cases per 1,000 ventilator days), including 31 patients with IVACs and 16 with possible VAP but none with probable VAP. Compared with patients without VACs, patients with VACs had longer ICU LOS (by 6.2 days), longer duration on MV (by 7.7 days), and higher hospital mortality rate (50.0% vs 27.3%). The mortality rate attributable to VACs was 11.7%. Compared with those with VACs alone, patients with IVACs had longer duration on MV and increased ICU LOS but no higher mortality rates. CONCLUSIONS: In China, surveillance of VACs and IVACs is able to identify MV patients with poorer outcomes. However, surveillance of possible and probable VAP can be problematic.

RIP1 modulates death receptor mediated apoptosis and autophagy in macrophages.

Macrophages are responsible for defending against diverse pathogens and play a crucial role in the innate immune system. Macrophage's lifespan is determined by homeostatic balance between survival and apoptosis. Here we report that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers both apoptosis and autophagy in human U937 cells. Inhibition of autophagy facilitates TRAIL-induced apoptosis, suggesting that autophagy of macrophages protects against TRAIL-induced apoptosis. TRAIL treatment influences the expression of death receptors, indicating that TRAIL-induced apoptosis and autophagy are mediated by death receptors. RIP1 ubiquitination and expression regulate apoptosis and autophagy. Furthermore, expression and bioactivity of the p43/41-caspase-8 variant are critical to TRAIL-induced autophagy and apoptosis. Knockdown of RIP1 suppresses autophagy in macrophage. These data demonstrate that RIP1 is essential for the regulation of death receptor mediated autophagy and apoptosis. The results in this study contribute to understanding the regulation of autophagy and apoptosis in macrophages, and shed lights on death receptor-targeted therapy for cancer, inflammation and autoimmune diseases.

Vitamin D therapy in experimental allergic encephalomyelitis could be limited by opposing effects of sphingosine 1-phosphate and gelsolin dysregulation.

Several studies support a protective effect of vitamin D on multiple sclerosis and experimental allergic encephalomyelitis (EAE), but the mechanisms of these favorable effects are unclear. Our study demonstrates that sphingosine 1-phosphate (S1P) is upregulated in the serum and spinal cords of EAE rats, but that vitamin D reverses the upregulation to alleviate inflammation. Vitamin D, however, cannot prevent the disease process, suggesting that other factors may be involved. To identify additional factors that might limit vitamin D efficacy, we assessed the effects of vitamin D on plasma gelsolin (pGSN), a regulator of S1P that is downregulated in the CSF of MS patients. Our results show that pGSN is downregulated in the serum of EAE rats, whereas its cellular form, cytoplasmic gelsolin (cGSN), is upregulated in the spinal cord of EAE rats. Importantly, vitamin D causes a downregulation of both pGSN and cGSN, which may counteract the positive effects of S1P decrease. Furthermore, 48 and 42 kDa caspase-3 cleavage products of cGSN are detected in EAE spinal cords, suggesting enhanced apoptotic activity, but these cleaved products undergo a similar decrease upon vitamin D treatment. To directly test the role of cGSN in the apoptotic process, we performed RNA interference in PC-12, a rat sympathetic nerve cell line. Results verify that cGSN suppresses apoptosis induced by TNF-α. Collectively, these results support a therapeutic effect of vitamin D that is derived from its ability to reduce S1P, but is limited by its simultaneous effect in reducing pGSN and cGSN. Based on these observations, we postulate that combined therapy with recombinant human pGSN and vitamin D may produce more beneficial effect in treating multiple sclerosis.

Human T-cell leukemia virus type 1 Tax-deregulated autophagy pathway and c-FLIP expression contribute to resistance against death receptor-mediated apoptosis.

