Immune Stimulatory Properties Research Articles

THU-204 - Nucleic Acid-Based Polymers Effective against Hepatitis B Virus Infection in Patients Do Not Harbour Immune Stimulatory Properties in Primary Isolated Blood or Liver Cells

THU-204 - Nucleic Acid-Based Polymers Effective against Hepatitis B Virus Infection in Patients Do Not Harbour Immune Stimulatory Properties in Primary Isolated Blood or Liver Cells

Amphipathic, nucleic acid-based polymers optimized to treat hepatitis B virus infection in patients do not harbour immune stimulatory properties in primary isolated blood or liver cells

Amphipathic, nucleic acid-based polymers optimized to treat hepatitis B virus infection in patients do not harbour immune stimulatory properties in primary isolated blood or liver cells

Foxo 3a Signaling Mediates Apoptosis in Lymphoma Cells By the Diterpenoid Lactone Andrographolide (Andro), the Active Component of Andrographis Paniculata

Introduction: Andrographolide is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an herbal medicine used in Asia. It has been reported to have anti-inflammatory, antihypertensive, antiviral, and immune-stimulant properties. Furthermore, it has been shown to inhibit cancer cell proliferation and induce apoptosis in lymphoma, leukemia and other solid tumor cell lines. We have shown that Andro caused ROS-dependent apoptosis in lymphoma cell lines and in primary tumor samples that was mediated through mitochondrial pathways and enhanced by depletion of GSH and inhibited by NAC or the pan-caspase inhibitor Z-VAD-FMK (Yang et al Clin Cancer Res 2010; 16(19):4755). We hypothesized that the tumor suppressor, FOXO3a may be involved in signaling pathways that lead to apoptosis and to test that hypothesis we investigated the role of FOXO3A in Andro induced signaling in lymphoma.Methods: We studied the Burkitt p53-mutated Ramos cell line, the mantle cell lymphoma (MCL) line Granta, the transformed follicular lymphoma (FL) cell line HF-1, and the diffuse large B-cell lymphoma (DLBCL) cell line SUDHL4, as well as primary cells from patients with FL and MCL. We transfected shRNA FOXO3a by electroporation to build stable cells with constant knockdown of FOXO3a in Ramos and SUDHL4 cell lines. We then compared the cell viability (MTT and Golgi fragmentation), apoptosis (Annexin V by flow), c-MYC and Bcl2 expression, death receptors 4 (DR4) expression and cell cycle related proteins in wild type and FOXO3a knockdowns.Results: We found that Andro resulted in nuclear translocation of FOXO3a in Ramos at early time points. We found that shRNA stable knockdown of FOXO3a in Ramos and SUDHL4 cell lines protected cells (Ramos and SUDHL4) from Andro-induced apoptosis (Figure 1). Moreover, in multiple cell lines, we found that Andro decreased c-MYC expression, which was abrogated in part by FOXO3A knockdown compared with wild type cells. Similarly, reduction in mitochondrial membrane potential by Andro is abrogated in the FOXO 3a knockdown cells. These data suggest that FOXO3a regulates c-MYC stabilization by mitochondrial proteins (for example TFAM and MAD-1). In the Granta cell line, derived from Mantle Cell Lymphoma (MCL) and in an MCL patient sample, Andro reduced c-MYC expression. We also found that Andro induced Death Receptor 4 (DR4) at the mRNA and protein level in Granta cells in a dose-dependent manner. The cell cycle control proteins Aurora, p21, p27 (the latter 2 regulated by FOXO3a), are also increased by Andro. When cell death was measured by Golgi fragmentation and subsequent collapse, we found that Andro induced Golgi fragmentation in Granta and SUDHL4 cellsConclusion: Andro-induced lymphoma cell apoptosis is mediated through multiple signaling pathways, including FOXO3a, which appears to play a significant role, perhaps by regulating c-MYC stabilization and BCL2 expression and cell cycle proteins. These data suggest that this novel diterpenoid lactone compound deserves further pre-clinical and clinical testing in malignant lymphoma. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In vitro antibacterial activity of Chilean propolis against Helicobacter pylori

