Suppression Assays Research Articles

Calycosin enhances Treg differentiation for alleviating skin inflammation in atopic dermatitis

Ethnopharmacological relevanceAtopic dermatitis (AD) is a prevalent chronic inflammatory skin disorder that poses a significant global health challenge. There is a lack of safe and effective medications to treat AD. Astragalus membranaceous is a traditional Chinese medicine widely used in clinical treatment of skin diseases. Calycosin (CA), derived from the root of Astragalus membranaceous, exhibits dual attributes of anti-inflammatory and antioxidant properties, suggesting its promise for addressing cutaneous inflammation. Nonetheless, the precise mechanisms underlying CA's therapeutic actions in AD remain elusive. Aim of the studyThis study aimed to evaluate the efficacy and safety of CA in treating AD while also delving into the mechanistic underpinnings of CA's action in AD. Materials and methodsThe cell viability and anti-inflammatory impacts of CA in vitro were first gauged using CCK-8 and RT-qPCR. The potential mechanisms of CA were then probed using modular pharmacology. Flow cytometry was employed to ascertain the differentiation of Treg and Th17 cells derived from naïve T cells, as well as the proportions and mean fluorescence intensity (MFI) of human iTreg cells. The expressions of IL-10 and TGF-β1 were measured and Treg suppression assay was performed. The in vivo therapeutic efficacy of topical CA application was assessed using a calcipotriol (MC903)-induced AD mouse model. The expression metrics of inflammatory cytokines, IL-17A, FOXP3, and RORγt were authenticated via immunohistochemistry, RT-qPCR, Western blot, and ELISA. ResultsCA exhibited a favorable safety profile and reduced the mRNA expressions of Th2 inflammatory cytokines in HaCaT cells. Modular pharmacology analysis pinpointed Th17 differentiation as the pivotal mechanism behind CA's therapeutic effect on AD. In vitro, CA fostered the differentiation of naïve T cells into Tregs while inhibiting their differentiation into Th17 cells. Furthermore, CA augmented the proliferation of human iTregs. In vivo, CA alleviated skin manifestations and decreased the levels of inflammatory mediators (IL-4, IL-5, IL-13, TSLP, and NF-κB related cytokines) in AD-like mouse models. Simultaneously, it regulated Treg/Th17 balance through suppressing IL-17A and RORγt expressions and bolstering FOXP3 expression. ConclusionsThe study provides insights into the mechanistic pathways through which CA exerts its anti-inflammatory effects, particularly through promoting Treg cell differentiation and inhibiting Th17 cell differentiation. Furthermore, CA emerges as an alternative or adjunctive treatment strategy for managing AD.

Neuronal knockdown of Cullin3 as a Drosophila model of autism spectrum disorder

Mutations in Cullin-3 (Cul3), a conserved gene encoding a ubiquitin ligase, are strongly associated with autism spectrum disorder (ASD). Here, we characterize ASD-related pathologies caused by neuron-specific Cul3 knockdown in Drosophila. We confirmed that neuronal Cul3 knockdown causes short sleep, paralleling sleep disturbances in ASD. Because sleep defects and ASD are linked to metabolic dysregulation, we tested the starvation response of neuronal Cul3 knockdown flies; they starved faster and had lower triacylglyceride levels than controls, suggesting defects in metabolic homeostasis. ASD is also characterized by increased biomarkers of oxidative stress; we found that neuronal Cul3 knockdown increased sensitivity to hyperoxia, an exogenous oxidative stress. Additional hallmarks of ASD are deficits in social interactions and learning. Using a courtship suppression assay that measures social interactions and memory of prior courtship, we found that neuronal Cul3 knockdown reduced courtship and learning compared to controls. Finally, we found that neuronal Cul3 depletion alters the anatomy of the mushroom body, a brain region required for memory and sleep. Taken together, the ASD-related phenotypes of neuronal Cul3 knockdown flies establish these flies as a genetic model to study molecular and cellular mechanisms underlying ASD pathology, including metabolic and oxidative stress dysregulation and neurodevelopment.