UNLABELLED: The human T-cell leukemia virus type 1 (HTLV-1) Tax protein is considered to play a central role in the process that leads to adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 Tax-expressing cells show resistance to apoptosis induced by Fas ligand (FasL) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). The regulation of Tax on the autophagy pathway in HeLa cells and peripheral T cells was recently reported, but the function and underlying molecular mechanism of the Tax-regulated autophagy are not yet well defined. Here, we report that HTLV-1 Tax deregulates the autophagy pathway, which plays a protective role during the death receptor (DR)-mediated apoptosis of human U251 astroglioma cells. The cellular FLICE-inhibitory protein (c-FLIP), which is upregulated by Tax, also contributes to the resistance against DR-mediated apoptosis. Both Tax-induced autophagy and Tax-induced c-FLIP expression require Tax-induced activation of IκB kinases (IKK). Furthermore, Tax-induced c-FLIP expression is regulated through the Tax-IKK-NF-κB signaling pathway, whereas Tax-triggered autophagy depends on the activation of IKK but not the activation of NF-κB. In addition, DR-mediated apoptosis is correlated with the degradation of Tax, which can be facilitated by the inhibitors of autophagy. IMPORTANCE: Our study reveals that Tax-deregulated autophagy is a protective mechanism for DR-mediated apoptosis. The molecular mechanism of Tax-induced autophagy is also illuminated, which is different from Tax-increased c-FLIP. Tax can be degraded via manipulation of autophagy and TRAIL-induced apoptosis. These results outline a complex regulatory network between and among apoptosis, autophagy, and Tax and also present evidence that autophagy represents a new possible target for therapeutic intervention for the HTVL-1 related diseases.

TRAIL receptor deficiency sensitizes mice to dextran sodium sulphate-induced colitis and colitis-associated carcinogenesis.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor (TRAIL-R) play important roles in immune regulation and cancer cell death. Although TRAIL has been shown to induce chemokine release in various tumour cells, the function of TRAIL-R in the development of colitis and colitis-associated carcinogenesis has not been explored. In this study, we found that TRAIL-R-deficient mice exhibited a higher incidence of colitis and colitis-associated cancer than that of wild-type (WT) mice, and TRAIL-R expression was down-regulated in WT mice that were fed dextran sulphate sodium. Chemokines, including CCL2 and CXCL1, were highly expressed in the serum and inflammatory colon tissues of TRAIL-R(-/-) mice compared with WT mice, and TRAIL-R(-/-) mice showed a marked infiltration of immune cells during colitis. Hyperactivation of Janus kinase and nuclear factor-κB in colon epithelial cells was also observed, which correlated with the severity of colonic inflammation in TRAIL-R(-/-) mice. These data suggest that TRAIL-R plays a protective role in chemical-induced colon injury and negatively regulates mucosal immune responses.

TRAIL-induced miR-146a expression suppresses CXCR4-mediated human breast cancer migration.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered a promising agent for cancer therapy, as this molecule induces apoptosis specifically in various cancer cells. Apart from apoptosis, TRAIL also induces non-apoptotic signals, such as those for autophagy, proliferation and metastasis in cancer cells. In the present study, we report that TRAIL suppressed CXCR4-mediated human breast cancer MDA-MB-231 cell migration by up-regulating miR-146a expression through NF-κB. TRAIL receptor 1 (TRAIL-R1, DR4) was highly expressed in TRAIL-treated MDA-MB-231 cells. A neutralization antibody against DR4 specifically blocked TRAIL-induced NF-κB activation and miR-146a expression. These results were confirmed in a human breast cancer xenograft mouse model, suggesting that TRAIL significantly enhanced miR-146a expression and suppressed CXCR4 expression, indicating that TRAIL-induced miR-146a up-regulation is negatively associated with CXCR4 expression. These findings suggest that TRAIL-induced miR-146a expression suppresses CXCR4-mediated human breast cancer migration, and provide further insight into the non-apoptotic function of TRAIL in the prevention of metastasis as a therapy for breast cancer.

Vitamin D-binding protein in cerebrospinal fluid is associated with multiple sclerosis progression.