Propolis is a natural product derived from beekeeping. It has anesthetic, anti-inflammatory, immune-stimulant and antibacterial properties on grampositive and gramnegative bacteria. However, little is known regarding its activity on Helicobacter pylori. This bacteria colonizes about half of the world's population and is associated with chronic gastritis, peptic ulcer and gastric cancer. The aim of this study was to evaluate the inhibitory activity of 22 propolis extracts from nine of the 11 beekeeping Chilean regions on 10 strains of H. pylori isolated from gastric mucosa. The antibacterial activity of the extracts was determined using the well diffusion method and diffusion disks. 100% of the extracts were active on the tested strains, showing inhibition halos equal to or greater than 15 mm by both methods. Our results show an effective anti H. pylori activity of propolis. However, additional microbiological studies are needed before a potential clinical utility of these natural products is warranted.

Abstract 2472: SEA-CD40, a sugar engineered non-fucosylated anti-CD40 antibody with improved immune activating capabilities

Abstract SEA-CD40 is a non-fucosylated, humanized IgG1 monoclonal antibody directed against human CD40, a co-stimulatory receptor of the TNF receptor superfamily. SEA-CD40 is derived from dacetuzumab, a humanized IgG1 previously developed and studied for B-lineage malignancies. Glycosylation of the antibody Fc is essential for Fc receptor-mediated activity and non-fucosylated antibodies show improved efficacy, particularly via increased binding to low affinity FcγRIIIa. Enhanced functionality of SEA-CD40 was determined through FcγRIIIa binding affinity, antibody-dependent cellular cytotoxicity (ADCC) activity, activation of the immune response, and induction of antigen-specific T-cells. While SEA-CD40 and the parent antibody dacetuzumab bind to CD40 with similar affinity, the non-fucosylated SEA-CD40 binds equally well to the low (158F) and high (158V) affinity versions of FcγRIIIa with higher affinity than dacetuzumab. The consequence of enhanced SEA-CD40/FcγRIIIa binding is potent ADCC activity against a CD40+ lymphoma B cell line and improved agonistic signaling to antigen presenting cells (APCs). SEA-CD40 treatment of human PBMCs elicits a robust immune response as measured by increased cytokine production and up-regulation of maturation markers on APCs with maintained activity at antibody concentrations as low as 10 ng/ml. The immune stimulatory properties of SEA-CD40 were observed in vivo as increased activity in xenograft and syngenic tumor models as well as induction of cytokine production in cynomolgus monkeys. Both in-vitro and in-vivo activity of SEA-CD40 was significantly greater than with dacetuzumab. The increased functionality of SEA-CD40 occurs through the non-fucosylated Fc domain as a F(ab’)2 version loses the ability to induce ADCC, stimulate cytokines, or up-regulate APC activation markers. SEA-CD40 induction of antigen specific T-cells was assessed using human peripheral blood mononuclear cells (PBMCs) exposed to influenza antigen. In the presence of SEA-CD40 influenza specific T-cells mount a robust antigen-specific response characterized by tetramer staining and elevated production of IFNγ. SEA-CD40 activity on PBMCs from donors with melanoma, pancreatic, or breast cancer was assessed and tumor antigen specific T-cell proliferation and IFNγ production was observed. SEA-CD40 is a non-fucosylated agonistic anti-CD40 antibody that shows enhanced binding to FcγRIIIa resulting in amplified cytokine production, co-stimulatory molecule up regulation, and ultimately stimulation of antigen specific T-cell responses to viral and tumor antigens. Citation Format: Shyra J. Gardai, Angela Epp, Germein Linares, Lori Westendorf, May Sutherland, Haley Neff-LaFord, Stanford L. Peng, Che-Leung Law. SEA-CD40, a sugar engineered non-fucosylated anti-CD40 antibody with improved immune activating capabilities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2472. doi:10.1158/1538-7445.AM2015-2472

Proliferative and Inhibitory Activity of Siberian ginseng (Eleutherococcus senticosus) Extract on Cancer Cell Lines; A-549, XWLC-05, HCT-116, CNE and Beas-2b.

