High Levels Of Th2 Cytokines Research Articles

Effect of Cyclosporine A on Th1/Th2 Cytokine Production by Decidual Stromal Cells Mediated by Trophoblast-derived Galectin-9.

This study aimed to investigate the effect of cyclosporine A (CsA) on secretion of Th1 and Th2 cytokines by decidual stromal cells (DSCs) mediated by galectin (Gal)-9.HTR8/SVneo cells and primary trophoblasts were used for in vitro studies. Gal-9 expression was measured using quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, CsA was used to regulate Gal-9 expression in trophoblasts. DSCs were treated with trophoblast supernatant and changes in Th1 and Th2 cytokine levels were analyzed. Changes in DSC levels of the T-cell immunoglobulin mucin receptor 3 (TIM-3) levels in DSCs after treatment with Gal-9 were assessed. Western blotting and ERK and AKT inhibitors were used to assess the involvement of the corresponding signaling pathways. Gal-9 was expressed by both primary trophoblasts and HTR8/SVneo cells. CsA treatment increased Gal-9 secretion by trophoblasts, which in turn increased IL-6 (Th2 cytokine) and decreased TNF-α and IFN-γ (Th1 cytokines) secretion in DSCs. Upon downregulation of trophoblast Gal-9 secretion, DSCs secreted lower levels of Th2 cytokines and higher levels of Th1 cytokines, and the effect was reversed by addition of CsA. TIM-3 expression changed in parallel with Gal-9 secretion. CsA treatment upregulated expression of Gal-9 in trophoblasts, promoted secretion of Th2 cytokines, and inhibited secretion of Th1 cytokines via ERK signaling.

Computational design and evaluation of mRNA- and protein-based conjugate vaccines for influenza A and SARS-CoV-2 viruses

BackgroundIsrael confirmed the first case of “flurona”—a co-infection of seasonal flu (IAV) and SARS-CoV-2 in an unvaccinated pregnant woman. This twindemic has been confirmed in multiple countries and underscores the importance of managing respiratory viral illnesses. ResultsThe novel conjugate vaccine was designed by joining four hemagglutinin, three neuraminidase, and four S protein of B-cell epitopes, two hemagglutinin, three neuraminidase, and four S proteins of MHC-I epitopes, and three hemagglutinin, nine neuraminidase, and five S proteins of MHC-II epitopes with linkers and adjuvants. The constructed conjugate vaccine was found stable, non-toxic, non-allergic, and antigenic with 0.6466 scores. The vaccine contained 14.87% alpha helix, 29.85% extended strand, 9.64% beta-turn, and 45.64% random coil, which was modeled to a 3D structure with 94.7% residues in the most favored region of the Ramachandran plot and Z-score of −3.33. The molecular docking of the vaccine with TLR3 represented −1513.9 kcal/mol of binding energy with 39 hydrogen bonds and 514 non-bonded contacts, and 1.582925e-07 of eigenvalue complex. Immune stimulation prediction showed the conjugate vaccine could activate T and B lymphocytes to produce high levels of Th1 cytokines and antibodies. ConclusionThe in silico-designed vaccine against IAV and SARS-CoV-2 showed good population coverage and immune response with predicted T- and B-cell epitopes, favorable molecular docking, Ramachandran plot results, and good protein expression. It fulfilled safety criteria, indicating potential for preclinical studies and experimental clinical trials.

Impact of Echinococcus granulosus Antigens on Monocyte Development and Dendritic Cell Differentiation.

Different subtypes of dendritic cells (DCs) can induce different types of immune responses. Our previous study found that Echinococcus granulosus (E. granulosus) antigens (Eg.ferritin, Eg.mMDH and Eg.10) stimulated DC differentiation to different subtypes and produced different immune responses. To further understand whether Eg.ferritin, Eg.mMDH and Eg.10 affect the DC-mediated immune response by promoting the differentiation of monocytes to DCs. Bone marrow-derived monocytes were exposed to three antigens of E. granulosus on days 0, 3, 5, and 7. The percentage of monocyte-derived DCs (moDCs), DCs subsets, and the expression of surface molecules of DCs at different time points in different groups were assessed by flow cytometry. The levels of cytokines of IL-1β, IL-4, IL-6, IL-10, IL-13, IFN-γ, TNF-α, IL-12p70, IL-18, IL-23, and IL-27 in the cell culture supernatant were detected by multi-factorial detection technology. The percentage of moDCs revealed that none of the three antigens blocked monocyte differentiation to DCs. The monocytes of 7-day-old cultures showed increased sensitivity to these antigens. The Eg.ferritin induced more mature DCs, which expressed high levels of MHC II and costimulatory molecules, and secreted Th1 cytokines. Eg10 and Eg.mMDH induced lower degrees of DC maturation, however differentiated DCs were in a semi-mature state due to low expression of MHC II and costimulatory molecules and secretion of higher Th2 and lower Th1 cytokines. Eg.ferritin promotes full maturation of DCs and induces Th1 immune response, whereas Eg.10 and Eg.mMDH induce semi-mature DCs producing higher levels of Th2 cytokines.