The FOXP3+ Pro-Inflammatory T Cell: A Potential Therapeutic Target in Crohn’s Disease

Background and aimsThe incidence of Crohn’s disease (CD) continues to increase worldwide. The contribution of CD4+ cell populations remains to be elucidated. Here, we aim to provide an in-depth transcriptional assessment of CD4+ T cells driving chronic inflammation in CD. MethodsWe performed single cell RNA-sequencing in CD4+ T cells isolated from ileal biopsies of CD patients compared to healthy individuals. Cells underwent clustering analysis, followed by analysis of gene signalling networks. We overlapped our differentially expressed genes with publicly available microarray datasets and performed functional in vitro studies, including an in vitro suppression assay and organoid systems, to model gene expression changes observed in CD TREGs and to test predicted therapeutics. ResultsWe identified five distinct FOXP3+ regulatory T (Treg) subpopulations. Tregs isolated from healthy controls represent the origin of pseudotemporal development into inflammation-associated subtypes. These proinflammatory Tregs displayed a unique responsiveness to TNFα signaling with impaired suppressive activity in vitro and an elevated cytokine response in an organoid co-culture system. As predicted in silico, the histone deacetylase inhibitor Vorinostat normalized gene expression patterns, rescuing the suppressive function of FOXP3+ cells in vitro. ConclusionWe identified a novel, proinflammatory FOXP3+ T cell subpopulation in CD patients and developed a pipeline to specifically target these cells using the FDA-approved drug Vorinostat.

Targeting the activin receptor 1C on CD4+ T cells for cancer immunotherapy

ABSTRACT Activins, members of the TGF-beta superfamily, have been isolated and identified in the endocrine system, but have not been substantially investigated in the context of the immune system and endocrine-unrelated cancers. Here, we demonstrated that tumor-bearing mice had elevated systemic activin levels, which correlated directly with tumor burden. Likewise, cancer patients have elevated plasma activin levels compared to healthy controls. We observed that both tumor and immune cells could be sources of activins. Importantly, our in vitro studies suggest that activins promote differentiation of naïve CD4+ cells into Foxp3-expressing induced regulatory T cells (Tregs), particularly when TGF-beta was limited in the culture medium. Database and qRT-PCR analysis of sorted major immune cell subsets in mice revealed that activin receptor 1c (ActRIC) was uniquely expressed on Tregs and that both ActRIC and ActRIIB (activin receptor 2b) were highly upregulated during iTreg differentiation. ActRIC-deficient naïve CD4+ cells were found to be defective in iTreg generation both in vitro and in vivo. Treg suppression assays were also performed, and ActRIC deficiency did not change the function or stability of iTregs. Mice lacking ActRIC or mice treated with monoclonal anti-ActRIC antibody were more resistant to tumor progression than wild-type controls. This phenotype was correlated with reduced expression of Foxp3 in CD4+ cells in the tumor microenvironment. In light of the information presented above, blocking activin-ActRIC signaling is a promising and disease-specific strategy to impede the accumulation of immunosuppressive iTregs in cancer. Therefore, it is a potential candidate for cancer immunotherapy.

Distinct characteristics of unique immunoregulatory canine non-conventional TCRαβpos CD4negCD8αneg double-negative T cell subpopulations.

Conventional CD4pos regulatory T (Treg) cells characterized by expression of the key transcription factor forkhead box P3 (FoxP3) are crucial to control immune responses, thereby maintaining homeostasis and self-tolerance. Within the substantial population of non-conventional T cell receptor (TCR)αβpos CD4negCD8αneg double-negative (dn) T cells of dogs, a novel FoxP3pos Treg-like subset was described that, similar to conventional CD4pos Treg cells, is characterized by high expression of CD25. Noteworthy, human and murine TCRαβpos regulatory dn T cells lack FoxP3. Immunosuppressive capacity of canine dn T cells was hypothesized based on expression of inhibitory molecules (interleukin (IL)-10, cytotoxic T-lymphocyte associated protein 4, CTLA4). Here, to verify their regulatory function, the dnCD25pos (enriched for FoxP3pos Treg-like cells) and the dnCD25neg fraction, were isolated by fluorescence-activated cell sorting from peripheral blood mononuclear cells (PBMC) of Beagle dogs and analyzed in an in vitro suppression assay in comparison to conventional CD4posCD25pos Treg cells (positive control) and CD4posCD25neg T cells (negative control). Canine dnCD25pos T cells suppressed the Concanavalin A-driven proliferation of responder PBMC to a similar extent as conventional CD4posCD25pos Treg cells. Albeit to a lesser extent than FoxP3-enriched dn and CD4posCD25pos populations, even dnCD25neg T cells reduced the proliferation of responder cells. This is remarkable, as dnCD25neg T cells have a FoxP3neg phenotype comparable to non-suppressive CD4posCD25neg T cells. Both, CD25pos and CD25neg dn T cells, can mediate suppression independent of cell-cell contact and do not require additional signals from CD4posCD25neg T cells to secrete inhibitory factors in contrast to CD4posCD25pos T cells. Neutralization of IL-10 completely abrogated the suppression by dnCD25pos and CD4posCD25pos Treg cells in a Transwell™ system, while it only partially reduced suppression by dnCD25neg T cells. Taken together, unique canine non-conventional dnCD25pos FoxP3pos Treg-like cells are potent suppressor cells in vitro. Moreover, inhibition of proliferation of responder T cells by the dnCD25neg fraction indicates suppressive function of a subset of dn T cells even in the absence of FoxP3. The identification of unique immunoregulatory non-conventional dn T cell subpopulations of the dog in vitro is of high relevance, given the immunotherapeutic potential of manipulating regulatory T cell responses in vivo.

