High-affinity Receptor Research Articles

Cyclic adenosine monophosphate-elevating agents inhibit amyloid-beta internalization and neurotoxicity: their action in Alzheimer's disease prevention.

Cyclic adenosine monophosphate-elevating agents inhibit amyloid-beta internalization and neurotoxicity: their action in Alzheimer's disease prevention.

Wykorzystanie sekwencyjnego wirtualnego skriningu opartego na uczeniu maszynowym w poszukiwaniu nowych ligandów receptora 5-HT6

5-HT6 receptor takes part in learning and memory processes. For this reason, the use of ligands of this receptor in the treatment of neurodegenerative diseases such as Alzheimer's disease, depression or autism is being investigated. The development of machine learning (ML) and access to large compound databases allow for the increasing use of these methods in search of new drugs. The use of ML in pre-clinical tests allows for a reduction in time and costs of drug discovery. In this study, we used a sequential virtual screening approach in search of new structures with potential high affinity for the 5-HT6 receptor. Data from the ChEMBL database containing ligand binding affinities, measured as an inhibition constant (Ki), was used as the training dataset. Each step of the screening was based on machine learning models, the task of which was to classify compounds as potentially active and inactive. The first step included a ligand-based drug discovery (LBDD) approach, in which, using Klekota-Roth fingerprints and descriptors describing the chemical structure of the ligands, a classification model was developed to select a preliminary group of candidates from the Otava chemical compound database. In the second step, a structure-based drug discovery (SBDD) approach was used. For this purpose, compounds were docked to the homology model of the 5-HT6 receptor, developed using the AlphaFold algorithm and optimized by Induced-Fit Docking tool and molecular dynamics. Docking poses were scored by a trained Extra Trees classifier. Interactions of a reference ligand with 14 binding site residues were used as features for the trained model. The use of machine learning as a scoring function allowed to improve the virtual screening parameters compared to the Glide GScore scoring function. Based on the obtained model, it was also confirmed that the location of a ligand near the Ser5.43 and Phe5.38 residues is important for binding the compound to the receptor. The procedure has allowed to select 20 candidates with new chemical structures compared to known ligands. In addition, the obtained compounds had a relatively low basic pKa compared to known ligands and thus may be suspected to have a low affinity for hERG channels and good brain penetration.

Abstract 1866: D02, a novel, non-blocking antibody targeting IL2RA, exhibits significant anti-tumor activity in IL2RA-humanized mice

Abstract Regulatory T cells, or Tregs, are a subset of CD4+ T cells that negatively regulate immune responses to promote their resolution and protect against autoimmunity. Infiltration of Tregs in the tumor microenvironment (TME), and a low ratio of effector T cells to Tregs, is frequently associated with tumor progression and poor prognosis. The alpha subunit of the IL-2 cytokine receptor (IL2RA), a component of the high-affinity IL-2 receptor along with the β and γ subunits (IL2Rβγ), is constitutively expressed on Tregs and transiently expressed on effector T cells. IL-2 is a key cytokine for effector T cell survival and cytolytic responses. We aimed to discover an IL2RA-targeting antibody capable of depleting Tregs but permitting IL-2 stimulation of effector T cells in the TME. We developed a fully human IgG1 antibody specific to IL2RA, D02, from our proprietary RenMab™ mice, which contain the entire human immunoglobulin variable domain. D02 binds both human and cynomolgus monkey IL2RA with high affinity without hindering IL2RA-IL-2 binding or inhibiting IL-2 signaling. To evaluate the safety and efficacy of D02 in vivo, we established a syngeneic mouse tumor model in IL2RA-humanized mice. We observed that D02 potently inhibited MC38 (colon adenocarcinoma) tumor growth at 10 mg/kg. Further studies will aim to characterize the effect of D02 on the ratio of effector T cells to Tregs in the TME. These data indicate that D02 is a novel, IL2RA-specific monoclonal antibody that uniquely exhibits significant anti-tumor activity in vivo while preserving IL-2 signaling to effector T cells. Citation Format: Shuzhen Cao, Wenjiao Zhang, Qingcong Lin. D02, a novel, non-blocking antibody targeting IL2RA, exhibits significant anti-tumor activity in IL2RA-humanized mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1866.