Multiple sclerosis is a neurological disorder that presents with symptoms including inflammation, neurodegeneration, and demyelination of the central nervous system (CNS). Secondary progressive multiple sclerosis (SPMS) manifests with serious physical disability. To quantitatively analyze differential protein expression in patients with SPMS, we performed two-dimensional fluorescence difference in-gel electrophoresis, followed by mass spectrometry on the cerebrospinal fluid of these patients and patients with other neurological diseases. Vitamin D-binding protein (DBP), gelsolin, albumin, etc. showed more than a 1.5-fold difference between the two groups. Based on these results, an experimental allergic encephalomyelitis (EAE) model of multiple sclerosis in Lewis rats was used to investigate DBP's role in the disease. Protein levels, mRNA transcripts, and ligands of DBP in different regions of the CNS were evaluated under various vitamin D intake levels. Here, DBP levels increased in the experimental rat groups compared to the control groups regardless of vitamin D intake. Moreover, DBP mRNA levels varied in different parts of the CNS including spinal cords in the experimental groups. The observed differences between DBP protein and mRNA levels in the experimental groups' spinal cords could be derived from the disruption of the blood-brain barrier. Furthermore, an interaction between DBP and actin was confirmed using coimmunoprecipitation and western blot. These results indicate a role for DBP in the actin scavenge system. Moreover, in the experimental group that received oral vitamin D3 supplement, we observed both delayed onset and diminished severity of the disease. When DBP was upregulated, however, the benefits from the vitamin D3 supplements were lost. Thus, we inferred that high levels of DBP were adverse to recovery. In conclusion, here we observed upregulated DBP in the cerebrospinal fluid could serve as a specific diagnostic biomarker for the progression of multiple sclerosis. Next, we demonstrate the vital function of increased levels of free vitamin D metabolites for multiple sclerosis treatment. Finally, vitamin D supplements may be particularly beneficial for SPMS patients.

TRAIL suppresses tumor growth in mice by inducing tumor-infiltrating CD4(+)CD25 (+) Treg apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a promising and novel anticancer cytokine, specifically kills numerous tumor cells by apoptosis. However, some malignancies are resistant to TRAIL treatment in clinical trials, thus limiting its therapeutic potential. In the present study, the TRAIL-resistant murine hepatocellular carcinoma cell line Hepa1-6 was used to elucidate the physiological significance of TRAIL resistance, especially with respect to the immune regulatory function of TRAIL. Hepa1-6 cells were resistant to TRAIL-induced apoptosis in vitro; however, intratumoral injection of recombinant soluble TRAIL inhibited tumor growth and prolonged survival time in tumor-bearing mice. Local TRAIL treatment decreased the number of intratumoral CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) but did not affect CD4(+)CD25(+)Foxp3(+) Tregs in the draining lymph nodes and spleen. Further investigation showed that TRAIL induced apoptosis of tumor-activated CD4(+)CD25(+)Foxp3(+) Tregs, but not of CD4(+)CD25(-) T cells. Moreover, mouse TRAIL receptor DR5 expression was detected on the surface of the tumor-infiltrating CD4(+)CD25(+)Foxp3(+) Tregs, but not on naïve CD4(+)CD25(+)Foxp3(+) Tregs. Interestingly, intratumoral injection of TRAIL not only decreased the number of CD4(+)CD25(+)Foxp3(+) Tregs but also increased the number of tumor-specific CD8(+) CTL and augmented their cytotoxicity to the tumor cells. These data provide the novel evidence for an immune regulatory function of TRAIL and may shed light on the clinical application of TRAIL.

[Molecular mechanism of hydroxyurea enhances K562 cell apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand].