Indoor aeroallergens from American cockroaches and mites initiate atopic march via cutaneous contact in a murine model.

The progression of allergic diseases from atopic dermatitis in childhood to other allergic conditions such as asthma in later life is often referred to as the atopic march. In order to study the relationship between cutaneous sensitization by aeroallergen and atopic march, we established a mouse model to test the hypothesis using American cockroaches and house dust mites as the model allergens. Mice were sensitized via skin with native cockroach extract (CraA) or recombinant Per a 2 and Der p 2 proteins without adjuvant. Each mouse was subjected to a total of three 1-week patching sensitizations with a 2-week interval in between each application. The resulting immunological variables in sera, scratching behavior, airway hyperresponsiveness (AHR), and pathology of skin lesions and nasal mucosa were evaluated. In mice, application of CraA, rPer a 2, and rDer p 2 aeroallergens through skin patching induced significantly high levels of both total IgE and specific IgEs. The epicutaneous sensitization after a subsequent allergen challenge showed a significant increase in scratch bouts, AHR, epidermal thickness, and eosinophil counts in the skin compared with the control mice. In addition, stimulation of murine splenocytes with allergens increased higher levels of Th2 cytokines, anti-inflammatory cytokines, and chemokines excretion. Our study provides evidence supporting that epicutaneous sensitization to aeroallergens also led to nasal and airway symptoms comparable to atopic march as described in humans. We hope this new allergy model will be useful in the development of new preventive and therapeutic strategies aimed at stopping the atopic march.

Heterologous prime-boost immunisation with mRNA- and AdC68-based 2019-nCoV variant vaccines induces broad-spectrum immune responses in mice.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or 2019-nCoV) variants has been associated with the transmission and pathogenicity of COVID-19. Therefore, exploring the optimal immunisation strategy to improve the broad-spectrum cross-protection ability of COVID-19 vaccines is of great significance. Herein, we assessed different heterologous prime-boost strategies with chimpanzee adenovirus vector-based COVID-19 vaccines plus Wuhan-Hu-1 (WH-1) strain (AdW) and Beta variant (AdB) and mRNA-based COVID-19 vaccines plus WH-1 strain (ARW) and Omicron (B.1.1.529) variant (ARO) in 6-week-old female BALB/c mice. AdW and AdB were administered intramuscularly or intranasally, while ARW and ARO were administered intramuscularly. Intranasal or intramuscular vaccination with AdB followed by ARO booster exhibited the highest levels of cross-reactive IgG, pseudovirus-neutralising antibody (PNAb) responses, and angiotensin-converting enzyme-2 (ACE2)-binding inhibition rates against different 2019-nCoV variants among all vaccination groups. Moreover, intranasal AdB vaccination followed by ARO induced higher levels of IgA and neutralising antibody responses against live 2019-nCoV than intramuscular AdB vaccination followed by ARO. A single dose of AdB administered intranasally or intramuscularly induced broader cross-NAb responses than AdW. Th1-biased cellular immune response was induced in all vaccination groups. Intramuscular vaccination-only groups exhibited higher levels of Th1 cytokines than intranasal vaccination-only and intranasal vaccination-containing groups. However, no obvious differences were found in the levels of Th2 cytokines between the control and all vaccination groups. Our findings provide a basis for exploring vaccination strategies against different 2019-nCoV variants to achieve high broad-spectrum immune efficacy.

Biomarker Profiles Associated with COVID-19 Severity and Mortality.

The aim of this study was to analyze biomarkers that might predict the severity and progression of the SARS-CoV-2 infection, both in the acute phase and after recovery. Unvaccinated patients infected with the original strain of COVID-19 requiring ward (Group 1, n = 48) or ICU (Group 2, n = 41) admission were included. At the time of admission (visit 1), a clinical history was acquired, and blood samples were obtained. One and six months after discharge from the hospital (visits 2 and 3, respectively), a clinical history, lung function tests, and blood samples were carried out. At visit 2, patients also underwent a chest CT scan. Different cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17A, G-CSF, GM-CSF, IFN-ɣ, MCP-1, MIP-1β, and TNF-α) and lung fibrosis biomarkers (YKL-40 and KL-6) were measured in blood samples obtained at visits 1, 2, and 3. At visit 1, IL-4, IL-5, and IL-6 levels were higher in Group 2 (p = 0.039, 0.011, and 0.045, respectively), and IL-17 and IL-8 levels were higher in Group 1 (p = 0.026 and 0.001, respectively). The number of patients in Groups 1 and 2 who died during hospitalization was 8 and 11, respectively. YKL-40 and KL-6 levels were higher in patients who died. Serum YKL-40 and KL-6 levels determined at visit 2 correlated negatively with FVC (p = 0.022 and p = 0.024, respectively) and FEV1 (p = 0.012 and p = 0.032, respectively) measured at visit 3. KL-6 levels also correlated negatively with the diffusing capacity of the lungs for carbon monoxide (DLCO, p = 0.001). Patients who required ICU admission had higher levels of Th2 cytokines, while patients admitted to the ward showed an innate immune response activation, with IL-8 release and Th1/Th17 lymphocyte contribution. Increased levels of YKL-40 and KL-6 were associated with mortality in COVID-19 patients.

