Enhanced indigenous consortia for the remediation of uranium-contaminated groundwater by bioaugmentation: Reducing and phosphate-solubilizing consortia.

To investigate the strengthening effects and mechanisms of bioaugmentation on the microbial remediation of uranium-contaminated groundwater via bioreduction coupled to biomineralization, two exogenous microbial consortia with reducing and phosphate-solubilizing functions were screened and added to uranium-contaminated groundwater as the experimental groups (group B, reducing consortium added; group C, phosphate-solubilizing consortium added). ß-glycerophosphate (GP) was selected to stimulate the microbial community as the sole electron donor and phosphorus source. The results showed that bioaugmentation accelerated the consumption of GP and the proliferation of key functional microbes in groups B and C. In group B, Dysgonomonas, Clostridium_sensu_stricto_11 and Clostridium_sensu_stricto_13 were the main reducing bacteria, and Paenibacillus was the main phosphate-solubilizing bacteria. In group C, the microorganisms that solubilized phosphate were mainly unclassified_f_Enterobacteriaceae. Additionally, bioaugmentation promoted the formation of unattached precipitates and alleviated the inhibitory effect of cell surface precipitation on microbial metabolism. As a result, the formation rate of U-phosphate precipitates and the removal rates of aqueous U(VI) in both groups B and C were elevated significantly after bioaugmentation. The U(VI) removal rate was poor in the control group (group A, with only an indigenous consortium). Propionispora, Sporomusa and Clostridium_sensu_stricto_11 may have played an important role in the removal of uranium in group A. Furthermore, the addition of a reducing consortium promoted the reduction of U(VI) to U(IV), and immobilized uranium existed in the form of U(IV)-phosphate and U(VI)-phosphate precipitates in group B. In contrast, U was present mainly as U(VI)-phosphate precipitates in groups A and C. Overall, bioaugmentation with an exogenous consortium resulted in the rapid removal of uranium from groundwater and the formation of U-phosphate minerals and served as an effective strategy for improving the treatment of uranium-contaminated groundwater in situ.

Combination immunotherapy of glioblastoma with dendritic cell cancer vaccines, anti-PD-1 and poly I:C.

Glioblastoma (GBM) is a lethal cancer with limited therapeutic options. Dendritic cell (DC)-based cancer vaccines provide a promising approach for GBM treatment. Clinical studies suggest that other immunotherapeutic agents may be combined with DC vaccines to further enhance antitumor activity. Here, we report a GBM case with combination immunotherapy consisting of DC vaccines, anti-programmed death-1 (anti-PD-1) and poly I:C as well as the chemotherapeutic agent cyclophosphamide that was integrated with standard chemoradiation therapy, and the patient remained disease-free for 69 months. The patient received DC vaccines loaded with multiple forms of tumor antigens, including mRNA-tumor associated antigens (TAA), mRNA-neoantigens, and hypochlorous acid (HOCl)-oxidized tumor lysates. Furthermore, mRNA-TAAs were modified with a novel TriVac technology that fuses TAAs with a destabilization domain and inserts TAAs into full-length lysosomal associated membrane protein-1 to enhance major histocompatibility complex (MHC) class I and II antigen presentation. The treatment consisted of 42 DC cancer vaccine infusions, 26 anti-PD-1 antibody nivolumab administrations and 126 poly I:C injections for DC infusions. The patient also received 28 doses of cyclophosphamide for depletion of regulatory T cells. No immunotherapy-related adverse events were observed during the treatment. Robust antitumor CD4+ and CD8+ T-cell responses were detected. The patient remains free of disease progression. This is the first case report on the combination of the above three agents to treat glioblastoma patients. Our results suggest that integrated combination immunotherapy is safe and feasible for long-term treatment in this patient. A large-scale trial to validate these findings is warranted.

Clinically approved combination immunotherapy: Current status, limitations, and future perspective.