Siberian ginseng (Eleutherococcus senticosus) is used primarily as an adaptogen herb and also for its immune stimulant properties in Western herbal medicine. Another closely related species used in East Asian medicine systems i.e. Kampo, TCM (Manchuria, Korea, Japan and Ainu of Hokkaido) and also called Siberian ginseng (Acanthopanax senticosus) also displays immune-stimulant and anti-cancer properties. These may affect tumour growth and also provide an anti-fatigue effect for cancer patients, in particular for those suffering from lung cancer. There is some evidence that a carbohydrate in Siberian ginseng may possess not only immune stimulatory but also anti-tumour effects and also display other various anti-cancer properties. Our study aimed to determine the inhibitory and also proliferative effects of a methanol plant extract of Siberan ginseng (E. senticosus) on various cancer and normal cell lines including: A-549 (small cell lung cancer), XWLC-05 (Yunnan lung cancer cell line), CNE (human nasopharyngeal carcinoma cell line), HCT-116 (human colon cancer) and Beas-2b (human lung epithelial). These cell lines were treated with an extract from E. senticosus that was evaporated and re- constituted in DMSO. Treatment of A-549 (small cell lung cancer) cells with E. senticosus methanolic extract showed a concentration-dependent inhibitory trend from 12.5 - 50μg/mL, and then a plateau, whereas at 12.5 and 25 μg/mL, there is a slight growth suppression in QBC-939 cells, but then a steady suppression from 50, 100 and 200μg/mL. Further, in XWLC-05 (Yunnan lung cancer cell line), E. senticosus methanolic extract displayed an inhibitory effect which plateaued with increasing dosage. Next, in CNE (human nasopharyngeal carcinoma cell line) there was a dose dependent proliferative response, whereas in Beas-2 (human lung epithelial cell line), an inhibitory effect. Finally in colon cancer cell line (HCT-116) we observed an initially weak inhibitory effect and then plateau.

Extensive in vitro gastrointestinal digestion markedly reduces the immune-toxicity of Triticum monococcum wheat: implication for celiac disease.

The ancient diploid Triticum monococcum is of special interest as a candidate low-toxic wheat species for celiac disease patients. Here, we investigated how an in vitro gastro-intestinal digestion, affected the immune toxic properties of gliadin from diploid compared to hexaploid wheat. Gliadins from Triticum monococcum, and Triticum aestivum cultivars were digested using either a partial proteolysis with pepsin-chymotrypsin, or an extensive degradation that used gastrointestinal enzymes including the brush border membrane enzymes. The immune stimulatory properties of the digested samples were investigated on T-cell lines and jejunal biopsies from celiac disease patients. The T-cell response profile to the Triticum monococcum gliadin was comparable to that obtained with Triticum aestivum gliadin after the partial pepsin-chymotrypsin digestion. In contrast, the extensive gastrointestinal hydrolysis drastically reduced the immune stimulatory properties of Triticum monococcum gliadin. MS-based analysis showed that several Triticum monococcum peptides, including known T-cell epitopes, were degraded during the gastrointestinal treatment, whereas many of Triticum aestivum gliadin survived the gastrointestinal digestion. The pattern of Triticum monococcum gliadin proteins is sufficiently different from those of common hexaploid wheat to determine a lower toxicity in celiac disease patients following in vitro simulation of human digestion.

Immune-stimulating properties of 80% methanolic extract of Ludwigia octovalvis against Shiga toxin-producing E. coli O157:H7 in Balb/c mice following experimental infection

Ethnopharmacological relevanceLudwigia octovalvis is an aquatic plant widely distributed throughout the tropical and sub-tropical regions. It is commonly consumed as a health drink and traditionally used for treating various ailments such as dysentery, diarrhea, diabetes, nephritisn and headache. No information is available on its in vivo antibacterial activity against an important foodborne pathogen, Shiga toxin producing Escherichia coli O157:H7. Materials and methodsMale Balb/c mice were orally administered with the extract at doses of 200 or 400mg/kg body weight for one week before the infection with E. coli O157:H7 and continued for 14 consecutive days after infection. Serum antibody (IgA, IgG and IgM) levels were quantified at days 7 and 14 post-challenge by an ADVIA® 2400 Clinical Chemistry Auto Analyzer. Nitroblue tetrazolium (NBT) and Ceruloplasmin, as nonspecific immune parameters, were determined enzymatically. ResultsA significant increase (p<0.05) in IgA serum level was indicated on the 7th day post-challenge with the pathogen in the experimental group received 400mg/kg of the extract in comparison with other groups. Total IgA serum levels on day 7 post-challenge in groups of PBS negative control, E. coli O157:H7 positive control, E. coli O157:H7+200mg/kg extract group and E. coli O157:H7+400mg/kg extract group were 709.4±149.6, 1655.8±139.7, 1728.6±64.3 and 1971.4±135.6µg/ml, respectively. Serum IgG and IgM did not significantly change among different groups. The extract administered orally to infected Balb/c mice did not affect the NBT as well as ceruloplasmin levels. ConclusionsThe extract of L. octovalvis contains biologically active principles which increased systemic immune response to E. coli O157:H7 via potentiating the synthesis of IgA antibodies.