P4 protein of an Indian isolate of rice tungro bacilliform virus modulates gene silencing.

Plant hosts and their viral pathogens are engaged in a constant cycle of defense and counter-defense as part of a molecular arms race, principal among them being the plant RNAi defense and the viral RNAi suppressor counter-defense. Rice tungro bacilliform virus (RTBV), member of the family Caulimoviridae, genus Tungrovirus, species Tungrovirus oryzae, infects rice in South- and Southeast Asia and causes severe symptoms of stunting, yellow-orange discoloration and twisting of leaf tips. To better understand the possible counter-defensive roles of RTBV against the host RNAi defense system, we explored the ability of the P4 protein of an Indian isolate of RTBV to act as a possible modulator of RNAi. Using a transient silencing and silencing suppression assay in Nicotiana benthamiana, we show that P4 not only displays an RNAi suppressor function, but also potentially enhances RNAi. The results also suggests that the N-terminal 168 amino acid residues of P4 are sufficient to maintain RNAi suppressor activity. Taken together with the earlier reports this work strengthens the view that the P4 protein carries out RNAi suppressor and a potential RNAi enhancer function.

Tramadol suppresses growth of orthotopic liver tumors via promoting M1 macrophage polarization in the tumor microenvironment.

Tumor-associated macrophages (TAMs) are major infiltrating immune cells in liver cancer. They are polarized to anti-tumor M1 type or tumor-supporting M2 type in a dynamic changing state. Tramadol, a synthetic opioid, exhibits tumor-suppressing effect in several cancers, but whether it plays a role in TAMs polarization is uncertain. In the present study, the potential influence of tramadol on TAMs polarization was explored in liver cancer. An orthotopic murine Hepa 1-6 liver cancer model was constructed. The potential function of tramadol was evaluated by cell viability assay, EdU incorporation assay, flow cytometry, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) assay, T cell proliferation and suppression assays and western blot. We found that tramadol suppressed proliferation and tumor formation of murine Hepa 1-6 cells in vitro and in vivo. Tramadol reprogramed the immune microenvironment to favor M1 macrophage polarization in orthotopic Hepa 1-6 tumors. Moreover, tramadol facilitated M1 macrophage polarization and inhibited M2 macrophage polarization of bone marrow-derived macrophages (BMDMs) and human THP-1 macrophages in vitro. Furthermore, tramadol-treated BMDMs promoted proliferation and activation of splenic CD4+ and CD8+ T cells. Tramadol induced cellular ROS production and mitochondrial dysfunction of BMDMs. Finally, tramadol activated NF-κB signaling in BMDMs and THP-1 macrophages, while inhibition of NF-κB signaling by JSH-23 attenuated the influence of tramadol on macrophage polarization. In conclusion, these data elucidated a novel anti-tumor mechanism of tramadol in liver cancer. Tramadol might be a promising treatment strategy for liver cancer patients.

