scholarly journals Foxp3 Silencing with Antisense Oligonucleotide Improves Immunogenicity of an Adjuvanted Recombinant Vaccine against Sporothrix schenckii

2021 ◽  
Vol 22 (7) ◽  
pp. 3470
Author(s):  
Alexander Batista-Duharte ◽  
Luis Sendra ◽  
Maria José Herrero ◽  
Deivys Leandro Portuondo ◽  
Damiana Téllez-Martínez ◽  
...  
Keyword(s):  
Immune Responses ◽  
Sporothrix Schenckii ◽  
Recombinant Vaccine ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Murine Splenocytes ◽  
Treg Depletion ◽  
In Vivo Experiments

Background: In recent years, there has been great interest in developing molecular adjuvants based on antisense oligonucleotides (ASOs) targeting immunosuppressor pathways with inhibitory effects on regulatory T cells (Tregs) to improve immunogenicity and vaccine efficacy. We aim to evaluate the immunostimulating effect of 2′OMe phosphorothioated Foxp3-targeted ASO in an antifungal adjuvanted recombinant vaccine. Methods: The uptake kinetics of Foxp3 ASO, its cytotoxicity and its ability to deplete Tregs were evaluated in murine splenocytes in vitro. Groups of mice were vaccinated with recombinant enolase (Eno) of Sporothix schenckii in Montanide Gel 01 adjuvant alone or in combination with either 1 µg or 8 µg of Foxp3 ASO. The titers of antigen-specific antibody in serum samples from vaccinated mice (male C57BL/6) were determined by ELISA (enzyme-linked immunosorbent assay). Cultured splenocytes from each group were activated in vitro with Eno and the levels of IFN-γ and IL-12 were also measured by ELISA. The results showed that the anti-Eno antibody titer was significantly higher upon addition of 8 µM Foxp3 ASO in the vaccine formulation compared to the standard vaccine without ASO. In vitro and in vivo experiments suggest that Foxp3 ASO enhances specific immune responses by means of Treg depletion during vaccination. Conclusion: Foxp3 ASO significantly enhances immune responses against co-delivered adjuvanted recombinant Eno vaccine and it has the potential to improve vaccine immunogenicity.

scholarly journals Development of a flow cytometric bead immunoassay and its assessment as a possible aid to potency evaluation of enterotoxaemia vaccines

2014 ◽  
Vol 85 (1) ◽  
Author(s):  
Angela Buys ◽  
Raynard Macdonald ◽  
Jannie Crafford ◽  
Jacques Theron
Keyword(s):  
Cost Effective ◽  
In Vitro Assays ◽  
Enzyme Linked Immunosorbent Assay ◽  
In Vitro Tests ◽  
Serum Samples ◽  
Type D ◽  
Epsilon Toxin ◽  
Bead Immunoassay

Enterotoxaemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens type D. The only practical means of controlling the occurrence of enterotoxaemia is to immunise animals by vaccination. The vaccine is prepared by deriving a toxoid from the bacterial culture filtrate and the potency of the vaccine is tested with the in vivo mouse neutralisation test (MNT). Due to ethical, economic and technical reasons, alternative in vitro assays are needed. In this study an indirect cytometric bead immunoassay (I-CBA) was developed for use in vaccine potency testing and the results were compared with those obtained using an indirect enzyme-linked immunosorbent assay (I-ELISA) and the MNT. Sera were collected from guinea pigs immunised with three different production batches of enterotoxaemia vaccine and the levels of anti-epsilon toxin antibodies were determined. Although the intra- and inter-assay variability was satisfactory, epsilon antitoxin levels determined by both the I-ELISA and indirect cytometric bead immunoassay (I-CBA) tests were higher than those of the MNT assay. In contrast to the MNT, all of the serum samples were identified as having antitoxin levels above the required minimum (not less than 5 U/mL). These results indicate that the respective in vitro tests in their current formats are not yet suitable alternatives to the in vivo MNT. The growing demand for a more humane, cost-effective and efficient method for testing the potency of enterotoxaemia vaccines, however, provides a strong impetus for further optimisation and standardisation of the I-CBA assay but further analytical research is required.