Abstract 1801: STK-012, an a/b-selective IL-2 activates tumor antigen specific CD25+ CD8 T cells to reject tumors without acute vascular toxicity

Abstract High-dose Interleukin-2 (IL-2) monotherapy induces complete responses in cancer patients but its use is limited by acute vascular toxicities including capillary leak syndrome and severe hypotension 1,2. IL-2 activates lymphocytes and NK-cells through the intermediate-affinity dimeric IL-2 receptor, IL-2Rβ/γ (CD122/CD132), while antigen activated T-cells and regulatory T-cells (Tregs) have increased sensitivity to IL-2 by expressing the high-affinity trimeric IL-2 receptor, IL-2Rα/β/γ (CD25/CD122/CD132)3. CD25-independent IL-2s (“non-α-IL-2s”) aim to increase the therapeutic efficacy of IL2 in cancer patients by avoiding Treg activation through selective binding to IL-2Rβ/γ 4. However, those molecules still are reported to induce fever and hypotension and have limited efficacy as a monotherapy or in combination with anti-PD-1 5,6. Here we show that a novel α/β-IL-2 agonist that was designed to preferentially bind to the IL-2Rα/β/γ receptor highly upregulated on antigen activated T-cells can greatly improve on the efficacy of IL-2 while avoiding the vascular toxicity commonly associated with IL-2 treatment. In syngeneic tumor models, this α/β-IL-2 agonist significantly reduced exhaustion of tumor infiltrating T cells compared to WT-IL-2 or a non-α-IL-2 leading to improved expansion of tumor antigen specific CD25+PD-1+CD8+ T cells systemically and in the tumor microenvironment. This resulted in complete responses and tumor immune memory with α/β-IL-2 monotherapy as well as improved outcomes in combination with anti-PD-1 therapy in PD-1 refractory syngeneic tumors. In contrast, WT-IL-2 reduced T cell exhaustion and drove antigen specific T cell responses to a lesser degree, resulting in reduced combinatorial efficacy with anti-PD-1, while the non-α-IL-2 failed to do either. Furthermore, the α/β-IL-2 agonist reduced intratumoral Tregs compared to treatment with WT-IL-2 or PBS improving the intratumoral CD8 to Treg ratio. In non-human primates and mice, WT-IL-2 and a non-α-IL-2 led to broad extravasation of lymphocytes and NK cells and activation of intra-pulmonal T cells resulting in systemic tissue inflammation and NK cell-mediated lethal capillary leak syndrome whereas the α/β-IL-2 agonist, which avoids binding the dimeric IL-2Rβ/γ expressed on NK cells, avoided systemic lymphocyte activation which facilitated continuous treatment without acute vascular toxicities. Overall, through selective engagement of CD25+ T cells, this α/β-IL-2 agonist demonstrated improved efficacy and tolerability of IL-2 in preclinical tumor models. Clinical trials with STK-012, a human α/β-IL-2 agonist, are in progress.1 Atkins, et al.; JCO 1999, 2 Dutcher, et al.; JITC 2014, 3 Liao, et al.; Immunity 2013, 4 Levin, et al.; Nature 2012, 5 Janku, et al.; Cancer Research 2021; 6 Diab, et al.; Cancer Disc. 2020 Citation Format: Martin Oft, Navneet Ratti, Sandro Vivona, Jan Emmerich, Romina Riener, Ievgen O. Koliesnik, Scott McCauley, Michele Bauer, Marie Semana, Deepti Rokkam, Bhargavi Jayaraman, Rene de Waal Malefyt, Paul-Joseph Aspuria, Michael Totagrande, Anita Mehta-Damani, Patrick J. Lupardus, Rob A. Kastelein. STK-012, an a/b-selective IL-2 activates tumor antigen specific CD25+ CD8 T cells to reject tumors without acute vascular toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1801.

Abstract 3782: Targeting cancer cells using folate conjugated to a fully modified version of miR-34a (FolamiR-34a) to produce enhanced and sustained anti-tumor activity