Abstract A009: CD103+CD56+ innate lymphoid cells are associated with an immunosuppressive microenvironment in ovarian cancer

Abstract Background: Regulatory innate lymphoid cells (ILCregs) are an emerging heterogenous subset of innate lymphoid cells that suppress immunity in several healthy and diseased contexts, including cancer. Previously, we identified CD56+ ILCregs from in vitro expanded tumour-infiltrating lymphocyte (TIL) cultures in patients with epithelial ovarian carcinoma (EOC). However, lineage-defining surface markers or transcription factors that distinguishes these regulatory populations from other conventional NK cells have not been identified. The objective of this study is to characterize markers that define ILCregs directly from primary tumours and investigate its role within the tumour microenvironment (TME). Methods: Women with suspected abdominal mass were recruited and consented at Gynecology Cancer Clinic at Princess Margaret Hospital. Surgically resected EOC tumours were processed and analyzed by flow cytometry and single-cell RNA-sequencing. Suppression assays were performed by co-culture of tumour-infiltrating CD103+CD56+ ILCregs with autologous TILs for 4 days in vitro with soluble αCD3. Results: We found that CD103 expressing CD56+ ILCregs were associated with poor clinical prognosis in patients with ovarian carcinoma. CD103+CD56+ ILCregs displayed reduced expression of cytolytic related markers (i.e., GZMB, CD107a, CD16) compared to CD103-CD56+ NK cells and had a distinct transcriptomic profile from NK cells. Interestingly, CD103+CD56+ ILCregs displayed distinct surface markers (i.e., CD49a, CD69, GITR) and transcription factor networks that were associated with Treg development, differentiation, and suppressive function. Intratumoural CD103+CD56+ ILCregs suppressed autologous CD8+ T cells by downregulation of granzyme B in vitro. Concordantly, the abundance of CD103+CD56+ ILCregs in patient tumours were associated with reduced activated CD8+ T cells within the TME. Conclusion: This study identified CD103+CD56+ ILCregs in the TME of patients with EOC that are associated with poor clinical prognosis and plays a key role in regulating T cells. Further investigations into underlying immunoregulatory networks, including ILCregs, within the TME may provide novel targets to improve prognosis for patients with EOC. Citation Format: Douglas C Chung, Jehan Vakharia, Kathrin Warner, Nicolas Jacquelot, Azin Sayad, SeongJun Han, Maryam Ghaedi, Carlos R Garcia-Batres, Alisha Elford, Ben X Wang, Linh T Nguyen, Patricia A Shaw, Blaise A Clarke, Marcus Q Bernardini, Sarah E Ferguson, Pamela S Ohashi. CD103+CD56+ innate lymphoid cells are associated with an immunosuppressive microenvironment in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A009.

A novel anti-galectin-9 immunotherapy limits the early progression of pancreatic neoplastic lesions in transgenic mice.

Pancreatic adenocarcinoma (PDAC) is a devastating disease with an urgent need for therapeutic innovation. Immune checkpoint inhibition has shown promise in a variety of solid tumors, but most clinical trials have failed to demonstrate clinical efficacy in PDAC. This low efficacy is partly explained by a highly immunosuppressive microenvironment, which dampens anti-tumor immunity through the recruitment or induction of immunosuppressive cells, particularly regulatory T cells (Tregs). In this context, our laboratory has developed a novel immunotherapeutic strategy aimed at inhibiting the suppressive activity of Tregs, based on a patented (EP3152234B1) monoclonal antibody (mAb) targeting galectin-9 (LGALS9). CD4+ conventional T cells (TCD4 or Tconv), Treg ratio, and LGALS9 expression were analyzed by immunohistochemistry (IHC) and cytometry in blood and pancreas of K-rasLSL.G12D/+;Pdx-1-Cre (KC) and K-rasWildType (WT);Pdx1-Cre (WT) mice aged 4-13 months. Pancreatic intraepithelial neoplasm (PanIN) progression and grade were quantified using FIJI software and validated by pathologists. The anti-galectin-9 mAb was validated for its use in mice on isolated murine C57BL/6 Treg by immunofluorescence staining and cytometry. Its specificity and functionality were validated in proliferation assays on rLGALS9-immunosuppressed murine Tconv and in suppression assays between murine Treg and Tconv. Finally, 2-month-old KC mice were treated with anti-LGALS9 and compared to WT mice for peripheral and infiltrating TCD4, Treg, and PanIN progression. IHC and cytometry revealed a significant increase in LGALS9 expression and Treg levels in the blood and pancreas of KC mice proportional to the stages of precancerous lesions. Although present in WT mice, LGALS9 is expressed at a basal level with low and restricted expression that increases slightly over time, while Treg cells are few in number in their circulation and even absent from the pancreas over time. Using our anti-LGALS9 mAb in mice, it is shown that (i) murine Treg express LGALS9, (ii) the mAb could target and inhibit recombinant murine LGALS9, and (iii) neutralize murine Treg suppressive activity. Finally, the anti-LGALS9 mAb in KC mice reduced (i) LGALS9 expression in pancreatic cancer cells, (ii) the Treg ratio, and (iii) the total surface area and grade of PanIN. We demonstrate for the first time that an anti-LGALS9 antibody, by specifically targeting endogenous LGALS9 tumor and exogenous LGALS9 produced by Treg, was able to limit the progression of pancreatic neoplastic lesions in mice, opening up new prospects for its use as an immunotherapeutic tool in PDAC.