Developing a multiepitope vaccine for the prevention of SARS-CoV-2 and monkeypox virus co-infection: A reverse vaccinology analysis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and monkeypox virus (MPXV) severely threaten human health; however, currently, no vaccine can prevent a co-infection with both viruses. Five antigens were selected to predict dominant T and B cell epitopes screened for immunogenicity, antigenicity, toxicity, and sensitization. After screening, all antigens joined in the construction of a novel multiepitope vaccine. The physicochemical and immunological characteristics, and secondary and tertiary structures of the vaccine were predicted and analyzed using bio- and immunoinformatics. Finally, codon optimization and cloning in-silico were performed. A new multiepitope vaccine, named S7M8, was constructed based on four helper T lymphocyte (HTL) epitopes, six cytotoxic T lymphocyte (CTL) epitopes, five B cell epitopes, as well as Toll-like receptor (TLR) agonists. The antigenicity and immunogenicity scores of the S7M8 vaccine were 0.907374 and 0.6552, respectively. The S7M8 vaccine was comprised of 26.96% α-helices, the optimized Z-value of the tertiary structure was -5.92, and the favored area after majorization in the Ramachandran plot was 84.54%. Molecular docking showed that the S7M8 vaccine could tightly bind to TLR2 (-1100.6kcal/mol) and TLR4 (-950.3kcal/mol). In addition, the immune stimulation prediction indicated that the S7M8 vaccine could activate T and B lymphocytes to produce high levels of Th1 cytokines and antibodies. S7M8 is a promising biomarker with good antigenicity, immunogenicity, non-toxicity, and non-sensitization. The S7M8 vaccine can trigger significantly high levels of Th1 cytokines and antibodies and may be a potentially powerful tool in preventing SARS-CoV-2 and MPXV.

Immunization with EmCRT-Induced Protective Immunity against Echinococcus multilocularis Infection in BALB/c Mice

Alveolar echinococcosis (AE) is a severe parasitic zoonosis caused by the larval stage of Echinococcus multilocularis. The identification of the antigens eliciting acquired immunity during infection is important for vaccine development against Echinococcus infection. Here, we identified that E. multilocularis calreticulin (EmCRT), a ubiquitous protein with a Ca2+-binding ability, could be recognized by the sera of mice infected with E. multilocularis. The native EmCRT was expressed on the surface of E. multilocularis larvae as well as in the secreted products of metacestode vesicles and protoscoleces (PSCs). The coding DNA for EmCRT was cloned from the mRNA of the E. multilocularis metacestode vesicles and a recombinant EmCRT protein (rEmCRT) was expressed in E. coli. Mice immunized with soluble rEmCRT formulated with Freund’s adjuvant (FA) produced a 43.16% larval vesicle weight reduction against the challenge of E. multilocularis PSCs compared to those that received the PBS control associated with a high titer of IgG, IgG1 and IgG2a antibody responses as well as high levels of Th1 cytokines (IFN-γ and IL-2) and Th2 cytokines (IL-4, IL-5 and IL-10), produced by splenocytes. Our results suggest that EmCRT is an immunodominant protein secreted by E. multilocularis larvae and a vaccine candidate that induces partial protective immunity in vaccinated mice against Echinococcus infection.

Immunomodulatory and Anticancer Activities of Barley Bran Grown in Jordan: An in vitro and in vivo Study.