Abstract Due to the pleiotropic nature of miR-34a, including its ability to downregulate multiple genes that drive resistance to currently used anti-cancer therapies, we hypothesize that the best use of miR-34a in a clinical setting would be in combination with standard-of-care treatments. For in vivo use, such a molecule would necessitate the following: i) a stabilized version of miR-34a that can withstand the harsh environment in circulation and intracellular nucleases, and ii) a robust, specific, and safe delivery vehicle. With regard to stability, we successfully generated the first fully modified version of miR-34a. This new chemically modified molecule is over 400-fold more stable than the previously used partially modified version and induces enhanced and sustained target gene repression. To assess in vivo activity of fully modified miR-34a requires a suitable delivery vehicle. Indeed, while various approaches have been used to restore miR-34a in tumors, clinically relevant mechanisms for delivery of miR-34a have been a challenge due to poor tumor uptake, unfavorable bioavailability, and unwanted toxicity. Previously, we determined that a partially modified version of miR-34a can be delivered specifically and robustly to cancer cells in a targeted, vehicle-free manner through direct conjugation to folate, a ligand of the high-affinity folate receptors. Thus, we conjugated our fully modified miR-34a (FM-miR-34a) to folate generating FM-FolamiR-34a. Following in vivo delivery of FM-FolamiR-34a to mice with breast cancer xenografts we observed enhanced and sustained target gene repression, in comparison to our first-generation partially modified folate-miR-34a conjugate (FolamiR). Critical miR-34a target genes involved in promoting resistance, including MET, AXL, and CD44 were downregulated over 90% for at least 120 hours following a single 1.5 nmol dose of FM-FolamiR-34a. Our final objective is to evaluate FM-FolamiR-34a in combination with standard-of-care agents in vivo. Citation Format: Shreyas Ganesan Iyer, Ahmed M. Abdelaal, Ikjot Singh Sohal, Sudarsan R. Kasireddy, Andrea L. Kasinski. Targeting cancer cells using folate conjugated to a fully modified version of miR-34a (FolamiR-34a) to produce enhanced and sustained anti-tumor activity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3782.

Abstract 4158: Selective targeting of CD122 combined with radiotherapy triggers CD8 and NK mediated immunity, abrogating metastasis in head and neck squamous cell carcinoma

Abstract The implementation of cancer immunotherapies, while revolutionary in certain cancer types, has seen limited clinical success in head and neck squamous cell carcinomas (HNSCC). This revelation has prompted investigation in alternative targets to invigorate anti-tumor immunity. IL-2 signaling governs the survival and functionality of lymphocytes, thus making IL-2 receptor signaling an attractive target for new immunotherapies. However, therapies to enhance IL-2 signaling are often limited in their efficacy by modulating the activation of regulatory T cells (Tregs), which preferentially binds IL-2 via the high affinity receptor IL-2Rα (CD25). Selectively targeting IL2Rβ (CD122), the intermediate affinity receptor, can be leveraged to induce anti-tumor immune responses in effector T cells and natural killer (NK) cells while limiting negative regulation conferred by Tregs. The novel bispecific immunocytokine PD1-IL2v preferentially activates IL-2 signaling through CD122 on PD-1 expressing cells, activating immune effectors, and preventing immune exhaustion by blockade of PD-1. However, PD-1 blockade can amplify immunosuppression by Tregs, necessitating their depletion using αCD25. Here, we observe that radiation therapy and combination of PD1-IL2v with an Fc-optimized αCD25 antibody induces systemic activation of effector CD4 and CD8 T cells, reducing overall tumor burden. Concordant with improved local tumor control, we also observed a decrease in metastatic spread using a newly developed Kras/smad4/p53 mutated metastatic tumor model. NK cells, which are known to play a central role in the control of tumor cell dissemination though direct cytotoxicity, displayed a phenotypic shift towards an activated state upon treatment with PD1-IL2v, highlighted by enhanced cytotoxicity, NK receptor diversity, and metabolic activity. Although relatively uncommon in comparison to other cancer types, those patients who present with metastases display a substantially poorer outcome, demonstrating the need for therapies which target this axis of cancer disease progression. In summary, we find that radiation therapy and PD1-IL2v imparts potent anti-tumor immunity, reducing local tumor growth and distant tumor spread, which taken together contribute to increased overall survival. Citation Format: Jacob Gadwa, Maria Amann, Thomas E. Bickett, Michael W. Knitz, Laurel B. Darragh, Miles Piper, Benjamin Van Court, Sanjana Bukkapatanam, Tiffany T. Pham, Xiao-Jing Wang, Laura Codarri Deak, Christian Klein, Pablo Umana, Angelo D'Alessandro, Sana Karam. Selective targeting of CD122 combined with radiotherapy triggers CD8 and NK mediated immunity, abrogating metastasis in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4158.