Comparative study of sodium bicarbonate- and magnesium hydroxide-based gastric antacids for the effectiveness of Salmonella delivered Brucella antigens against wild type challenge in BALB/c mice

2021 ◽  
Vol 79 (2) ◽  
Author(s):  
Chamith Hewawaduge ◽  
Amal Senevirathne ◽  
Myeon-Sik Yang ◽  
Tae-Won Jeong ◽  
Bumseok Kim ◽  
...  
Keyword(s):  
Immune Responses ◽  
Oral Administration ◽  
Sodium Bicarbonate ◽  
Magnesium Hydroxide ◽  
Buffering Capacity ◽  
Live Attenuated Vaccine ◽  
Ph Buffering ◽  
In Vivo Experiments

ABSTRACT We compared the effects of two antacid formulations based on sodium bicarbonate and magnesium hydroxide on a Salmonella-delivered oral Brucella live attenuated vaccine. We conducted a series of in vitro and in vivo experiments to investigate the pH buffering capacity, buffering longevity and the effects of these formulations on the survival of Salmonella under neutralized pH conditions and its impact on immune responses. Magnesium hydroxide had a greater, stable and prolonged buffering capacity than sodium bicarbonate and was safer when administered orally. Oral administration of sodium bicarbonate resulted in discomfort as reflected by mouse behavior and mild muscle tremors, whereas mice treated with magnesium hydroxide and PBS were completely normal. Gastric survival studies using BALB/c mice revealed that a higher number of Salmonella reached the intestine when the magnesium hydroxide-based antacid buffer was administrated. Co-administration with attenuated Salmonella secreting Brucella antigens, SodC and Omp19 along with individual antacid formulations, significantly enhanced the antigen-specific protective immune responses against virulent Brucella challenge. Together, our results indicated that the pre vaccinated oral administration of bicarbonate-citric acid or magnesium hydroxide-based neutralizing buffers significantly counteract stomach acidity by maintaining the viability of an oral enteric vaccine formulation.

scholarly journals A CTLA-4 nanobody improves the immunity of mice against challenges with Staphylococcus aureus and Streptococcus agalactiae

2021 ◽  
Author(s):  
Peng Wu ◽  
Ning ning Yang ◽  
Ming guo Xu ◽  
chuangfu chen
Keyword(s):  
Staphylococcus Aureus ◽  
Immune Responses ◽  
Streptococcus Agalactiae ◽  
Cytotoxic T Lymphocyte ◽  
Enzyme Linked Immunosorbent Assay ◽  
No Production ◽  
High Affinity ◽  
Mouth Disease Virus

Cytotoxic T lymphocyte associated antigen 4 (CTLA-4) , also known as CD152, is a transmembrane receptor and leukocyte differentiation antigen on T cells that participates in the negative regulation of immune responses. CTLA-4 Ig can effectively and specifically inhibit cellular and humoral immune responses in vivo and in vitro, and is therefore, considered to be a promising new immunosuppressive antibody . In this study, we investigated the role of CTLA-4 nanobody in immunity. We purified recombinant CTLA-4 protein and constructed a phage display nanobody library . After screening the library, we obtained a nanobody with high affinity for the CTLA-4 protein. The nanobody was expressed and purified and the specific high-affinity for CTLA-4 confirmed by enzyme-linked immunosorbent assay. The nanobody was shown to enhance the activity and nitric oxide (NO) production of bone marrow derived dendritic cells (DCs) as well as their ability to capture foot-and-mouth disease virus (FMDV). The CTLA-4 nanobody also improved the immunity of animals after challenge with Staphylococcus aureus and Streptococcus agalactiae , thus indicating the potential of the CTLA-4 nanobody to improve cellular immunity and enhance immune responses.

scholarly journals A Sensitive, Robust High-Throughput Electrochemiluminescence Assay for Rat Insulin

2004 ◽  
Vol 9 (1) ◽  
pp. 62-70 ◽  
Author(s):  
Rajasree Golla ◽  
Ramakrishna Seethala
Keyword(s):  
Dynamic Range ◽  
Enzyme Linked Immunosorbent Assay ◽  
Diabetes Research ◽  
Serum Samples ◽  
Biological Assays ◽  
In Vivo Experiments ◽  
Assay Performance ◽  
Homogeneous Assay ◽  
Z Value