The Mediterranean diet is regarded as one of the most healthful dietary patterns in the world, owing to a combination of foods high in antioxidants and anticancer constituents. Barley bran is one of the components of the Mediterranean diet. It has nutritional and beneficial effects in different pathological conditions. Many studies were achieved to assess the nutritious values of barley bran, but there is no research indicating immunomodulatory and anticancer activities of barley bran grown in Jordan. The present study aims to examine and assess the potential immunomodulatory and anti-tumor activities of ethanol, n-hexane, aqueous/methanol, and water extracts obtained from barley bran. The Maceration method was utilized to prepare ethanol, n-hexane, aqueous/methanol, and water extracts. Various phytochemical groups were determined by using qualitative phytochemical tests. The antiproliferative activity of extracts was determined against MCF-7, HCT-116, A549, and EMT6/p by the MTT assay. The Folin-Ciocalteu reagent was used to detect the total phenolic content in extracts. Furthermore, immunomodulatory activity was assessed by determining the effect of extracts on splenocytes proliferation in the presence and absence of mitogens. The nitro blue tetrazolium assay and the neutral red method were used to assess the effect of each extract on the phagocytic activity of macrophages and pinocytosis, respectively. For the in vivo part, three different concentrations (10, 20, and 30% w/v) of barley bran were used to test the prophylactic effect in four Balb/C mice groups inoculated with EMT6/p cell-line subcutaneously. Also, serum samples were collected to assess the effect on cytokines (IFN-gamma, IL-2, IL-4, and IL-10). Barley bran extracts inhibited cancer cell proliferation. According to immunoassays, n-hexane and aqueous/methanol extracts could significantly rise lymphocyte proliferation and pinocytosis activity of macrophages. The activity of phagocytosis was increased by n-hexane and ethanol extracts. For the in vivo part, the average tumor size and weight of mice given the 30% barley bran group was significantly reduced (p < 0.05) compared with the control group. During our study, higher levels of TH1 cytokines (IFN- γ, IL-2) and lower levels of TH2 cytokine (IL-4) and T regulatory cytokine (IL-10) were obtained due to consumption of barley bran in food. Barley bran can be used as a prophylactic agent because it has anti-cancer and immunomodulatory activities.

Effects of Prior Exposure to Tec Kinase(BTK/ITK) Inhibitors on Kte-X19 Products

Introduction:Frequent and durable responses were recently reported in relapsed or refractory (R/R) mantle cell lymphoma (MCL) patients treated with KTE-X19, an autologous CD19-targeted chimeric antigen receptor-engineered T-cell (CAR-T) product (Wang et al. N Engl J Med. 2020). Most patients enrolled had received at least one line of Tec kinase inhibitor prior to KTE-X19 manufacturing, either in the form of ibrutinib, a Bruton's tyrosine kinase (BTK) and Inducible T cell kinase (ITK) inhibitor, or acalabrutinib, a more selective BTK inhibitor. Pharmacokinetic expansion of KTE-X19 was higher in ibrutinib-treated patients relative to acalabrutinib-treated patients. We previously showed that prolonged exposure to ibrutinib enhanced T cell effector function and proliferation in patients with CLL (Fraietta et al, Blood, 2016). To assess the impact of Tec kinase inhibitor on KTE-X19 products and downstream clinical outcomes, we examined the phenotype, transcriptional profile and cytokine production of KTE-X19 infusion products and post-infusion lymphocytes from patients with R/R MCL treated on the Zuma-2 study.Study Design and Methods:We evaluated biospecimens from MCL patients who enrolled on the Zuma-2 clinical trial (NCT02601313) and who were previously treated with ibrutinib (n=14) or acalabrutinib (n=6). Samples analyzed consisted of KTE-X19 CAR T products and peripheral blood mononuclear cells (PBMC) collected 7 days after infusion. Lymphocytes were assessed for CAR expression, T cell phenotype, transcriptional profile and cytokine production. In addition, CAR T cell phenotypes and cytokines were profiled following co-culture of KTE-X19 with CD19 + Toledo cells (DLBCL).Results:Flow cytometric analysis of KTE-X19 demonstrated similar distributions of CD4+ and CD8+ T cells and comparable frequencies of central and effector memory populations in the CAR+ T cells derived from patients with prior exposure to ibrutinib vs. acalabrutinib. T helper subset analysis trended towards enrichment of Th1/Th17 populations within the CAR+ CD4+ cells of the ibrutinib cohort. This finding was further supported by transcriptional profiling of sorted CAR+ T cells from infusion products, where Th1/Th17, Jak/STAT and activation-related genes were enriched in the cohort with prior ibrutinib exposure. In addition, the Th1 phenotype was more frequent in PBMC of ibrutinib-exposed patients (8/14) compared to acalabrutinib-exposed patients (1/4). Interestingly, a shift from a central memory-dominant product towards an effector memory phenotype was observed in peripheral CD4+ and CD8+ CAR T cells in the ibrutinib cohort, whereas acalabrutinib post-infusion CAR T cells maintained a central memory phenotype. In vitro stimulation of KTE-X19 CAR-T infusion products with tumor cells resulted in a significant enrichment of the Th1 population in patients who had received ibrutinib compared to those that received acalabrutinib (p=0.0058). Following stimulation, CAR-T cells from the acalabrutinib cohort produced higher levels of Th2 cytokines, including IL-4, IL-5, and IL-13 as well as GM-CSF compared to the ibrutinib cohort.Conclusions:Analysis of KTE-X19 infusion products and day 7 post-infusion PBMC demonstrated that CAR T cells from patients with prior ibrutinib exposure have a Th1 predominant phenotype, suggesting that ibrutinib but not acalabrutinib promotes Th1 differentiation and effector function, potentially through the inhibition of ITK. Furthermore, our data suggest that inhibition of non-BTK targets such as ITK may play a role in driving a Th17 phenotype. Prior exposure to ibrutinib may increase CAR T cell effector function to a greater extent than exposure to acalabrutinib to enhance clinical outcome in patients with MCL. DisclosuresBudka: Kite Pharma: Current Employment. Sowrirajan: Kite Pharma: Current Employment. Nguyen: Kite Pharma: Current Employment. Shen: Gilead Sciences: Current equity holder in publicly-traded company; Kite, a Gilead Company: Current Employment, Other: Leadership role, Patents & Royalties; Atara: Current Employment, Current equity holder in publicly-traded company, Other: Leadership role, Patents & Royalties. Bot: Kite, a Gilead Company: Current Employment; Gilead Sciences: Consultancy, Current equity holder in publicly-traded company, Other: Travel support. Maus: Agenus: Consultancy; Arcellx: Consultancy; Astellas: Consultancy; AstraZeneca: Consultancy; Atara: Consultancy; Bayer: Consultancy; BMS: Consultancy; Cabaletta Bio (SAB): Consultancy; CRISPR therapeutics: Consultancy; In8bio (SAB): Consultancy; Intellia: Consultancy; GSK: Consultancy; Kite Pharma: Consultancy, Research Funding; Micromedicine: Consultancy, Current holder of stock options in a privately-held company; Novartis: Consultancy; Tmunity: Consultancy; Torque: Consultancy, Current holder of stock options in a privately-held company; WindMIL: Consultancy; Adaptimmune: Consultancy; tcr2: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months; century: Current equity holder in publicly-traded company; ichnos biosciences: Consultancy, Current holder of stock options in a privately-held company.