Immune-checkpoint inhibitor-based combination immunotherapy has become a first-line treatment for several major types of cancer including hepatocellular carcinoma (HCC), renal cell carcinoma, lung cancer, cervical cancer, and gastric cancer. Combination immunotherapy counters several immunosuppressive elements in the tumor microenvironment and activates multiple steps of the cancer-immunity cycle. The anti-PD-L1 antibody, atezolizumab, plus the anti-vascular endothelial growth factor antibody, bevacizumab, represents a promising class of combination immunotherapy. This combination has produced unprecedented clinical efficacy in unresectable HCC and become a landmark in HCC therapy. Advanced HCC patients treated with atezolizumab plus bevacizumab demonstrated impressive improvements in multiple clinical endpoints including overall survival, progress-free survival, objective response rate, and patient-reported quality of life when compared to current first-line treatment with sorafenib. However, atezolizumab plus bevacizumab first-line therapy has limitations. First, cancer patients falling into the criteria for the combination therapy may need to be further selected to reap benefits while avoiding some potential pitfalls. Second, the treatment regimen of atezolizumab plus bevacizumab at a fixed dose may require adjustment for optimal normalization of the tumor microenvironment to obtain maximum efficacy and reduce adverse events. Third, utilization of predictive biomarkers is urgently needed to guide the entire treatment process. Here we review the current status of clinically approved combination immunotherapies and the underlying immune mechanisms. We further provide a perspective analysis of the limitations for combination immunotherapies and potential approaches to overcome the limitations.

Antibody response and therapy in COVID-19 patients: what can be learned for vaccine development?

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions of people and caused tremendous morbidity and mortality worldwide. Effective treatment for coronavirus disease 2019 (COVID-19) due to SARS-CoV-2 infection is lacking, and different therapeutic strategies are under testing. Host humoral and cellular immunity to SARS-CoV-2 infection is a critical determinant for patients' outcomes. SARS-CoV-2 infection results in seroconversion and production of anti-SARS-CoV-2 antibodies. The antibodies may suppress viral replication through neutralization but might also participate in COVID-19 pathogenesis through a process termed antibody-dependent enhancement. Rapid progress has been made in the research of antibody response and therapy in COVID-19 patients, including characterization of the clinical features of antibody responses in different populations infected by SARS-CoV-2, treatment of COVID-19 patients with convalescent plasma and intravenous immunoglobin products, isolation and characterization of a large panel of monoclonal neutralizing antibodies and early clinical testing, as well as clinical results from several COVID-19 vaccine candidates. In this review, we summarize the recent progress and discuss the implications of these findings in vaccine development.

Preventing Mortality in COVID-19 Patients: Which Cytokine to Target in a Raging Storm?

Coronavirus disease 2019 (COVID-19) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in tremendous morbidity and mortality worldwide. A major underlying cause of COVID-19 mortality is a hyperinflammatory cytokine storm in severe/critically ill patients. Although many clinical trials are testing the efficacy of targeting inflammatory cytokines/chemokines in COVID-19 patients, the critical inflammatory mediator initiating COVID-19 patient death is undefined. Here we suggest that the immunopathological pathway leading to COVID-19 mortality can be divided into three stages with distinct clinical features that can be used to guide therapeutic strategies. Our interpretation of the recently published clinical trials from COVID-19 patients suggests that the clinical efficacy in preventing COVID-19 mortality using IL-1 blockade is subjected to notable caveats, while that for IL-6 blockade is suboptimal. We discuss critical factors in determining appropriate inflammatory cytokine/chemokine targets, timing, and combination of treatments to prevent COVID-19 mortality.

Enhanced Human T Lymphocyte Antigen Priming by Cytokine-Matured Dendritic Cells Overexpressing Bcl-2 and IL-12.

Dendritic cell (DC)-based vaccination is a promising immunotherapeutic strategy for cancer. However, clinical trials have shown only limited efficacy, suggesting the need to optimize protocols for human DC vaccine preparation. In this study, we systemically compared five different human DC vaccine maturation protocols used in clinical trials: (1) a four-cytokine cocktail (TNF-α, IL-6, IL-1ß, and PGE2); (2) an α-DC-cytokine cocktail (TNF-α, IL-1ß, IFN-α, IFN-γ, and poly I:C); (3) lipopolysaccharide (LPS)/IFN-γ; (4) TNF-α and PGE2; and (5) TriMix (mRNAs encoding CD40L, CD70, and constitutively active Toll-like receptor 4 electroporated into immature DCs). We found that the four-cytokine cocktail induced high levels of costimulatory and HLA molecules, as well as CCR7, in DCs. Mature DCs (mDCs) matured with the four-cytokine cocktail had higher viability than those obtained with the other protocols. Based on these features, we chose the four-cytokine cocktail protocol to further improve the immunizing capability of DCs by introducing exogenous genes. We showed that introducing exogenous Bcl-2 increased DC survival. Furthermore, introducing IL-12p70 rescued the inhibition of IL-12 secretion by PGE2 without impairing the DC phenotype. Introducing both Bcl-2 and IL-12p70 mRNAs into DCs induced enhanced cytomegalovirus pp65-specific CD8+ T cells secreting IFN-γ and TNF-α. Taken together, our data suggest that DC matured by the four-cytokine cocktail combined with exogenous Bcl-2 and IL-12p70 gene expression represents a promising approach for clinical applications in cancer immunotherapy.