In diabetes research, mouse and rat models are used for in vivo experiments, and quantification of insulin in serum samples under different pathophysiological conditions and after treatment with compounds is essential. There are few commercial radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) assay kits to determine the rat/mouse plasma levels of insulin. However, reliability in insulin measurements using the available biological assays is a great concern. The authors report a robust, extremely sensitive electrochemiluminescence (ECL) insulin assay using the Origen technology platform. The assay performance, as judged by the Z′ value of 0.82±0.03 and the signal-to-background (S/B) ratio of 133, suggests that this is a robust and reliable assay. The intra-assay and interassay variation is less than 5%. The dynamic range of detection for insulin is 5 pg to 5 ng in the ECL assays. Recovery of insulin was about 100% when different volumes of serum were spiked with exogenous insulin. These results suggest that the ECL insulin assay is an extremely sensitive, robust, nonradioactive homogeneous assay and can be used successfully to determine the insulin levels in rodent serum samples. ( Journal of Biomolecular Screening 2004:62-70)

scholarly journals 846 Neospora caninum – an immunotherapeutic protozoan against solid cancers

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A899-A899
Author(s):  
Louis Lantier ◽  
Agathe Poupee-Beauge ◽  
Louis Lantier ◽  
Céline Ducournau ◽  
Anne Di Tommaso ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Immune Responses ◽  
Neospora Caninum ◽  
Tumor Development ◽  
Molecular Therapy ◽  
List Type ◽  
In Vivo Experiments ◽  
Attenuated Viruses

BackgroundImmunotherapy induces, provides, and/or reactivates anti-tumor immune responses. Some microorganisms also can initiate response that lyzes infected tumor and/or stimulates systemic immunity. Attenuated viruses or bacteria are well studied as oncotherapeutics, but not protozoa except Toxoplasma gondii.1 We assessed the effect on tumors of other protozoa that were naturally non-pathogenic to humans. Thus, we discovered the ability to use Neospora caninum (Nc) in a manner and form that demonstrated a synergistic array of pertinent immunotherapeutic characteristics against solid cancers. Our first Article on Neospora as Onco-immunotherapeutic is currently under revision after review by the JITC. We report on the most recent data notably from Nc engineered to secrete human IL-15 within the tumor.MethodsIn vitro, the immunostimulatory properties of Nc strains wildtype and engineered to secrete human IL-15 were studied. In vivo experiments of treatment with of Nc tachyzoites2 administered locally (intra and peri tumoral) or remotely (subcutaneous) in a murine thymoma EG7 tumor and in human Merkel cell carcinoma (MCC).ResultsWe demonstrated that the treatment of thymoma EG7 by Nc strongly inhibited tumor development. Analysis of immune responses and interactions between Nc and tumor cells showed that Nc had the ability to lyze infected cancer cells, reactivated immune competence within the Tumor Microenvironment (TME), and activated the systemic immune system by promoting the recruitment of immune cells to the site of tumor. We also established in a NOD/SCID mouse model that Nc was able to induce a strong regression of human MCC. Recently, to further enhance oncotherapeutic effect, we engineered an Nc strain to secrete human IL-15 (cross reactive with mouse cells), associated with alpha subunit of IL-15 receptor, increasing its stability.3 This strain induced proliferation of human PBMCs and their secretion of IFN-γ. In the EG7 model, human IL-15 secreting Nc showed greater protection against tumor development, confirming enhancement of immunotherapy by engineering Nc to deliver/secrete IL-15.ConclusionsThese results highlight Neospora caninum as a potentially extremely efficient, and non-toxic anti-cancer agent, capable of being engineered to express at its surface or to secrete bio-drugs, like human IL-15 cytokine. Our work has identified the broad clinical possibilities of using N. caninum as an oncolytic protozoan in human medicine capable of vectoring molecular therapy, overcoming TME defenses.ReferencesFox BA, Butler KL, Guevara RB, Bzik DJ. Cancer therapy in a microbial bottle: Uncorking the novel biology of the protozoan Toxoplasma gondii. PLoS Pathog 2017;13(9):e1006523. https://doi.org/10.1371/journal.ppat.1006523Bjerkas I, Jenkins MC, Dubey JP. Identification and characterization of Neospora caninum tachyzoite antigens useful for diagnosis of neosporosis. Clin Diagn Lab Immunol 1994;1(2):214-221.Article for publication in the Journal of Immunotherapy of Cancer, under revision on September 20, 2020.