Lack of WDFY4 Aggravates Ovalbumin-Induced Asthma via Enhanced Th2 Cell Differentiation

Background: Asthma is a chronic inflammatory airway disease, and Th2 cells play an important role in asthma. WDFY4 (WDFY family member 4) is a susceptibility gene in several autoimmune diseases. Objective: In this study, the roles of WDFY4 in Th2 cell differentiation and Th2-dependent asthma were investigated. Methods: Naïve CD4⁺ T cells were isolated from wild-type and WDFY4-deficient mice and induced to differentiate in vitro. Subsequently, a mouse model of asthma was established by sensitization with ovalbumin. Results: Study results showed that WDFY4 deficiency could promote the differentiation of Th2 cells and the production of Th2 cytokines. WDFY4-deficient asthmatic mice showed higher levels of Th2 cytokines in the lungs and bronchoalveolar lavage fluid than wild-type mice. Moreover, infiltration of inflammatory cells, hyperplasia of goblet cells, production of mucus, and deposition of collagen were enhanced in WDFY4-deficient asthmatic mice. Conclusions: Our study demonstrates the pivotal role of WDFY4 in the pathogenesis of asthma and in Th2 cell differentiation.

HCV Core/NS3 Protein Immunization with “N-Terminal Heat Shock gp96 Protein (rNT (gp96))” Induced Strong and Sustained Th1-Type Cytokines in Immunized Mice

Feeble cellular responses induced by T cell-based vaccines are a major challenge for the development of an effective vaccine against Hepatitis C virus (HCV) infection. To address this challenge, the potential of N-terminal fragment of gp96 heat shock protein (rNT (gp96) as an adjuvant was evaluated and compared to that of the CpG (as a recognized Th1-type adjuvant) in the formulation of HCV core/NS3 antigens in three immunization strategies of protein/protein, DNA/DNA, and DNA/protein. Immunized mice were evaluated for elicited immune responses in week 3 (W3) and 11 post-immunizations. Our results demonstrated that the protein (subunit) vaccine formulated with rNT (gp96) in protein/protein strategy (core/NS3 + gp96) was significantly more efficient than CpG oligodeoxynucleotides (CpG ODN) formulation and all other immunization strategies in the induction of Th1-type cytokines. This group of mice (core/NS3 + gp96) also elicited a high level of anti-Core-NS3 total immunoglobulin G (IgG) with dominant IgG2a isotype at W3. Thus, the co-administration of recombinant NT (gp96) protein with rHCV proteins might be a promising approach in the formulation of HCV subunit vaccine candidates for induction of high levels of Th1 cytokines and humoral responses.

Fasciola hepatica co-infection enhances Th1 immune response in the adventitial layer of non-fertile Echinococcus granulosus cysts