Evaluation of the presence of reduced nicotinamide adenine dinucleotide phosphate in bacterial metabolites used as immunostimulators and its role in nitric oxide induction.

Bacterial metabolites that act as immunostimulators have aroused interest because of their therapeutic potential in several immune disorders. These metabolites are complex, heterogeneous, and comprise numerous immune-boosting biomolecules. To better understand their immune stimulatory properties, characterization of their components is essential. An ether extract of metabolites from nine bacterial species was analyzed for the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) or other fluorophores. This metabolite in combination with bile lipids is a licensed immune stimulatory drug. Excitation of the extract at 340 nm resulted in fluorescence with an emission maximum of around 410 nm, which is fairly specific for NADH and NADPH. Reverse-phase-HPLC and electro-spray ionization-mass analysis confirmed the presence of NADPH in the bacterial metabolites. Quantification by glutathione reductase assay indicated 11.90 ± 0.01 µM of NADPH in the metabolites. Further characterization of the individual bacterial extracts of the metabolite confirmed the presence of NADPH. Subsequently, studies were performed to evaluate the role/s of NADPH in immune-stimulatory drugs. NADPH is known to be involved in production of nitric oxide (NO), which has versatile roles in the immune system. The biological function of NADPH in NO induction by RAW 264.7 (mouse macrophage) cells was evaluated and it was found that bacterial NADPH has a significant role in inducing NO and that NADPH from individual bacterial extracts is capable of inducing NO. Investigation on the stability and biological potency of NADPH in bacterial metabolites is important because of NADPH's wide therapeutic applications, most of which are associated with its role in NO induction.

439. Oncolytic Newcastle Disease Virus Iraqi Virulent Strain Induce Apoptosis In Vitro through intrinsic Pathway and Association of Both Intrinsic and Extrinsic Pathways In Vivo

Newcastle disease virus is very promising antitumor agent. Iraqi virulent strain showed pronounced oncolytic activity. This study aimed to investigate the specific pathway of apoptosis induced by Iraqi strain of Newcastle disease virus (NDV) on tumor cells In vitro and In vivo. Several cell lines were tested (Human Rhabdomyosarcoma cell line (RD), Human Glioblastoma multiforme culture (AMGM) and Murine mammary adenocarcinoma cell line (AMN3) for ability of Iraqi NDV strain to induce apoptosis and to give deep look about the main pathway that induce apoptosis in tumor cell lines by the virus. The cell lines were infected with Newcastle disease virus and investigated for apoptotic pathway by using immunocytochemistry for caspase 3, 8, and 9. The result of this in vitro experiment revealed that NDV induce apoptosis in tumor cell lines when compared to control group with marked increase in the percentage of cells expressing caspase 9 with low percentage of cells expressing caspase 8 in NDV treated group at 24,48hr. These results indicated the role of NDV Iraqi strain in inducing apoptosis through intrinsic mitochondrial pathway in vitro. The in vivo experiment included female mice transplanted with mammary adenocarcinoma transplantable tumor line (AM3), after tumor growth; tumor bearing mice randomly divided into groups, first group treated by NDV by intratumoral injection, second group treated by NDV intrapretonially. Caspase 3, caspase 8, and caspase 9 expression in tumor sections were checked after 24hrs, 48hrs, 72hrs, 7 and 14 days of treatment with NDV. The results revealed that NDV induce apoptosis significantly in treated tumors by both ways IT and IP when compared with control group. There is significant (p<0.05) marked increase in the mean percentage of cells expressing caspase 9 in NDV treated groups compared with mean percentage of cells expressing caspase 8 and untreated control group at 24,48,72, 1week, 2 weeks post single injection treatment. Moreover, Caspase-8 expression in IT and IP groups was significantly higher than control untreated group. These results revealed that NDV had a powerful effect on inducing mitochondrial (intrinsic) apoptotic pathway in vivo in association of extrinsic pathway. This association can explained by NDV induction for antitumor immune response and this immune response can trigger tumor killing by extrinsic pathway. The current work suggest that Iraqi oncolytic virulent strain of NDV induce apoptosis through intrinsic pathway directly as the results of in vitro study and induce extrinsic pathway indirectly through immune stimulation properties of NDV.