OBJECTIVE: To explore the molecular mechanism via which the chemotherapeutic drug hydroxyurea (HU) enhances K562 cell apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). METHODS: Chronic myelogenous leukemia-derived K562 and SVT-35 cells were treated with recombinant soluble TRAIL (rsTRAIL) alone or combined with HU for a time course, and the cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-4-sulfophenyl-2H-tetrazolium-phenazine methosulphate assay. Western blot was performed to analyze the activation of apoptosis-related protein kinases and the expression of apoptosis inhibitor molecules. RESULTS: The survival rates of SVT-35 and K562 cells treated with 1 µg/ml rsTRAIL for 24 hours were 32% and 93%, respectively. HU significantly increased the sensitivity of K562 cells to rsTRAIL cytotoxicity. Combination of rsTRAIL and HU resulted in the phosphorylation of rat sarcoma (RAS), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase and in the significant reduction of apoptosis-inhibited molecule Fas associated death domain protein-like interleukin-1 beta-convening enzyme inhibitory protein and cellular inhibitor of apoptosis protein-1 in K562 cells. CONCLUSIONS: HU enhanced K562 cell sensitivity to rsTRAIL is mediated by Ras-MEK-ERK signaling pathway. Expression of antiapoptotic proteins cellular Fas associated death domain protein-like interleukin-1 beta-convening enzyme inhibitory protein and cellular inhibitor of apoptosis protein-1 is also down-regulated during this process. These results may through light on the therapeutic study of human chronic myelogenous leukemia.

A novel anti-DR5 chimeric antibody and epirubicin synergistically suppress tumor growth.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of tumor cells. TRAIL receptor 2 (DR5) expression is high in tumor cells, transformed cells, and clinical tumor specimens and is low in most normal cells and tissues; therefore, DR5 is considered an attractive target for cancer therapy. In this study, HMCAZ5, a novel mouse-human chimeric antibody based on AD5-10, was generated and stably expressed in CHO-dhfr(-) cells. Highly purified HMCAZ5 exhibits a high affinity for the receptor that is equal to the parental mouse antibody, induces apoptosis in various cancer cells but not in normal hepatocytes, and elicits both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in various human cancer cells. The anthracycline anticancer drug epirubicin (EPB) synergizes the cytotoxicity of HMCAZ5 in cancer cells by upregulating DR5 expression on the cell surfaces, enhancing p53 expression, Bid cleavage, and JNK phosphorylation and downregulating c-FLIP expression and Akt phosphorylation. Moreover, HMCAZ5 alone suppresses tumor growth, and EPB augments the tumoricidal activity in human colorectal and hepatocellular tumor xenografts in athymic nude mice. These data suggest that the anti-DR5 chimeric antibody HMCAZ5 may have a clinical use and represents a useful immunological strategy, in combination with chemotherapy, for the treatment of cancer.

Dysregulated expression of miR-146a contributes to age-related dysfunction of macrophages.

Age-associated immune dysfunction, characterized by increased systemic levels of cytokines, manifests as an increased susceptibility to infections. Thus, understanding these negative regulators of the immune response has paved the way to delineating signaling pathways that impact immune senescence. In the present study, we found that miR-146a, which negatively regulated the expression of IL-1ß and IL-6, was highly expressed in aged mice. However, there was a lack of response to the stimulation of lipopolysaccharide (LPS) and proinflammatory cytokines in macrophages of aged mice. As a result, the negative feedback regulation loop with miR-146a involving down-regulation of inflammation factors was interrupted in aged mice. Aberrant NF-κB binding to the miR-146a promoter was demonstrated to be associated with the abnormal expression of miR-146a in aged mice. The DNA methyltransferase inhibitor (5-aza-2-deoxycytidine) and the histone deacetylase inhibitor [trichostatin A (TSA)] both significantly up-regulated miR-146a transcriptional activation by altering the DNA-binding activity of NF-κB in macrophages isolated from aged mice, which suggests that DNA methylation and histone acetylation are involved in the suppression of age-dependent miR-146a expression. Additionally, high levels of histone deacetylase (HDACs) expressions contributed to the inhibition of miR-146a expression in LPS-stimulated macrophages from aged mice in vitro. While the suppression of HDACs activities by TSA could improve LPS-induced inflammatory responses owing to up-regulation of miR-146a expression in macrophages from aged mice. These data indicate that the dysregulated expression of miR-146a results in the age-associated dysfunction of macrophages, and miR-146a may be a good target for the treatment of age-related inflammatory diseases.