Cystic echinococcosis is a widespread zoonosis caused by the larval stage of the tapeworm Echinococcus granulosus. In intermediary hosts, two types of echinococcal cysts can be found: fertile, which produce protoscoleces, the infective form of the parasite to dogs; and infertile, that do not present protoscoleces and therefore are not able to continue with the parasite life cycle. The adventitial layer, the local immune response against the cyst, plays an important role in cyst fertility. Grazing cattle can often feature Fasciola hepatica co-infection, a parasite known to modulate the host systemic immune response. In this work the cellular Th1/Th2 immune profiles were evaluated in the adventitial layer of fertile and non-fertile cysts with and without co-infection with Fasciola hepatica. Measuring with immunohistochemistry and qPCR in adventitial layer, we report that non-fertile cysts present higher levels of Th1 cytokines (IFN-γ (P < 0.0001) and TNF-α (P < 0.05)), and fertile cysts have higher levels of Th2 cytokines (IL-4 (P < 0.001)). Co-infection with Fasciola hepatica is associated with a decrease in the expression of IL-4 (P < 0.05) and an increase in the expression of IFN-γ (P < 0.0001) in the adventitial layer of fertile cysts. Non-fertile cysts were associated with higher levels of Th1 cytokines in the adventitial layer, with IFN-γ expression enhanced by F. hepatica co-infection (P < 0.0001), confirming that polyparasitism should be considered in the treatment and control of naturally infected cattle.

MicroRNA Targets for Asthma Therapy.

Asthma is a chronic inflammatory obstructive lung disease that is stratified into endotypes. Th2 high asthma is due to an imbalance of Th1/Th2 signaling leading to abnormally high levels of Th2 cytokines, IL-4, IL-5, and IL-13 and in some cases a reduction in type I interferons. Some asthmatics express Th2 low, Th1/Th17 high phenotypes with or without eosinophilia. Most asthmatics with Th2 high phenotype respond to beta-adrenergic agonists, muscarinic antagonists, and inhaled corticosteroids. However, 5-10% of asthmatics are not well controlled by these therapies despite significant advances in lung immunology and the pathogenesis of severe asthma. This problem is being addressed by developing novel classes of anti-inflammatory agents. Numerous studies have established efficacy of targeting pro-inflammatory microRNAs in mouse models of mild/moderate and severe asthma. Current approaches employ microRNA mimics and antagonists designed for use in vivo. Chemically modified oligonucleotides have enhanced stability in blood, increased cell permeability, and optimized target specificity. Delivery to lung tissue limits clinical applications, but it is a tractable problem. Future studies need to define the most effective microRNA targets and effective delivery systems. Successful oligonucleotide drug candidates must have adequate lung cell uptake, high target specificity, and efficacy with tolerable off-target effects.

Palliative Radiofrequency Ablation Accelerates the Residual Tumor Progression Through Increasing Tumor-Infiltrating MDSCs and Reducing T-Cell-Mediated Anti-Tumor Immune Responses in Animal Model.

Previous studies showed that radiofrequency ablation (RFA) has a favorable treatment efficacy for hepatocellular carcinoma (HCC) or colorectal liver metastases (CRLMs). Palliative RFA (pRFA) resulting from larger HCC or multiple CRLMs further accelerated the progression of potential residual tumor, yet its mechanism was still unknown. This study investigated the influence of myeloid-derived suppressor cells (MDSCs) on T-cell immune responses and tumor recurrence after pRFA. CT26 tumor models were used. The percentage of MDSCs in peripheral blood was analyzed by flow cytometry after pRFA. The level of Th1 and Th2 cytokines were measured by ELISA through different treatments (n = 4/group). The tumor-infiltrating MDSCs, dendritic cells, and intracellular cytokines level were analyzed by IHC staining after different treatments. The functional CD8+ T cells were confirmed by the co-localization immunofluorescence staining. The long-term outcomes were also evaluated through CT26 and 4T1 tumor models. The results showed that tumor models treated with pRFA displayed significant increases in the percentage of MDSCs of peripheral blood and tumor infiltration. The expression level of TGF-β and IL-6 after pRFA was higher than that before pRFA by ELISA and IHC staining. After depleting MDSCs by combining with Abs, the pRFA + Abs group achieved a higher level of Th1 cytokines and greatly enhanced the percentage of tumor-infiltrating functional CD8+ T cells when compared with pRFA alone. The depletion of MDSCs through combination with Abs also resulted in tumor regression. In conclusion, pRFA accelerates the residual tumor progression through increasing tumor-infiltrating MDSCs and reducing T-cell-mediated anti-tumor immune responses, which could provide a potential approach for delaying tumor recurrence caused by pRFA.

Enhancement of Anti-allergic Effect of Diethylcarbamazine Citrate in Asthmatic Mouse Model: Testing of Anti-drug Antibodies and Quercetin.