Abstract ES6-1: Immunotherapeutic approaches to breast cancer

Abstract There has been increasing interest in the field of immunooncology with the approval of a therapeutic cancer vaccine for prostate cancer and checkpoint inhibitors targeting CTLA-4 and PDL-1 for the treatment of advanced stage melanoma and lung cancer. These therapies stimulate the “adaptive” immune system. Adaptive immunity requires T-cells and antibodies to directly recognize specific proteins expressed on tumor cells. Recognition of immunogenic cancer associated proteins, known as “antigens”, allows specificity of the immune response to the tumor. Harnessing the immune system to treat cancer has several major advantages over other standard therapies. The T-cells capable of recognizing and killing tumor cells will persist in lymph nodes for years and will be poised to attack antigen expressing tumor when it recurs. T-cells, once primed to react to specific antigens, will traffic out of the circulation and can penetrate into tumor, killing tumor cells as long as they are present. Breast cancer is an immunogenic tumor. In fact, many of the favorable immune characteristics that are present in more classically immunogenic tumors, such as melanoma, are operative in breast cancer. Numerous tumor antigens have been identified in breast cancer. Antibodies directed against tumor antigens are generated very early in the disease process. Breast cancer patients mount immune responses against such proteins as HER-2/neu, IGF-IR, and topoisomerase II alpha to name a few. Further, large numbers of specific types of lymphocytes infiltrating breast cancers have been shown to have prognostic importance in some subtypes of the disease. However, mechanisms of tumor immune escape are also prevalent in breast cancer. Regulatory T-cells which secrete cytokines to prevent the function of cytotoxic T-cells are increased in the tumors of breast cancer patients. High levels of T-regulatory cells indicate poorer outcomes particularly in patients with estrogen receptor positive disease. Type II immune responses, associated with antibody production inhibit the function of Type I T-cells including cytotoxic T-cells needed for tumor eradication. Breast cancers have been shown to up regulate PD-L1 an immune checkpoint protein that prevents T-cells from effectively recognizing and eliminating tumors. A variety of immune therapies are being actively explored for the treatment of breast cancer. However, a more detailed understanding of the breast cancer immune microenvironment must guide how we approach the clinical application of immune therapy. Only a small fraction of patients have robust levels of tumor infiltrating T-cells. Therapeutic approaches aimed at blocking immune checkpoints may be more effective in these patients than in the majority of individuals with little or no pre-existent immunity. Many of the standard therapies that are used in the treatment of breast cancer today have potent immune stimulatory properties. This realization, along with a better understanding of the endogenous immune response in breast cancer, may allow more effective application of combination immunotherapeutic approaches for this disease. Citation Format: Mary L Disis. Immunotherapeutic approaches to breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr ES6-1.

Influenza virus-like particles engineered by protein transfer with tumor-associated antigens induces protective antitumor immunity.

Delivery of antigen in particulate form using either synthetic or natural particles induces stronger immunity than soluble forms of the antigen. Among naturally occurring particles, virus-like particles (VLPs) have been genetically engineered to express tumor-associated antigens (TAAs) and have shown to induce strong TAA-specific immune responses due to their nano-particulate size and ability to bind and activate antigen-presenting cells. In this report, we demonstrate that influenza VLPs can be modified by a protein transfer technology to express TAAs for induction of effective antitumor immune responses. We converted the breast cancer HER-2 antigen to a glycosylphosphatidylinositol (GPI)-anchored form and incorporated GPI-HER-2 onto VLPs by a rapid protein transfer process. Expression levels on VLPs depended on the GPI-HER-2 concentration added during protein transfer. Vaccination of mice with protein transferred GPI-HER-2-VLPs induced a strong Th1 and Th2-type anti-HER-2 antibody response and protected mice against a HER-2-expressing tumor challenge. The Soluble form of GPI-HER-2 induced only a weak Th2 response under similar conditions. These results suggest that influenza VLPs can be enriched with TAAs by protein transfer to develop effective VLP-based subunit vaccines against cancer without chemical or genetic modifications and thus preserve the immune stimulating properties of VLPs for easier production of antigen-specific therapeutic cancer vaccines.