Tumor-associated antigen-based personalized dendritic cell vaccine in solid tumor patients.

Tumor-associated antigens (TAAs) have been tested in various clinical trials in cancer treatment but the patterns of specific T cell response to personalized TAA immunization remains to be fully understood. We report antigen-specific T cell responses in patients immunized with dendritic cell vaccines pulsed with personalized TAA panels. Tumor samples from patients were first analyzed to identify overexpressed TAAs. Autologous DCs were then transfected with pre-manufactured mRNAs encoding the full-length TAAs, overexpressed in the patients' tumors. Patients with glioblastoma multiforme (GBM) or advanced lung cancer received DC vaccines transfected with personalized TAA panels, in combination with low-dose cyclophosphamide, poly I:C, imiquimod and anti-PD-1 antibody. Antigen-specific T cell responses were measured. Safety and efficacy were evaluated. A total of ten patients were treated with DC vaccines transfected with personalized TAA panels containing 3-13 different TAAs. Among the seven patients tested for anti-TAA T cell responses, most of the TAAs induced antigen-specific CD4+ and/or CD8+ T cell responses, regardless of their expression levels in the tumor tissues. No Grade III/IV adverse events were observed among these patients. Furthermore, the treated patients were associated with favorable overall survival when compared to patients who received standard treatment in the same institution. Personalized TAA immunization-induced-specific CD4+ and CD8+ T cell responses without obvious autoimmune adverse events and was associated with favorable overall survival. These results support further studies on DC immunization with personalized TAA panels for combined immunotherapeutic regimens in solid tumor patients.Trial registration ClinicalTrials.gov, NCT02709616 (March, 2016), NCT02808364 (June 2016), NCT02808416 (June, 2016).

Establishing a Cell-Based High-Content Screening Assay for TCM Compounds with Anti-Renal Fibrosis Effects.

Renal fibrosis is thought to be the final common pathway leading to chronic kidney disease (CKD) and end-stage renal failure. Except for renal replacement therapy, no adequate treatment regimen is available; therefore studies on the treatment of renal fibrosis have attracted significant interest. In recent years, studies have shown that traditional Chinese medicine (TCM) may represent an attractive source to produce drugs with antifibrosis effects. The aim of this study was to establish a robust cell-based high-content screening (HCS) approach to identify TCM compounds with antifibrosis effects in NRK49F cells following TGF-ß1 exposure. When designing the model, one of the most important steps involved the stability and reproducibility of this cell-based model. Therefore, we initially optimized the experimental parameters. Then, our HCS model was validated using SB525334, an inhibitor of the TGF-ß1 receptor, and curcumin and emodin, two TCM compounds with well-documented anti-renal fibrosis activity. Subsequently, the proven reliable HCS model was used to screen a standard TCM compound library, which included 344 TCM molecules. Based on our HCS algorithm, a total of 16 compounds were identified to have prospective inhibitory activity. These compounds were further validated by verification experiments. Strikingly, eight compounds have been shown to inhibit renal fibrosis; six of them had rarely been described in the literature, namely, Ligustrazine, Glycyrrhizic acid, Astragaloside iv, Hydroxysafflor Yellow A, Crocin, and Gypenosides. To the best of our knowledge, this is the first study in which a HCS assay was performed to identify TCM compounds with anti-renal fibrosis effects. The HCS approach was successfully applied to screen active compounds and will be propitious to further anti-renal fibrosis drugs discovery research. Meanwhile, it may offer possibilities for identifying lead compounds for treating other diseases from registered Chinese herbal medicines.

DanHong injection targets endothelin receptor type B and angiotensin II receptor type 1 in protection against cardiac hypertrophy.