Diethylcarbamazine citrate (DEC) is known as an effective treatment for bronchial asthma because of its ability to reduce eosinophil trafficking to the lung tissue. The current study aimed to potentiate the anti-allergic effect of the drug by passive immunization of the asthmatic model with anti-DEC antibody or prior treatment with quercetin (Qur). Eight mice groups were categorized into control, the model of lung asthma, treated with DEC, passively immunized with anti(α)-bovine serum albumin Ab, anti-DEC Ab, prior exposure to 10, 20, or 40 mg Qur/Kg. b.wt. Both eosinophil peroxidase (EPO) and eotaxin2 in the lung tissues were performed. Serum levels of cytokines, bronchoalveolar lavage fluid (BALF) IgE, rabbit anti-bovine serum albumin (anti-BSA), and DEC IgG in lung tissue homogenates were assayed by ELISA. Regarding the effect of anti-DEC Ab and Qur on DEC-induced recovery of histopathological alterations showed that the Ova group had peri-bronchial hyperplasia, mononuclear leukocyte infiltration, thickening in the wall of alveoli, and congested blood vessels. However, the reduction of inflammatory cells and thickened alveolar walls was dependent on the Qur dose. Qur40 enhanced the anti-allergic effect of DEC. Moreover, the present data revealed high levels of Th2 cytokines (IL-4 and IL-5) and IgE in the Ova group. An increased leukocyte infiltration/thickening of the alveolar wall and lung tissue EPO/eotaxin2 were also observed. Qur-40 could show an enhancement effect on DEC for the reduction of IL-4, IL-5, IgE, EPO, and eotaxin 2. Consequently, the IFN-γ/IL-4 ratio was increased. Qur at 40 mg/Kg could be recommended to enhance the DEC effect suggesting a novel approach for treatment.

Anti-tumor and immune modulating activity of T cell induced tumor-targeting effectors (TITE)

Adoptive transfer of Bispecific antibody Armed activated T cells (BATs) showed promising anti-tumor activity in clinical trials in solid tumors. The cytotoxic activity of BATs occurs upon engagement with tumor cells via the bispecific antibody (BiAb) bridge, which stimulates BATs to release cytotoxic molecules, cytokines, chemokines, and other signaling molecules extracellularly. We hypothesized that the release of BATs Induced Tumor-Targeting Effectors (TITE) by this complex interaction of T cells, bispecific antibody, and tumor cells may serve as a potent anti-tumor and immune-activating immunotherapeutic approach. In a 3D tumorsphere model, TITE showed potent cytotoxic activity against multiple breast cancer cell lines compared to control conditioned media (CM): Tumor-CM (T-CM), BATs-CM (B-CM), BiAb Armed PBMC-CM (BAP-CM) or PBMC-CM (P-CM). Multiplex cytokine analysis showed high levels of Th1 cytokines and chemokines; phospho-protein signaling array data suggest that the prominent JAK1/STAT1 pathway may be responsible for the induction and release of Th1 cytokines/chemokines in TITE. In xenograft breast cancer models, IV injections of 10× concentrated TITE (3×/week for 3weeks; 150μl TITE/injection) was able to inhibit tumor growth significantly (ICR/scid, p < 0.003; NSG p < 0.008) compared to the control mice. We tested the key components of the TITE for immune activating and anti-tumor activity individually and in combinations, the combination of IFN-γ, TNF-α and MIP-1β recapitulates the key activities of the TITE. In summary, master mix of active components of BATs-Tumor complex-derived TITE can provide a clinically controllable cell-free platform to target various tumor types regardless of the heterogeneous nature of the tumor cells and mutational tumor.

Myeloid Cell Expression of LACC1 Is Required for Bacterial Clearance and Control of Intestinal Inflammation

Loss-of-function variants in the laccase domain containing 1 (LACC1) gene are associated with immune-mediated diseases, including inflammatory bowel disease. It is not clear how LACC1 balances defenses against intestinal bacteria vs intestinal inflammation or what cells are responsible for this balance in humans or mice. Lacc1-/- mice and mice with myeloid-specific disruption of Lacc1 (Lacc1Δmye) were given oral Salmonella Typhimurium or dextran sodium sulfate. CD45RBhiCD4+T cells were transferred to Lacc1-/-Rag2-/- mice to induce colitis. Organs were collected and analyzed by histology and protein expression. Bone marrow-derived macrophages and dendritic cells, lamina propria macrophages, and mesenteric lymph node dendritic cells were examined. We performed assays to measure intestinal permeability, cell subsets, bacterial uptake and clearance, reactive oxygen species, nitrite production, autophagy, signaling, messenger RNA, and cytokine levels. Lacc1-/- mice developed more severe T-cell transfer colitis than wild-type mice and had an increased burden of bacteria in intestinal lymphoid organs, which expressed lower levels of T helper (Th) 1 and Th17 cytokines and higher levels of Th2 cytokines. Intestinal lymphoid organs from mice with deletion of LACC1 had an increased burden of bacteria after oral administration of S Typhimurium and after administration of dextran sodium sulfate compared with wild-type mice. In macrophages, expression of LACC1 was required for toll-like receptor-induced uptake of bacteria, which required PDK1, and of mitogen-activated protein kinase (MAPK)- and nuclear factor κB-dependent induction of reactive oxygen species, reactive nitrogen species, and autophagy. Expression of LACC1 by dendritic cells was required for increasing expression of Th1 and Th17 cytokines and reducing expression of Th2 cytokines upon coculture with CD4+ T cells. Mice with LACC1-deficient myeloid cells had an increased burden of bacteria and altered T-cell cytokines in intestinal lymphoid organs, similar to Lacc1-/- mice. Complementation of cytokines produced by myeloid cells to cocultures of LACC1-deficient myeloid cells and wild-type CD4+ T cells restored T-cell cytokine regulation. When S Typhimurium-infected Lacc1Δmye mice were injected with these myeloid cell-derived cytokines, intestinal tissues increased production of Th1 and Th17 cytokines, and bacteria were reduced. Disruption of Lacc1 in mice increases the burden of bacteria in intestinal lymphoid organs and intestinal inflammation after induction of chronic colitis. LACC1 expression by myeloid cells in mice is required to clear bacteria and to regulate adaptive T-cell responses against microbes.