Aripiprazole: Examining the Clinical Implications of D2 Affinity

AbstractBackgroundAripiprazole has high binding affinity for the dopamine D2 receptor, which is thought to be responsible for the antipsychotic effect, though aripiprazole is not the most potent of the second-generation antipsychotics. Theoretically, aripiprazole could displace or outcompete more potent antipsychotics, prompting decreased antipsychotic effect. We describe a case of aripiprazole potentially worsening psychiatric symptoms by blocking paliperidone.CaseMs. A is a 43-year-old woman with schizophrenia, multiple inpatient hospitalizations, and a history of court-ordered treatment. She historically has had good response to oral and long-acting formulations of risperidone and paliperidone. Ms. A requested a medication change and was transitioned to aripiprazole lauroxil injection with plan for bimonthly administration. Approximately 1 month after receiving her aripiprazole lauroxil injection, Ms. A presented to our CPEP due to symptoms of psychosis and was admitted to our inpatient unit. She was restarted on oral paliperidone, titrated up to her previously effective dose, and was transitioned to paliperidone palmitate LAI. In contrast to prior admissions, she did not respond well to paliperidone and displayed continued and worsened psychosis.DiscussionPrior studies have examined how adding aripiprazole to another, more potent D2 antagonist can cause a relapse in psychotic symptoms; however, few studies have investigated the inverse relationship or mechanism. Those that have proposed mechanisms typically refer to aripiprazole’s partial agonist activity as the causative factor, rather than an impediment to antipsychotic binding which we have described. Prescribers should be aware of this potential interaction and carefully consider initiating long-acting injectable forms of aripiprazole to avoid this phenomenon.FundingNo Funding

372 IPSC-Derived NK cells Exhibit Potent In Vitro and In Vivo Tumorcidal Activity Against Patient-Derived Glioblastoma Stem Cells (GSCs)

INTRODUCTION: Natural Killer (NK) cells play pivotal roles in cancer immune surveillance and harbor potent anti-tumor activities. We have engineered an induced pluripotent stem cells (iPSCs) derived, NK cell (termed FT538) that express 1) a high-affinity non-cleavable CD16 Fc receptor to facilitate antibody engagement 2) a membrane-bound IL-15/IL-15 receptor fusion to enhance NK activity and persistence and 3) a knock-out of CD38 to enhance NK metabolic fitness. FT538 is produced in a GMP-compliant manner as an off-the-shelf immunotherapy. Clinical experiences with FT538 have shown evidence of durable anti-tumor immunity after single administration in select lymphoma patients. METHODS: Laboratory characterization and murine modeling. RESULTS: Without specific antibody to engage CD16, FT538 exhibited potent in vitro activity against ∼80% of a panel of isocitrate dehydrogenase wild-type patient derived xenograft (PDX) glioblastoma lines, including MGG8. When directly injected into the murine brain, 50% of the FT538 persisted for >2 weeks without addition of exogenous cytokine. Moreover, the mice exhibited no evidence of neurologic compromise, and brains harvested after injection show no signs of FT538 activation. In vivo intratumoral injection of FT538 into an orthotopically established MGG8 glioblastoma caused an ∼20 fold increase in granzyme B release by FT538, with a comparable increase of cleaved Caspase 3 in the tumor proper. In independent experiments, >90% of mice orthotopically implanted MGG8 survived beyond 100 days after intra-tumoral FT538 while all mice treated with mock injection died by 60 days. For glioblastoma PDX refractory to FT538, such as MGG4, tumoricidal activity of FT583 can be induced in vitro and in vivo by the addition of a tri-specific engager targeting B7H3, a surface protein highly expressed in glioblastomas. CONCLUSIONS: Our results suggest that FT538 great promise as a glioblastoma therapy, with plans for translation into a first-in-human clinical trial.

Unveiling chronic spontaneous urticaria pathophysiology through systems biology

Chronic spontaneous urticaria (CSU) is a rare, heterogeneous, severely debilitating, and often poorly controlled skin disease resulting in an itchy eruption that can be persistent. Antihistamines and omalizumab, an anti-IgE mAb, are the only licensed therapies. Although CSU pathogenesis is not yet fully understood, mast cell activation through the IgE:high-affinity IgE receptor (FcεRI) axis appears central to the disease process. We sought to model CSU pathophysiology and identify in silico the mechanism of action of different CSU therapeutic strategies currently in use or under development. Therapeutic performance mapping system technology, based on systems biology and machine learning, was used to create a CSU interactome validated with gene expression data from patients with CSU and a CSU model that was used to evaluate CSU pathophysiology and the mechanism of action of different therapeutic strategies. Our models reflect the known role of mast cell activation as a central process of CSU pathophysiology, as well as recognized roles for different therapeutic strategies in this and other innate and adaptive immune processes. They also allow determining similarities and differences between them; anti-IgE and Bruton tyrosine kinase inhibitors play a more direct role in mast cell biology through abrogation of FcεRI signaling activity, whereas anti-interleukins and anti-Siglec-8 have a role in adaptive immunity modulation. In silico CSU models reproduced known CSU and therapeutic strategies features. Our results could help advance understanding of therapeutic mechanisms of action and further advance treatment research by patient profile.