ANTIMICROBIAL ACTIVITY OF CINNAMALDEHYDE, VANILLIN AND KIGELIA AFRICANA FRUIT EXTRACTS AGAINST FISH-ASSOCIATED CHRYSEOBACTERIUM AND MYROIDES SPP. ISOLATES

Background: Members of the family Flavobacteriaceae exhibit intrinsic multi-drug resistance, which hampers their effective eradication. Phytochemicals are being explored as alternatives to the use of antimicrobial agents in aquaculture since they have growth-promoting, immune-stimulating, and antimicrobial properties. &#x0D; Materials and Methods: The susceptibility of 36 Chryseobacterium and seven Myroides spp. isolates from salmon, tilapia and trout as well as 19 selected Flavobacteriaceae type strains to cinnamaldehyde, vanillin and four crude Kigelia africana extracts (ethyl acetate, dichloromethane, methanol and hexane), was assessed using disc diffusion assays and compared to standard antimicrobial agents, ampicillin and tetracycline using activity indices. &#x0D; Results: Cinnamaldehyde (≥250 µg/ml) was the most effective (77. 8 – 100% susceptibility) while vanillin was the least effective with inhibitory activity only at 1000 µg/ml. The K. africana hexane extract (4 mg/ml) was the most effective, with only 11.3% of isolates displaying resistance, while 94.4% of isolates demonstrated resistance to ampicillin and 38.9% susceptibility to tetracycline. K. africana extract inhibitory efficacy decreased in the following order: hexane &gt; ethyl acetate &gt; dichloromethane &gt; methanol. Cinnamaldehyde and K. africana EX 4 activity indices ≥ 1 were obtained for 83.3 - 97.2% and 25% of Chryseobacterium spp. isolates, respectively, relative to tetracycline. &#x0D; Conclusions: Cinnamaldehyde and K. africana fruit hexane extracts are promising candidates to be tested for their efficacy in the treatment of Chryseobacterium/Myroides-associated fish infections. These phytochemicals might serve as environmentally-friendly, cost-effective alternatives to the use of antimicrobial agents in aquaculture farms, with a lesser chance of resistance development.

Inhibition of microglial activation by elderberry extracts and its phenolic components.

Elderberry (Sambucus spp.) is one of the oldest medicinal plants noted for its cardiovascular, anti-inflammatory, and immune-stimulatory properties. In this study, we investigated the anti-inflammatory and anti-oxidant effects of the American elderberry (Sambucus nigra subsp. canadensis) pomace as well as some of the anthocyanins (cyanidin chloride and cyanidin 3-O-glucoside) and flavonols (quercetin and rutin) in bv-2 mouse microglial cells. The bv-2 cells were pretreated with elderberry pomace (extracted with ethanol or ethyl acetate) or its anthocyanins and flavonols and stimulated by either lipopolysaccharide (LPS) or interferon-γ (IFNγ). Reactive oxygen species (ROS) and nitric oxide (NO) production (indicating oxidative stress and inflammatory response) were measured using the ROS detection reagent DCF-DA and the Griess reaction, respectively. Analysis of total monomeric anthocyanin (as cyanidin 3-O-glucoside equivalents) indicated five-fold higher amount in the freeze-dried ethanol extract as compared to that of the oven-dried extract; anthocyanin was not detected in the ethyl acetate extracts. Elderberry ethanol extracts (freeze-dried or oven-dried) showed higher anti-oxidant activities and better ability to inhibit LPS or IFNγ-induced NO production as compared with the ethyl acetate extracts. The phenolic compounds strongly inhibited LPS or IFNγ-induced ROS production, but except for quercetin, they were relatively poor in inhibiting NO production. These results demonstrated differences in anti-oxidative and anti-inflammatory effects of elderberry extracts depending on solvents used. Results further identified quercetin as the most active component in suppressing oxidative stress and inflammatory responses on microglial cells.