84-OR: Laminin-Islet Interactions Protect against Cytokine-Mediated Beta-Cell Death via Down-Regulation of Protein Kinase C Delta

Islets are surrounded by a specialized extracellular matrix (ECM). Interactions with the peri-islet ECM, specifically laminin, promote islet viability and function. In T1D during immune infiltration the peri-islet ECM is broken down and β-cell death occurs in part through production of high levels of Th1 cytokines. Recent work has shown that cytokines alter islet function via protein kinase C Δ (PKCΔ) dependent mechanisms and that islet interactions with laminin reduce PKCΔ activity. We hypothesize that laminin-islet interactions protect against cytokine-mediated β-cell death via downregulation of PKCΔ. To test our hypothesis, we used a 3D reverse thermal gel (RTG) scaffold with either laminin-10 (RTG-Lam) or lysine (RTG-Lys) as a control. Islets were isolated from WT or mice with an inducible β-cell specific PKCΔ knockout (PKCΔ-βKO) and encapsulated in RTG-Lam and RTG-Lys for 24hrs with or without cytokines (10ng/mL TNF-α, 5ng/mL IL-1β, 100ng/mL IFN-γ). Viability, PKCΔ translocation, and PKCΔ activity were measured in encapsulated islets in situ using fluorescent biosensors or by western blot. PKCΔ-βKO islets were protected against cytokine-induced death compared to controls (p=0.003). Cytokines increased β-cell death in RTG-Lys (p&amp;lt;0.001), while islets encapsulated in RTG-Lam were protected against cytokine-mediated death (p&amp;lt;0.04). Islets encapsulated in RTG-Lam had reduced PKCΔ activity at the cell membrane compared to RTG-Lys and were also protected against cytokine-mediated increases in PKCΔ activity at the cell membrane. In summary, islet interactions with laminin protect against cytokine-induced β-cell death via downregulation of PKCΔ activity at the cell membrane. Our results suggest that loss of islet interactions with laminin, as occurs in T1D, may make islets more susceptible to cytokine-mediated death. This mechanism of β-cell death provides a novel therapeutic target to prevent β-cell death and halt the progression of T1D. Disclosure N.L. Farnsworth: None. R.A. Piscopio: None. R.K. Benninger: None. Funding JDRF (3-APF-2019-749-A-N, 1-FAC-2020-891-A-N); Colorado Clinical and Translational Sciences Institute (CO-M-19-133)

Toxoplasma gondii ADSL Knockout Provides Excellent Immune Protection against a Variety of Strains.

Toxoplasma gondii is a protozoan parasite, occurring worldwide, endangers human health and causes enormous economic losses to the Ministry of Agriculture. A safe and effective vaccination is needed to handle these problems. In addition, ideal vaccine production is a challenge in the future. In this study, we knocked out the adenylosuccinate lyase (ADSL) gene and found that the gene reduces the growth rate of T. gondii tachyzoites in vitro under standard growth conditions by plaque or replication experiments. Furthermore, mice that were immunized with tachyzoites of the ME49ΔADSL strain induced 100% protection efficacy against challenge with the type 1 strain RH, type 2 strain ME49 and type 3 strain VEG. All mice that were immunized with ME49ΔADSL had a survival rate of 100% when they were reinfected with wild-type strains, either 30 days or 70 days after immunization, and immunization was also protective against homologous infection with 50 T. gondii ME49 tissue cysts. In addition, the level of Toxoplasma-specific IgG was significantly elevated at 30 and 70 days after immunization. ME49ΔADSL induced high levels of Th1 cytokines (interferon gamma (IFN-γ), interleukin (IL)-12) at 4 weeks after immunization and spleen cell cultures from mice vaccinated for 150 days were able to produce robust INF-γ and IL-12 levels in the supernatant. The results of the present study showed that ΔADSL vaccination induced a T. gondii-specific cellular immune response against further infections. These results suggest that the ADSL-deficient vaccine can induce anti-Toxoplasma gondii humoral and cellular immune responses and has 100% immune protection against post-challenge by the type 1 strain RH, type 2 strain ME49 and type 3 strain VEG. It will be used as an excellent candidate for live vaccines and may contribute in a positive meaning to control human toxoplasmosis.