205 Liquid Biopsy Biomarker for Differentiating Neurofibroma/Schwannoma From Other Solid Mass Mimics

INTRODUCTION: Interleukin13 receptor alpha 2 (IL13Ra2) is a high-affinity receptor for the interleukin 13 (IL13) cytokine. With the exception of the testes, in normal conditions, IL13Ra2 demonstrates little normal cell expression. Prior work in our laboratory has demonstrated that IL13Ra2 is expressed on benign and malignant peripheral nerve sheath tumors in sporadic and syndromic cases. We have shown in in vitro and in murine MPST models that the receptor can be utilized to facilitate the treatment of malignant peripheral nerve sheath tumors. While mice have membrane-bound and soluble forms of IL13Ra2, humans lack the soluble form. We have previously shown the detection of IL13Ra2 exosomes in the cerebrospinal fluid and plasma of patients with glioblastoma. METHODS: IL13Ra2 levels were obtained from plasma and tumor specimens of 17 patients with diagnoses of Schwannoma/Neurofibroma (n = 13) and Lipoma/AVM (n = 4). Plasma and Tumor specimens were collected pre-operatively on the day of tumor resection. Plasma levels of IL13Ra2 were measured with ELISA. The tumor specimen was placed in NP40 buffer for extraction then levels were measured with ELISA. RESULTS: The plasma level of IL13Ra2 is significantly elevated in patients with schwannoma/neurofibroma compared to patients with lipomas/AVMs. Increased level of IL13Ra2 in tumors correlates with the increased levels of IL13Ra2 in the plasma. CONCLUSIONS: Plasma level of IL13Ra2 has the potential for diagnosis and may serve as a liquid biopsy biomarker to help differentiate solid tumors like neurofibroma/schwannoma from lipomas /arteriovenous malformations.

Ligelizumab: A novel molecule in the management of chronic spontaneous urticaria

The next-generation high affinity monoclonal immunoglobulin E (IgE) inhibitor Ligelizumab is being tested in clinical studies to treat chronic spontaneous urticaria (CSU). In 2020, the US Food and Drug Administration (FDA) designated Ligelizumab (QGE031) as a breakthrough therapy for patients with chronic idiopathic urticaria; however, FDA clearance has not yet been granted, despite the phase 3 trial’s completion. Recent clinical investigations have shown that it has a considerable affinity for the high-affinity IgE receptor (FcRI) on the surface of immune cells, which is quite beneficial in the management of the distressing symptoms of CSU. Studies have been done comparing Ligelizumab to Omalizumab, the current standard of care for treating CSU that is refractory. While omalizumab is more effective than Ligelizumab at inhibiting IgE: CD23 interactions, Ligelizumab exhibits stronger inhibition of IgE binding to FcRI, basophil activation, IgE synthesis by B cells, and passive systemic anaphylaxis. To learn about this molecule’s extended characteristics, long-term efficacy and safety, and potential uses in other dermatological disorders, more clinical data is needed.

GRK2 differentially regulates FcεRI and MRGPRB2-mediated responses in mast cells.