Cardiac hypertrophy (CH) is an independent risk factor for cardiovascular diseases (CVDs). Mitigating or preventing CH is the most effective strategy for the treatment of CVDs. DanHong injection (DH) is a Chinese herbal medicine preparation (CHMP) widely used in clinical treatment of several CVDs in China. However, the direct targets and cellular mechanisms for these protective effects remain unclear. This study was designed to illustrate the direct targets of DH in protecting against CH and investigate CH molecular pathogenesis. A hypertrophic cell model was induced by endothelin-1 (ET-1) on human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). Real time cellular analysis (RTCA) cardio system and high content analysis (HCA) were used to detect the changes in contractile function, morphology and protein level of hypertrophic hiPS-CMs. Agonist and antagonist assay on receptors were performed using calcium mobilization high-throughput screening (HTS). DH significantly attenuated CH by modulating myocardial contractility, suppressing cell area enlargement and down-regulating ET-1-induced brain natriuretic peptide (BNP), actinin alpha 2 (ACTN2) and cardiac muscle troponin T (TNNT2) protein expression (P < 0.05). Endothelin receptor type B (ETBR) and angiotensin II receptor type 1 (AT1R) were DH direct targets, with IC50 value of 25.67 µL/mL and 1.10 µL/mL, respectively. Proteomics analysis showed that proteins involved in cell cycle inhibition, RNA processing, mitochondrial translation and cytoskeleton are significant regulated by DH treatment. These data revealed that ETBR and AT1R are DH direct targets on protecting against CH, providing a strategy to explore direct targets of CHMPs.

New monoterpenoid oxindole alkaloid derivatives from the stems of Uncaria hirsuta Havil. and their cytotoxicity and tandem mass spectrometric fragmentation.

Four new alkaloids, comprising three 3-oxo-3,7-seco-oxindole alkaloids (hirsutanine D-F, 1-3) and one oxindole alkaloid N-oxide (uncarine B N-oxide, 4), together with four known heteroyohimbine-type oxindole alkaloids, were isolated from the stems of Uncaria hirsuta Havil. Structures of 1-4 were elucidated by extensive NMR and HR-ESIMS data analyses. Compound 3 is the first 3-oxo-3,7-seco-oxindole alkaloid with ring B opened and degraded isolated from the Uncaria genus. Compounds 1-3 exhibited slight inhibition effect on the proliferation of the breast cancer cell MDA-MB-231. The positive mode collision-induced dissociation of the 3-oxo-3,7-seco-oxindole alkaloids (1-3) was featured by the ß-cleavage and α-cleavage of the amido bond, while the N-oxide (4) showed characteristic neutral eliminations of ·OH and H2O.

Autocrine Complement Inhibits IL10-Dependent T-cell-Mediated Antitumor Immunity to Promote Tumor Progression.

UNLABELLED: In contrast to its inhibitory effects on many cells, IL10 activates CD8(+) tumor-infiltrating lymphocytes (TIL) and enhances their antitumor activity. However, CD8(+) TILs do not routinely express IL10, as autocrine complement C3 inhibits IL10 production through complement receptors C3aR and C5aR. CD8(+) TILs from C3-deficient mice, however, express IL10 and exhibit enhanced effector function. C3-deficient mice are resistant to tumor development in a T-cell- and IL10-dependent manner; human TILs expanded with IL2 plus IL10 increase the killing of primary tumors in vitro compared with IL2-treated TILs. Complement-mediated inhibition of antitumor immunity is independent of the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) immune checkpoint pathway. Our findings suggest that complement receptors C3aR and C5aR expressed on CD8(+) TILs represent a novel class of immune checkpoints that could be targeted for tumor immunotherapy. Moreover, incorporation of IL10 in the expansion of TILs and in gene-engineered T cells for adoptive cell therapy enhances their antitumor efficacy. SIGNIFICANCE: Our data suggest novel strategies to enhance immunotherapies: a combined blockade of complement signaling by antagonists to C3aR, C5aR, and anti-PD-1 to enhance anti-PD-1 efficacy; a targeted IL10 delivery to CD8(+) TILs using anti-PD-1-IL10 or anti-CTLA4-IL10 fusion proteins; and the addition of IL10 in TIL expansion for adoptive cellular therapy. Cancer Discov; 6(9); 1022-35. ©2016 AACR.See related commentary by Peng et al., p. 953This article is highlighted in the In This Issue feature, p. 932.

A high-throughput screening system for G-protein-coupled receptors using ß-lactamase enzyme complementation technology.