CsMYB67 participates in the flavonoid biosynthesis of summer tea leaves.

Flavonoids are important compounds in tea leaves imparting bitter and astringent taste, which also play key roles in tea plants responding to environmental stress. Our previous study showed that the expression level of CsMYB67 was positively correlated with the accumulation of flavonoids in tea leaves as exposed to sunlight. Here, we newly reported the function of CsMYB67 in regulating flavonoid biosynthesis in tea leaves. CsMYB67 was localized in the nucleus and responded to temperature. The results of transient expression assays showed the co-transformation of CsMYB67 and CsTTG1 promoted the transcription of CsANS promoter in the tobacco system. CsTTG1 was bound to the promoter of CsANS based on the results of yeast one-hybrid (Y1H) and transient expression assays, while CsMYB67 enhanced the transcription of CsANS through protein interaction with CsTTG1 according to the results of yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC). Thus, CsMYB67-CsTTG1 module enhanced the anthocyanin biosynthesis through up-regulating the transcription of CsANS. Besides, CsMYB67 also enhanced the transcription of CsFLS and CsUFGT through forming transcription factor complexes. The function of CsMYB67 on flavonoid biosynthesis in tea leaves was validated by gene suppression assay. As CsMYB67 was suppressed, the transcriptional level of CsFLS was greatly reduced, leading to a significant increase in the contents of total catechins and total anthocyanidins. Hence, CsMYB67 plays an important role in regulating the downstream pathway of flavonoid biosynthesis in summer tea leaves.

A community effort in SARS-CoV-2 drug discovery.

The COVID-19 pandemic continues to pose a substantial threat to human lives and is likely to do so for years to come. Despite the availability of vaccines, searching for efficient small-molecule drugs that are widely available, including in low- and middle-income countries, is an ongoing challenge. In this work, we report the results of an open science community effort, the "Billion molecules against COVID-19 challenge", to identify small-molecule inhibitors against SARS-CoV-2 or relevant human receptors. Participating teams used a wide variety of computational methods to screen a minimum of 1 billion virtual molecules against 6 protein targets. Overall, 31 teams participated, and they suggested a total of 639,024 molecules, which were subsequently ranked to find 'consensus compounds'. The organizing team coordinated with various contract research organizations (CROs) and collaborating institutions to synthesize and test 878 compounds for biological activity against proteases (Nsp5, Nsp3, TMPRSS2), nucleocapsid N, RdRP (only the Nsp12 domain), and (alpha) spike protein S. Overall, 27 compounds with weak inhibition/binding were experimentally identified by binding-, cleavage-, and/or viral suppression assays and are presented here. Open science approaches such as the one presented here contribute to the knowledge base of future drug discovery efforts in finding better SARS-CoV-2 treatments.

Evidence of antagonism in vitro and in vivo of extracts from Banisteriopsis laevifolia (A. Juss) B. Gates against the rice blast fungus