In addition to high-affinity IgE receptor (FcεRI), a subtype of mouse mast cells (MCs) expresses a G protein-coupled receptor known as Mas-related G protein-coupled receptor (GPCR)-B2 (MRGPRB2; human ortholog MRGPRX2). GPCR kinase 2 (GRK2) is a Serine/Threonine kinase that phosphorylates GPCRs to promote their desensitization and internalization. We previously showed that silencing GRK2 expression in mouse bone marrow-derived MCs (BMMCs) blocks IgE-mediated degranulation. Compound 48/80 (C48/80), substance P (SP) and LL-37 cause degranulation in human and mouse MCs via MRGPRX2 and MRGPRB2, respectively. We also reported that C48/80 and SP cause desensitization and internalization of MRGPRX2, but LL-37 does not. Here, we generated mice with MC-specific deletion of Grk2 (Cpa3Cre+/Grk2fl/fl ) to determine its role on IgE-mediated responses and to assess whether it differentially regulates degranulation in response to LL-37, C48/80 and SP. Absence of GRK2 substantially inhibited IgE-mediated tyrosine phosphorylation of STAT5, calcium mobilization, and degranulation in mouse primary lung-derived MCs (PLMCs). By contrast, peritoneal MCs (PMCs) from Cpa3Cre+/Grk2fl/fl mice demonstrated significant enhancement of degranulation in response to C48/80 and SP, but not LL-37. Deletion of Grk2 in MCs attenuated IgE-mediated passive cutaneous anaphylaxis (PCA) and itch but not passive systemic anaphylaxis (PSA). Surprisingly, PSA was significantly reduced in Mrgprb2-/- mice. These findings suggest that GRK2 contributes to PCA and itch but not PSA. By contrast, GRK2 desensitizes MRGPRX2/B2-mediated responses to C48/80 and SP but not LL-37. However, IgE-mediated PSA likely involves the activation of MRGPRB2 by LL-37 or a similar agonist, whose function is resistant to modulation by GRK2.

Hapten-Decorated DNA Nanostructures Decipher the Antigen-Mediated Spatial Organization of Antibodies Involved in Mast Cell Activation.

The immunological response of mast cells is controlled by the multivalent binding of antigens to immunoglobulin E (IgE) antibodies bound to the high-affinity receptor FcεRI on the cell membrane surface. However, the spatial organization of antigen-antibody-receptor complexes at the nanometer scale and the structural constraints involved in the initial events at the cell surface are not yet fully understood. For example, it is unclear what influence the affinity and nanoscale distance between the binding partners involved have on the activation of mast cells to degranulate inflammatory mediators from storage granules. We report the use of DNA origami nanostructures (DON) functionalized with different arrangements of the haptenic 2,4-dinitrophenyl (DNP) ligand to generate multivalent artificial antigens with full control over valency and nanoscale ligand architecture. To investigate the spatial requirements for mast cell activation, the DNP-DON complexes were initially used in surface plasmon resonance (SPR) analysis to study the binding kinetics of isolated IgE under physiological conditions. The most stable binding was observed in a narrow window of approximately 16 nm spacing between haptens. In contrast, affinity studies with FcεRI-linked IgE antibodies on the surface of rat basophilic leukemia cells (RBL-2H3) indicated virtually no distance-dependent variations in the binding of the differently structured DNP-DON complexes but suggested a supramolecular oligovalent nature of the interaction. Finally, the use of DNP-DON complexes for mast cell activation revealed that antigen-directed tight assembly of antibody-receptor complexes is the critical factor for triggering degranulation, even more critical than ligand valence. Our study emphasizes the significance of DNA nanostructures for the study of fundamental biological processes.

CCL5-producing migratory dendritic cells guide CCR5+ monocytes into the draining lymph nodes.

Dendritic cells (DCs) and monocytes capture, transport, and present antigen to cognate T cells in the draining lymph nodes (LNs) in a CCR7-dependent manner. Since only migratory DCs express this chemokine receptor, it is unclear how monocytes reach the LN. In steady-state and following inhalation of several PAMPs, scRNA-seq identified LN mononuclear phagocytes as monocytes, resident, or migratory type 1 and type 2 conventional (c)DCs, despite the downregulation of Xcr1, Clec9a, H2-Ab1, Sirpa, and Clec10a transcripts on migratory cDCs. Migratory cDCs, however, upregulated Ccr7, Ccl17, Ccl22, and Ccl5. Migratory monocytes expressed Ccr5, a high-affinity receptor for Ccl5. Using two tracking methods, we observed that both CD88hiCD26lomonocytes and CD88-CD26hi cDCs captured inhaled antigens in the lung and migrated to LNs. Antigen exposure in mixed-chimeric Ccl5-, Ccr2-, Ccr5-, Ccr7-, and Batf3-deficient mice demonstrated that while antigen-bearing DCs use CCR7 to reach the LN, monocytes use CCR5 to follow CCL5-secreting migratory cDCs into the LN, where they regulate DC-mediated immunity.