2A peptide-based, lentivirus-mediated anti-death receptor 5 chimeric antibody expression prevents tumor growth in nude mice.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces tumor cell death via death receptors on target cells, without adverse effects on most normal cells. Its receptors are therefore an attractive target for antibody-mediated tumor therapy. Here, we report the creation of a lentivirus vector constructed by linking the heavy chain and the light chain of the antibody with a 2A/furin self-processing peptide in a single open reading frame that expresses a novel chimeric antibody (named as zaptuximab) with tumoricidal activity, which is consisted of the variable region of a mouse anti-human DR5 monoclonal antibody, AD5-10, and the constant region of human immunoglobulin G1. Lentivirus-expressed zaptuximab bound specifically to its antigen, DR5, and exhibited significant apoptosis-inducing activity in various tumor cell lines. The packaged recombinant virus lenti-HF2AL showed strong apoptosis-inducing activity in vitro. Meanwhile, inoculated subcutaneous human colon HCT116 tumor formation in nude mice were inhibited significantly. Moreover, there was a synergistic effect of mitomycin C (MMC) on the observed tumoricidal efficacy, prolonging the life span of nude mice with orthotopic human lung tumor cancers. These data suggest that lentivirus-mediated, 2A peptide-based anti-DR5 chimeric antibody expression may have clinical utility as an anticancer treatment and may represent a rational adjuvant therapy in combination with chemotherapy.

[Construction and stable expression of anti-human tumor necrosis factor-related apoptosis-inducing ligand receptor 2 chimeric antibody in CHO cells].

AIM: To establish an human-mouse chimeric antibody against tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2 (death receptor 5, DR5) in an eukaryotic cell line and analyse its tumoricidal activity. METHODS: The cDNAs encoding for the variable regions of heavy chain (V(H);) and light chain (V(L);) of AD5-10 were amplified by PCR and inserted into the human IgG heavy and light chain containing expression vector RpCI-neo, respectively. The recombinant plasmids were co-transfected into HEK293 and/or CHO cells. The production of anti-DR5 human-mouse chimeric antibody (hmAD5-10) and the antibody affinity for DR5 were identified by ELISA and Western blot assay. The tumoricidal activity of hmAD5-10 was demonstrated by MTS assay. The stable expression cells were selected and cultured in serum-free medium. RESULTS: Two stable CHO cells CHO-A5 and CHO-B11 with the chimeric antibody hmAD5-10 expression were established, in which the production of hmAD5-10 were reached at (0.36±0.11) mg/L and (0.16±0.01) mg/L, respectively. The hmAD5-10 secreted from the cells can well bind with DR5 and kill the cultured leukemia SVT35 cells by apoptosis remarkably. CONCLUSION: The human-mouse chimeric antibody hmAD5-10 was successfully expressed in the eukaryotic cells and resulted tumor cell death by apoptosis. This study lays a fundamental basis for the potential application of the recombinant chimeric antibody in cancer therapy.

HIV infection enhances TRAIL-induced cell death in macrophage by down-regulating decoy receptor expression and generation of reactive oxygen species.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) could induce apoptosis of HIV-1-infected monocyte-derived macrophage (MDM), but the molecular mechanisms are not well understood. METHODOLOGY/PRINCIPAL FINDINGS: By using an HIV-1 Env-pseudotyped virus (HIV-1 PV)-infected MDM cell model we demonstrate that HIV-1 PV infection down-regulates the expression of TRAIL decoy receptor 1 (DcR1) and 2 (DcR2), and cellular FLICE-inhibitory protein (c-FLIP), but dose not affect the expression of death receptor 4 and 5 (DR4, DR5), and Bcl-2 family members in MDM cells. Furthermore, recombinant soluble TRAIL and an agonistic anti-DR5 antibody, AD5-10, treatment stimulates reactive oxygen species (ROS) generation and JNK phosphorylation. CONCLUSIONS/SIGNIFICANCE: HIV infection facilitates TRIAL-induced cell death in MDM by down-regulating the expression of TRAIL decoy receptors and intracellular c-FLIP. Meanwhile, the agonistic anti-DR5 antibody, AD5-10, induces apoptosis synergistically with TRAIL in HIV-1-infected cells. ROS generation and JNK phosphorylation are involved in this process. These findings potentiate clinical usage of the combination of TRAIL and AD5-10 in eradication of HIV-infected macrophage and AIDS.