Rice blast is a common disease caused by Magnaporthe oryzae which affects rice production worldwide, including Brazil. To reduce the use of harmful pesticides, sustainable control options are necessary for managing rice blast. One possible option is to use plant extracts because they produce secondary metabolites capable of inhibiting the pathogen and controlling the disease. This study aimed to evaluate the potential of ethanolic extracts of leaves and flowers from Banisteriopsis laevifollia, a native Brazilian plant, to antagonise M. oryzae. In vitro antifungal activity assays were carried out, in which mycelium growth reduction, conidia germination, and appressorium formation of M. oryzae were evaluated. In addition, in vivo assay for suppression of leaf blast severity by extracts was done. We also explored the identification of secondary metabolites from ethanolic extracts of leaves and flowers by HPLC–HRMS. Results showed that both leaf and flower extracts inhibited mycelial growth in 21.72% and 30.49%, respectively, due to the inhibition of ergosterol production. Additionally, both extracts inhibited melanin production in the mycelium of M. oryzae by inhibiting tyrosine production. Both leaf and flower extracts significantly inhibited conidia germination and appressorium formation. Furthermore, rice leaves treated with the extracts suppressed by 18.17% and 18.97% leaf blast severity, with the 1.00 mg/mL concentration of flower extract and 0.75 mg/mL concentration of leaf extract, respectively, showing the most significant reduction of leaf blast. The extracts contain phenolic compounds, including 2,3,7,8-tetrahydroxy-chromeno-(5,4,3)-chromene-5,10-dione, 3-(4-hydroxyphenyl)-2-propenoic acid, 2-(3,4-dihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7-triol, 3,4′,5,7-tetrahydroxyflavone, 3′,4′,5,7-tetrahydroxyflavone, 4′,5,7-trihydroxyflavanone, 3,3′,4′,5,7-pentahydroxyflavone, 3,3′,4′,5,7-pentahydroxyflavone -3-O-glucoside, and 3,3′,4′,5,7-pentahydroxyflavone-3-O-rutinoside, which are likely responsible for the antifungal activity of the extracts. Overall, this study demonstrates the potential of plant extracts as a sustainable alternative for the management of rice blast.

A novel peanut allergy immunotherapy: Plant-based enveloped Ara h 2 Bioparticles activate dendritic cells and polarize T cell responses to Th1

IntroductionAs the only market-authorized allergen immunotherapy (AIT) for peanut allergy is accompanied by a high risk of side effects and mainly induces robust desensitization without sustained efficacy, novel treatment options are required. Peanut-specific plant-derived eBioparticles (eBPs) surface expressing Ara h 2 at high density have been shown to be very hypoallergenic. Here, we assessed the dendritic cell (DC)-activating and T cell polarization capacity of these peanut-specific eBPs. MethodsRoute and kinetics of eBP uptake were studied by (imaging) flow cytometry using monocyte-derived DCs incubated with fluorescently-labelled Ara h 2 eBPs or natural Ara h 2 (nAra h 2) in the presence or absence of inhibitors that block pathways involved in macropinocytosis, phagocytosis, and/or receptor-mediated uptake. DC activation was monitored by flow cytometry (maturation marker expression) and ELISA (cytokine production). T cell polarization was assessed by co-culturing DCs exposed to Ara h 2 eBPs or nAra h 2 with naïve CD4+ T cells, followed by flow cytometry assessment of intracellular IFNγ+ (Th1) and IL-13+ (Th2), and CD25+CD127-Foxp3+ regulatory T cells (Tregs). The suppressive activity of Tregs was tested using a suppressor assay. ResultsAra h 2 eBPs were taken up by DCs through actin-dependent pathways. They activated DCs demonstrated by an induced expression of CD83 and CD86, and production of TNFα, IL-6, and IL-10. eBP-treated DCs polarized naïve CD4+ T cells towards Th1 cells, while reducing Th2 cell development. Furthermore, eBP-treated DCs induced reduced the frequency of Foxp3+ Tregs but did not significantly affect T cell IL-10 production or T cells with suppressive capacity. In contrast, DC activation and Th1 cell polarization were not observed for nAra h 2. ConclusionAra h 2 eBPs activate DCs that subsequently promote Th1 cell polarization and reduce Th2 cell polarization. These characteristics mark Ara h 2 eBPs as a promising novel candidate for peanut AIT.

Évaluation of the anti-malarial properties of the combined aqueous and alcoholic extract of Heliotropium indicum and Parquentina nigrescencs

This research investigates the potential antimalarial efficacy of combined extracts derived from Heliotropium indicum and Parquetina nigrescens, considering their traditional applications in addressing various ailments, including malaria. The primary objective is to assess the suppressive antimalarial activity of the combined extracts and compare it with individual extracts, utilizing toxicity assessments and syrup formulations. The study includes LD50 assessments to confirm the safety of the extracts, employing doses beyond 5000mg/kg. Suppressive assays are conducted on mice, evaluating both individual and combined extracts, with varying doses. White mice from a reputable source are used for the experiments. The extracts are prepared as syrups, and different doses are administered to the mice for toxicity and antimalarial assessment. Parasitemia is evaluated through blood films, and statistical analysis is performed. The obtained data are analyzed using one-way ANOVA and Student-Newman Keul’s test to determine statistical significance. Results are expressed as mean ± SEM. LD50 assessments reveal the safety of the extracts, and suppressive assays demonstrate significant antimalarial effects for both individual extracts at distinct doses. The combined aqueous extract shows promising outcomes, indicating potential synergistic interactions. Traditional plant-based remedies, such as Heliotropium indicum and Parquetina nigrescens, hold promise for developing innovative antimalarial strategies. The combined extract exhibits potential as a complementary antimalarial intervention, warranting further exploration and research in the field of malaria management.