ANTITUMOR EFFECT OF COLLOIDAL SILVER BISILICATE IN EXPERIMENTAL IN VITRO AND IN VIVO STUDIES

2019 ◽  
Vol 65 (5) ◽  
pp. 760-765
Author(s):  
Margarita Tyndyk ◽  
Irina Popovich ◽  
A. Malek ◽  
R. Samsonov ◽  
N. Germanov ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Human Tumor ◽  
Significant Inhibition ◽  
In Vivo Studies ◽  
Ehrlich Carcinoma ◽  
In Vivo Experiments

The paper presents the results of the research on the antitumor activity of a new drug - atomic clusters of silver (ACS), the colloidal solution of nanostructured silver bisilicate Ag6Si2O7 with particles size of 1-2 nm in deionized water. In vitro studies to evaluate the effect of various ACS concentrations in human tumor cells cultures (breast cancer, colon carcinoma and prostate cancer) were conducted. The highest antitumor activity of ACS was observed in dilutions from 2.7 mg/l to 5.1 mg/l, resulting in the death of tumor cells in all studied cell cultures. In vivo experiments on transplanted Ehrlich carcinoma model in mice consuming 0.75 mg/kg ACS with drinking water revealed significant inhibition of tumor growth since the 14th day of experiment (maximally by 52% on the 28th day, p < 0.05) in comparison with control. Subcutaneous injections of 2.5 mg/kg ACS inhibited Ehrlich's tumor growth on the 7th and 10th days of the experiment (p < 0.05) as compared to control.

scholarly journals Lysosomotropic agents including azithromycin, chloroquine and hydroxychloroquine activate the integrated stress response

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ai-Ling Tian ◽  
Qi Wu ◽  
Peng Liu ◽  
Liwei Zhao ◽  
Isabelle Martins ◽  
...  
Keyword(s):  
Stress Response ◽  
Immune Responses ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Serine Residue ◽  
Eif2α Phosphorylation ◽  
Lysosomotropic Agents ◽  
Cultured Human Cells

AbstractThe integrated stress response manifests with the phosphorylation of eukaryotic initiation factor 2α (eIF2α) on serine residue 51 and plays a major role in the adaptation of cells to endoplasmic reticulum stress in the initiation of autophagy and in the ignition of immune responses. Here, we report that lysosomotropic agents, including azithromycin, chloroquine, and hydroxychloroquine, can trigger eIF2α phosphorylation in vitro (in cultured human cells) and, as validated for hydroxychloroquine, in vivo (in mice). Cells bearing a non-phosphorylatable eIF2α mutant (S51A) failed to accumulate autophagic puncta in response to azithromycin, chloroquine, and hydroxychloroquine. Conversely, two inhibitors of eIF2α dephosphorylation, nelfinavir and salubrinal, enhanced the induction of such autophagic puncta. Altogether, these results point to the unexpected capacity of azithromycin, chloroquine, and hydroxychloroquine to elicit the integrated stress response.

scholarly journals The impact of FGF19/FGFR4 signaling inhibition in antitumor activity of multi-kinase inhibitors in hepatocellular carcinoma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hiroaki Kanzaki ◽  
Tetsuhiro Chiba ◽  
Junjie Ao ◽  
Keisuke Koroki ◽  
Kengo Kanayama ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Progression Free Survival ◽  
Erk Signaling ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antitumor Effects ◽  
The Impact

AbstractFGF19/FGFR4 autocrine signaling is one of the main targets for multi-kinase inhibitors (MKIs). However, the molecular mechanisms underlying FGF19/FGFR4 signaling in the antitumor effects to MKIs in hepatocellular carcinoma (HCC) remain unclear. In this study, the impact of FGFR4/ERK signaling inhibition on HCC following MKI treatment was analyzed in vitro and in vivo assays. Serum FGF19 in HCC patients treated using MKIs, such as sorafenib (n = 173) and lenvatinib (n = 40), was measured by enzyme-linked immunosorbent assay. Lenvatinib strongly inhibited the phosphorylation of FRS2 and ERK, the downstream signaling molecules of FGFR4, compared with sorafenib and regorafenib. Additional use of a selective FGFR4 inhibitor with sorafenib further suppressed FGFR4/ERK signaling and synergistically inhibited HCC cell growth in culture and xenograft subcutaneous tumors. Although serum FGF19high (n = 68) patients treated using sorafenib exhibited a significantly shorter progression-free survival and overall survival than FGF19low (n = 105) patients, there were no significant differences between FGF19high (n = 21) and FGF19low (n = 19) patients treated using lenvatinib. In conclusion, robust inhibition of FGF19/FGFR4 is of importance for the exertion of antitumor effects of MKIs. Serum FGF19 levels may function as a predictive marker for drug response and survival in HCC patients treated using sorafenib.

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