AIM: to establish a system for monitoring the activation of G-protein-coupled receptors (GPCRs) using ß-lactamase enzyme fragment complementation (EFC) technology. METHODS: two inactive ß-lactamase deletion fragments, bla(a) and bla(b), were fused to ß-arrestin and GPCR, respectively. A stable cell line named HEK/293-ß2a2, which expressed two fusion proteins, GPCR/bla(b) and ß-arrestin2/bla(a), was generated under antibiotic selection. A natural compound library of high performance liquid chromatography (HPLC)-fractionated samples from the ethanol extracts of Chinese medicinal herbs was used for high-throughput screening (HTS) of ß2-adrenoceptor (ß2AR) agonists against the cell line HEK/293-ß2a2. The interested hits were validated by the measurement of second-messenger cyclic adenosine monophosphate (cAMP) production. RESULTS: the stable cell line HEK/293-ß2a2 responded to ß2AR agonist/antagonist in a dose-dependent manner. The EC(50) value obtained for isoproterenol was 15.5 nmol/L, and the IC(50) value obtained for propranolol was 51.9 nmol/L. Furthermore, HTS was performed to identify ß2AR agonists from the natural compound library we established. The Z' factor value was determined to be 0.68. Three hits were identified from primary screening and found to be as potent as isoproterenol in a camp assay. CONCLUSION: a cell-based high-throughput functional assay was established to directly monitor the activation of GPCRs based on the interaction between agonist-activated GPCR and ß-arrestin using ß-lactamase EFC technology, which can be used to search for leads in the natural compound library.

A system for screening agonists targeting beta2-adrenoceptor from Chinese medicinal herbs.

In order to develop a model for screening the agonists of human beta(2)-adrenoceptor from Chinese medicinal herbs extracts, we used a cell-based functional assay based on a common G protein-coupled receptor (GPCR) regulation mechanism and destabilized enhanced green fluorescent protein (d(2)EGFP) reporter gene technique. The positive cell clone was confirmed by real-time polymerase chain reaction (PCR) and imaging analysis. To assess the value of this model, we screened over 2000 high performance liquid chromatography (HPLC)-fractionated samples from the ethanol extracts of Chinese medicinal herbs. Six fractions (isolated from Panax japonicus, Veratrum nigrum, Phellodendron amurense, Fructus Aurantii Immaturus, Chaenomeles speciosa, and Dictamnus dasycarpus) showed significant effects on active reporter gene expression, three of which (isolated from Phellodendron amurense, Fructus Aurantii Immaturus, and Chaenomeles speciosa) were selected for further concentration response analysis and the half maximal effective concentration (EC(1/2 max)) values were 4.2, 2.7, and 4.8 microg/ml, respectively. Therefore, this reporter gene assay was suitable for screening beta(2)-adrenoceptor agonists. The results suggest that the six herbal extracts are the possible agonists of beta(2)-adrenoceptor.

Identification of natural compounds with antiviral activities against SARS-associated coronavirus.

More than 200 Chinese medicinal herb extracts were screened for antiviral activities against Severe Acute Respiratory Syndrome-associated coronavirus (SARS-CoV) using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay for virus-induced cytopathic effect (CPE). Four of these extracts showed moderate to potent antiviral activities against SARS-CoV with 50% effective concentration (EC50) ranging from 2.4 +/- 0.2 to 88.2 +/- 7.7 microg/ml. Out of the four, Lycoris radiata was most potent. To identify the active component, L. radiata extract was subjected to further fractionation, purification, and CPE/MTS assays. This process led to the identification of a single substance lycorine as an anti-SARS-CoV component with an EC50 value of 15.7 +/- 1.2 nM. This compound has a CC50 value of 14980.0 +/- 912.0 nM in cytotoxicity assay and a selective index (SI) greater than 900. The results suggested that four herbal extracts and the compound lycorine are candidates for the development of new anti-SARS-CoV drugs in the treatment of SARS.

[Effects of one Chinese herbs on improving cognitive function and memory of Alzheimer's disease mouse models].

OBJECTIVE: To investigate Chinese herbs with the effect on significantly improving the cognitive function and retention of patients with Alzheimer's disease. METHOD: AD mouse models were established by injecting poly-beta-AP25-35 into the cerebral ventricle of mice. The curative effects of traditional Chinese herbs Dangguishaoyaosan, Chaihujialonggumulitang and two herbs (CHP I and CHP II) developed by the authors on improving the memory and cognitive function of AD mouse models were studied by the detection of behavioral and histochemical tests, with piracetam serving as control. RESULT: CHP II has profound curative effects on improving the memory and cognitive function of AD-like animal model. CONCLUSION: The present study indicates that it has a satisfactory prospect to seek new drugs from Chinese herbs to treat AD.