Chemogenetic inhibition of TrkB signalling reduces phrenic motor neuron survival and size

Brain derived neurotrophic factor (BDNF) signalling through its high-affinity tropomyosin receptor kinase B (TrkB) is known to have potent effects on motor neuron survival and morphology during development and in neurodegenerative diseases. Here, we employed a novel 1NMPP1 sensitive TrkBF616 rat model to evaluate the effect of 14 days inhibition of TrkB signalling on phrenic motor neurons (PhMNs). Adult female and male TrkBF616 rats were divided into 1NMPP1 or vehicle treated groups. Three days prior to treatment, PhMNs in both groups were initially labeled via intrapleural injection of Alexa-Fluor-647 cholera toxin B (CTB). After 11 days of treatment, retrograde axonal uptake/transport was assessed by secondary labeling of PhMNs by intrapleural injection of Alexa-Fluor-488 CTB. After 14 days of treatment, the spinal cord was excised 100 μm thick spinal sections containing PhMNs were imaged using two-channel confocal microscopy. TrkB inhibition reduced the total number of PhMNs by ∼16 %, reduced the mean PhMN somal surface areas by ∼25 %, impaired CTB uptake 2.5-fold and reduced the estimated PhMN dendritic surface area by ∼38 %. We conclude that inhibition of TrkB signalling alone in adult TrkBF616 rats is sufficient to lead to PhMN loss, morphological degeneration and deficits in retrograde axonal uptake/transport.

The Ubiquitin Ligase Itch Skews Light Zone Selection in Germinal Centers.

Ig diversification occurs in peripheral lymphoid organs after establishment of central tolerance during B cell development. In germinal centers (GCs), somatic hypermutation of Ig genes occurs in dark zones, followed by selection of mutated clones in light zones (LZs). This generates high-affinity Ig receptors to pathogens but can also produce autoreactive Ig receptors, which are removed by selection mechanisms that are incompletely understood. The ubiquitin ligase Itch prevents the emergence of autoimmune disease and autoantibodies in humans and mice, and patients lacking Itch develop potentially fatal autoimmune diseases; yet, how Itch regulates GC B cells is not well understood. By studying Itch-deficient mice, we have recently shown that Itch directly limits the magnitude of GC responses. Proteomic profiling of GC B cells uncovered that Itch-deficient cells exhibit high mTORC1 and Myc activity, hallmarks of positive selection. Bone marrow chimera and adoptive transfer experiments revealed that B cell Itch restricts noncycling LZ cells. These results support, to our knowledge, a novel role for Itch in skewing selection of GC B cells to restrict LZ accumulation and shape GC-derived humoral immunity. Determining how B cells integrate cues within GCs to navigate through LZs and dark zones will aid in understanding how autoreactive clones emerge from GCs in people with autoimmune disease.

Impact of FCGR2A R131H, FCGR3A F158V and FCGR3B NA1/NA2 polymorphisms on response to fc-containing TNF inhibitors in Tunisian rheumatoid arthritis patients.

Single nucleotid polymorphisms (SNPs) of Fc-gamma receptors (FcgRs), by inducing a variation of their affinity to the Fc-region of immunoglobulins, might influence the efficacy of Fc-containing biologics prescribed in rheumatoid arthritis (RA). Our aim was to investigate associations of FCGR2A, FCGR3A and FCGR3B SNPs with TNF-inhibitors (TNFi)' response in Tunisian RA patients. A cross-sectional, observational and analytic multicentric cohort study was conducted in a group of 47 Tunisian RA patients treated with (etanercept [ETA], adalimumab [ADL] and infliximab [IFX]). Treatment outcome was evaluated after 6months. R131H-FCGR2A, F158V-FCGR3A and NA1/NA2-FCGR3B SNPs were genotyped. The analytic study including all types of TNFi showed that FCGR3A-F/F low-affinity receptor was associated with a greater decrease of DAS28, while FCGR3B-NA1/NA1 high-affinity receptor was associated with a lower decrease of DAS28 in ADL group. Furthermore, both of high affinity receptors FCGR3B-NA1/NA1 and FCGR3A-V/V were more prevalent in non-responders to ADL, according to EULAR criteria. Identifying reliable biomarkers of response to biologics in RA is necessary to improve responsiveness, preserve joints' functions and structure, and reduce treatment's cost. Our study showed that FCGR3A and FCGR3B polymorphisms might have an impact on TNFis' response in RA Tunisian patients since bad response was more frequent in homozygous carriers of high affinity alleles FCGR3A-V and FCGR3B-NA1.

Molecular docking of 5-o-benzoylpinostrobin derivatives from Boesenbergia pandurata roxb. as anti-inflammatory.