Crosstalk between the tRNA methyltransferase Trm1 and RNA chaperone La influences eukaryotic tRNA maturation

tRNAs undergo an extensive maturation process involving posttranscriptional modifications often associated with tRNA structural stability and promoting the native fold. Impaired posttranscriptional modification has been linked to human disease, likely through defects in translation, mitochondrial function, and increased susceptibility to degradation by various tRNA decay pathways. More recently, evidence has emerged that bacterial tRNA modification enzymes can act as tRNA chaperones to guide tRNA folding in a manner independent from catalytic activity. Here, we provide evidence that the fission yeast tRNA methyltransferase Trm1, which dimethylates nuclear- and mitochondrial-encoded tRNAs at G26, can also promote tRNA functionality in the absence of catalysis. We show that WT and catalytic-dead Trm1 are active in an invivo tRNA-mediated suppression assay and possess RNA strand annealing and dissociation activity invitro, similar to previously characterized RNA chaperones. Trm1 and the RNA chaperone La have previously been proposed to function synergistically in promoting tRNA maturation, yet we surprisingly demonstrate that La binding to nascent pre-tRNAs decreases Trm1 tRNA dimethylation invivo and invitro. Collectively, these results support the hypothesis for tRNA modification enzymes that combine catalytic and noncatalytic activities to promote tRNA maturation, as well as expand our understanding of how La function can influence tRNA modification.

Polymorphonuclear myeloid-derived suppressor cells and phosphatidylinositol-3 kinase gamma are critical to tobacco-mimicking oral carcinogenesis in mice

BackgroundOral squamous cell carcinoma (OSCC) is a devastating disease most often associated with tobacco consumption that induces a field of mutations from which a tumor arises. Identification of ways to...

Increased SLAMF7+CD8+ T cells are associated with the pathogenesis of experimental autoimmune pancreatitis in mice

BackgroundIgG4-related autoimmune pancreatitis (AIP) is considered to be a T cell-mediated autoimmune disease. However, CD8+ T cells have only received brief mention, and have yet to be completely studied. The study aimed to investigate the expression of signaling lymphocytic activation molecule family 7 (SLAMF7) on CD8+ T cells and the features of SLAMF7+CD8+ T cells in MRL/Mp mice with AIP. MethodsA murine model of AIP was established by intraperitoneal injection with polyinosinic:polycytidylic acid (poly I:C) for 8 weeks. Dexamethasone treatment was daily administrated for the last 2 weeks during a 6-week course of poly I:C. SLAMF7 expression on CD8+ T cells in the spleen and pancreas was detected by flow cytometry. Granzyme B (GZMB) and cytokines including IFN-γ, TNF-α, and IL-2, were monitored in an in vitro T cell activation assay. Dexamethasone suppression assays were performed to downregulate SLAMF7 expression on T cells upon T cell receptor stimulation. ResultsAIP in MRL/Mp mice was induced by repeated intraperitoneal administration of poly I:C and CD8+ T cells were increased in the inflamed pancreas. SLAMF7+CD8+ T cells were elevated in the spleen and pancreas of AIP mice. SLAMF7+CD8+ T subsets produced more GZMB, IFN-γ, TNF-α and IL-2 than SLAMF7−CD8+ T subsets. Dexamethasone treatment ameliorated pancreatic inflammatory and fibrosis of AIP. Dexamethasone could downregulate SLAMF7+CD8+ T cells and reduce GZMB, IFN-γ and TNF-α levels both in vitro and in vivo. ConclusionsIncreased SLAMF7+CD8+ T cells exhibit enhanced cytotoxicity and cytokines secretion capacity, which may be involved in the pathogenesis of AIP.