Evaluation of immunomodulatory activities of methanolic extract of khat (Catha edulis, Forsk) and cathinone in Swiss albino mice

BackgroundThe objective of this study was to explore the immunomodulatory effect of khat and its active component, cathinone, using mice.Materials and methodsFemale Swiss albino mice aged 7–8 weeks weighing 25-30 g were used in the study. Mice were randomized into eight groups of 6 each and oral treatment of khat crude extract and cathinone were given daily for four weeks. Physical, hematological, biochemical, and immunological parameters were measured. Immunological studies included humeral immunity (IgG and IgM), cellular immune response (delay type hypersensitivity), phagocytic activities of reticuloendothelial system, and determination of T-lymphocyte population: CD3+, CD4+, CD8+ count and CD4+/CD8+ status.ResultsFindings of this study showed that, khat and its major metabolite, cathinone, can positively affect immune system in dose dependent manner. When doses of crude khat extract and cathinone increase, the induction of humeral (IgG and IgM) and cellular immune responses were up-regulated significantly (P < 0.05), while at higher dose of khat (200 mg/kg) cellular immune response was suppressed. In support of this, as doses of the two test substances increased, the count of T helper cells (CD4+) was significantly increased (P < 0.05), while higher dose significantly reduced whole white blood cell (WBC), CD8+, and CD3+ counts.ConclusionAt relatively lower dose (50-100 mg/kg), crude khat extract has immune stimulating property, although higher dose (200 mg/kg) leads to suppression of cellular immune response. Cathinone also share all immune modulating property of its parent compound, khat, but with intense strength. Thus, it calls for further detailed investigation of khat for practical application of the same in human medication.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-015-0072-5) contains supplementary material, which is available to authorized users.

Enhanced immunostimulatory activity of cyclic dinucleotides on mouse cells when complexed with a cell-penetrating peptide or combined with CpG.

Recognition of pathogen-derived nucleic acids by immune cells is critical for the activation of protective innate immune responses. Bacterial cyclic dinucleotides (CDNs) are small nucleic acids that are directly recognized by the cytosolic DNA sensor STING (stimulator of IFN genes), initiating a response characterized by proinflammatory cytokine and type I IFN production. Strategies to improve the immune stimulatory activities of CDNs can further their potential for clinical development. Here, we demonstrate that a simple complex of cylic-di-GMP with a cell-penetrating peptide enhances both cellular delivery and biological activity of the cyclic-di-GMP in murine splenocytes. Furthermore, our findings establish that activation of the TLR-dependent and TLR-independent DNA recognition pathways through combined use of CpG oligonucleotide (ODN) and CDN results in synergistic activity, augmenting cytokine production (IFN-α/β, IL-6, TNF-α, IP-10), costimulatory molecule upregulation (MHC class II, CD86), and antigen-specific humoral and cellular immunity. Results presented herein indicate that 3'3'-cGAMP, a recently identified bacterial CDN, is a superior stimulator of IFN genes ligand than cyclic-di-GMP in human PBMCs. Collectively, these findings suggest that the immune-stimulatory properties of CDNs can be augmented through peptide complexation or synergistic use with CpG oligonucleotide and may be of interest for the development of CDN-based immunotherapeutic agents.

Expression of innate immunity genes in kuruma shrimp Marsupenaeus japonicus after in vivo stimulation with garlic extract (allicin)

In recent times, attention has focused on immunostimulants and plant products which could have beneficial effects in disease control. At present, the application of immunostimulants has been considered a more effective approach to health management in aquaculture through the enhancement of immune capability and disease resistance in shrimp. Garlic possesses bactericidal property against bacteria and can inhibit the growth of protozoa as well as gregarine infection in cultured shrimp. However, its effect on viral disease infection has not been well studied and requires further investigation. Garlic can also stimulate shrimp haemocytes as determined by cellular immune responses (phagocytic activity, superoxide anion production and phenoloxidase activity) suggesting that the immune-stimulatory properties of garlic will be useful for improvement of shrimp health. Here, we determined the expression of the immune-related genes Penaeidin, Crustin, Lysozyme, Toll-like, and tumour necrosis factor in kuruma shrimp, upon stimulation with allicin extract. The expression of these factors was measured for the first time and was found to be elevated in intestine and lymphoid organ after stimulation for 3, 12, 24 and 48 h. We conclude that garlic can be used in shrimp culture as an alternative to antibiotics or chemotherapeutic agents; however, further research is needed under field conditions.