Regulation of mouse exploratory behaviour by irradiance and cone-opponent signals

BackgroundAnimal survival depends on the ability to adjust behaviour according to environmental conditions. The circadian system plays a key role in this capability, with diel changes in the quantity (irradiance) and spectral content (‘colour’) of ambient illumination providing signals of time-of-day that regulate the timing of rest and activity. Light also exerts much more immediate effects on behaviour, however, that are equally important in shaping daily activity patterns. Hence, nocturnal mammals will actively avoid light and dramatically reduce their activity when light cannot be avoided. The sensory mechanisms underlying these acute effects of light are incompletely understood, particularly the importance of colour.ResultsTo define sensory mechanisms controlling mouse behaviour, we used photoreceptor-isolating stimuli and mice with altered cone spectral sensitivity (Opn1mwR), lacking melanopsin (Opn1mwR; Opn4−/−) or cone phototransduction (Cnga3−/−) in assays of light-avoidance and activity suppression. In addition to roles for melanopsin-dependent irradiance signals, we find a major influence of spectral content in both cases. Hence, remarkably, selective increases in S-cone irradiance (producing a blue-shift in spectrum replicating twilight) drive light-seeking behaviour and promote activity. These effects are opposed by signals from longer-wavelength sensitive cones, indicating a true spectrally-opponent mechanism. Using c-Fos-mapping and multielectrode electrophysiology, we further show these effects are associated with a selective cone-opponent modulation of neural activity in the key brain site implicated in acute effects of light on behaviour, the subparaventricular zone.ConclusionsCollectively, these data reveal a mechanism whereby blue-shifts in the spectrum of environmental illumination, such as during twilight, promote mouse exploratory behaviour.

Regulatory T cells in autoimmune primary adrenal insufficiency.

Primary adrenal insufficiency (PAI) is most often caused by an autoimmune destruction of the adrenal cortex resulting in failure to produce cortisol and aldosterone. The aetiology is thought to be a combination of genetic and environmental risk factors, leading to breakdown of immunological tolerance. Regulatory T cells (Tregs) are deficient in many autoimmune disorders, but it is not known whether they contribute to development of PAI. We aimed to investigate the frequency and function of naive and expanded Tregs in patients with PAI and polyendocrine syndromes compared to age- and gender-matched healthy controls. Flow cytometry was used to assess the frequency and characterize functional markers of blood Tregs in PAI (N = 15). Expanded Treg suppressive abilities were assessed with a flow cytometry based suppression assay (N = 20), while bulk RNA-sequencing was used to examine transcriptomic differences (N = 16) and oxygen consumption rate was measured by a Seahorse cell metabolic assay (N = 11). Our results showed that Treg frequency and suppressive capacity were similar between patients and controls. An increased expression of killer-cell leptin-like receptors and mitochondrial genes was revealed in PAI patients, but their expanded Tregs did not display signs of mitochondrial dysfunction. Our findings do not support a clear role for Tregs in the contribution of PAI development.

A target-based discovery from a parasitic helminth as a novel therapeutic approach for autoimmune diseases

Regulatory T cells (Tregs) can alleviate the development of autoimmune and inflammatory diseases, thereby proposing their role as a new therapeutic strategy. Parasitic helminths have co-evolved with hosts to generate immunological privilege and immune tolerance through inducing Tregs. Thus, constructing a "Tregs-induction"-based discovery pipeline from parasitic helminth is a promising strategy to control autoimmune and inflammatory diseases. The gel filtration chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC) were used to isolate immunomodulatory components from the egg extracts of Schistosoma japonicum. The extracted peptides were evaluated for their effects on Tregs suppressive functions using flow cytometry, ELISA and T cell suppression assay. Finally, we carried out colitis and psoriasis models to evaluate the function of Tregs induced by helminth-derived peptide invivo. Here, based on target-driven discovery strategy, we successfully identified a small 3kDa peptide (SjDX5-53) from egg extracts of schistosome, which promoted both human and murine Tregs production. SjDX5-53 presented immunosuppressive function by arresting dendritic cells (DCs) at an immature state and augmenting the proportion and suppressive capacity of Tregs. In mouse models, SjDX5-53 protected mice against autoimmune-related colitis and psoriasis through inducing Tregs and inhibiting inflammatory T-helper (Th) 1 and Th17 responses. SjDX5-53 exhibited the promising therapeutic effects in alleviating the phenotype of immune-related colitis and psoriasis. This study displayed a screening and validation pipeline of the inducer of Tregs from helminth eggs, highlighting the discovery of new biologics inspired by co-evolution of hosts and their parasites. This study was supported by the Natural Science Foundation of China (82272368) and Natural Science Foundation of Jiangsu Province (BK20211586).