Non-responsiveness to therapeutic tumor vaccination is overcome by functional modulation of myeloid derived suppressor cells

Therapeutic tumor vaccination represents a promising strategy to induce anti-tumor activity in cancer patients. However, usually only a subpopulation of patients benefits from tumor vaccines, whereas others display continuous tumor growth and metastatic spreading. Here we report a similar observation in a B16 melanoma model. Vaccinating these mice with a model tumor antigen and a combination of adjuvants, i.e. TLR-9-ligand CpG and NKT-cell ligand α-galactosylceramide, controlled the tumor in most animals (High-Responders), whereas others showed no clear benefit from the treatment (Non-Responders). No differences in numbers of B, NK or T cells were detected between both groups. However, Non-Responders showed significantly increased numbers of CD11b+Gr-1+myeloid-derived-suppressor-cells (MDSCs). Treatment with all-trans-retinoic-acid (atRA), which has been described to modulate MDSCs, effectively restored the tumor control in Non-responders. Frequencies neither of regulatory T cells nor MDSCs differed after treatment with atRA, but those cells from atRA-treated animals with a phenotype of monocytic MDSCs (CD11b+Ly6ChighLy6G–cells) had lost their suppressive capacity and displayed upregulation of MHC-Class-II, indicating acquisition of immune-stimulatory properties. Concomitantly, IFNγ production by CD8+ T cells was significantly higher in atRA-treated mice.

The molecular structure features-immune stimulatory activity of arabinoxylans derived from the pentosan faction of wheat flour

Recently, the immune stimulation properties of cereal arabinoxylans (AX) have been reported. The aims of this study were to identify the molecular features and potential immune stimulation activities of AX and enzymatic modified arabinoxylan (AXE) from the pentosan fraction of wheat flour.The results of molecular characterization of AX and AXE show that AXE contains a larger portion (85.7%) of low Mw arabinoxylans (≤25 kDa) and has a higher degree of branch substitution (0.81 ± 0.01) compared to AX (49.5% and 0.62 ± 0.02). In in vitro testing, the ability of AX and AXE to stimulate immune cells, as measured by NO2− production by U937 cells and IL-8 secretion by Caco-2 cells were found to be dose–dependent in the range tested (100–5000 μg/mL p ≤ 0.05). AXE showed a greater activity at each concentration (100–5000 μg/mML) than AX (p ≤ 0.05).In conclusion, the greater immune stimulating activity of AXE may be associated with its low Mw (≤25 kDa) and a higher degree of branch substitution (0.81 ± 0.01).

Recognition of Specified RNA Modifications by the Innate Immune System.

Microbial nucleic acids have been described as important activators of human innate immune responses by triggering so-called pattern recognition receptors (PRRs) that are expressed on innate immune cells, including plasmacytoid dendritic cells and monocytes. Although host and microbial nucleic acids share pronounced chemical and structural similarities, they significantly differ in their posttranscriptional modification profile, allowing the host to discriminate between self and nonself. In this regard, ribose 2'-O-methylation has been discovered as suppressor of RNA-induced PRR activation. Although 2'-O-methylation occurs with higher frequencies in eukaryotic than in prokaryotic RNA, the immunosuppressive properties of 2'-O-methylated nucleotides may be misused by certain bacteria as immune evasion mechanism. In the course of identifying inhibitory RNA modifications, our groups have synthesized and comparatively analyzed a series of differentially modified RNAs, so-called modivariants, for their immune stimulatory capacities. In this chapter, we will detail the protocols for the design and synthesis of RNA modivariants by molecular cut-and-paste techniques (referred to as molecular surgery) and describe testing of their immune stimulatory properties upon transfection into peripheral blood mononuclear cells.