The use of NSAIDs, also known as non-steroidal anti-inflammatory drugs, has numerous adverse effects and consequences. For this reason, it is necessary to develop rational drugs as safer anti-inflammatory drugs with fewer side effects. Temu Kunci rhizome contains Pinostrobin (5-hydroxy-7-methoxyflavanone), which is believed to have anti-inflammatory properties. This study aims to determine the strongest antiinflammatory activity at the cyclooxygenase-2 (COX-2) receptor through the 5-O-Benzoylpinostrobin derivative design. Methods: AutoDockTools on the COX-2 receptor (PDB code: 5IKR) were used in molecular docking in this study. The metrics employed were binding afinity (ΔG), inhibition constant (Ki), which serve as indicators of affinities, and amino acid residue similarity, which serves as a measure of the similarity of interactions. Predictive scores were confirmed by Molecular Docking Simulation. The top five 5-O-Benzoylpinostrobin derivatives show a high affinity for the COX-2 receptor compared to Pinostrobin as a marker compound of Boesenbergia pandurata Roxb and furthermore give the lowest inhibition constant (Ki) and the highest negative binding free energy (ΔG), 35.40, 45.21, 54.75, 64.43, 76.97 nM and -10.16, -10.02, -9.91, -9.81, -9.7 kcal/mol. Interestingly, the five 5-O-Benzoylpinostrobin derivatives also have higher affinity than the native ligand Mefenamic acid, which is known to be a non-selective COX-2 inhibitor. The highest predicted affinity was shown by 4-Nitro-5-O-benzoylpinostrobin for the COX-2 receptor (PDP ID: 5IKR), with a higher predicted affinity for Mefenamic acid. The five selected 5-O-Benzoylpinostrobin derivatives were potent modifications of pinostrobin as an antiinflammatory because they showed a higher affinity than Pinostrobin and Mefenamic acid. This study demonstrated that it is highly feasible to produce and test the novel 5-OBenzoylpinostrobin derivative in vivo, specifically 4-Nitro-5-Obenzoylpinostrobin.

Fingolimod ameliorates chronic experimental autoimmune neuritis by modulating inflammatory cytokines and Akt/mTOR/NF-κB signaling.

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an immune-mediated disease that targets the myelin sheaths of the peripheral nerves. Fingolimod is a sphingosine 1 phosphate (S1P) receptor antagonist with a high affinity for S1P receptors through the Akt-mTOR pathway, and prior research has suggested that it might be helpful in autoimmune illnesses. Chronic experimental autoimmune neuritis (c-EAN) was induced by immunizing Lewis rats with the S-palm P0(180-199) peptide, and then the treatment group was intraperitoneally injected with fingolimod (1mg/kg) daily. Hematoxylin and eosin staining was used to assess the severity of nerve injury. Immunohistochemistry staining showed that fingolimod's anti-inflammatory effects on c-EAN rats might be realized through the NF-κB signaling pathway. Tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), interleukin-1beta (IL-1β), interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), and intercellular adhesion molecule-1 (ICAM-1) were measured to evaluate the inflammation levels, and pAkt, p-S6, and p-p65 were used to measure the abundance of downstream activation markers to determine whether the Akt/mTOR/NF-κB signaling pathway was activated in the c-EAN model. Fingolimod treatment reduced the inflammatory reaction and the expression of NF-κB in sciatic nerves. It also decreased the mRNA levels of the proinflammatory cytokines TNF-α, IFN-γ, IL-1β, IL-6, iNOS, and ICAM-1 and pAkt, p-S6, and p-p65, representing the Akt/mTOR/NF-κB signaling pathway. Our data showed that fingolimod could improve the disease course, alleviate the decrease in inflammation, and reduce proinflammatory cytokines through the Akt/mTOR/NF-κB axis in c-EAN rats, which could be beneficial for the development of CIDP-related research.

The leukotriene B4 receptors BLT1 and BLT2 as potential therapeutic targets.

Leukotriene B4 (LTB4 ) was recognized as an arachidonate-derived chemotactic factor for inflammatory cells and an important drug target even before the molecular identification of its receptors. We cloned the high- and low-affinity LTB4 receptors, BLT1 and BLT2, respectively, and examined their functions by generating and studying gene-targeted mice. BLT1 is involved in the pathogenesis of various inflammatory and immune diseases, including asthma, psoriasis, contact dermatitis, allergic conjunctivitis, age-related macular degeneration, and immune complex-mediated glomerulonephritis. Meanwhile, BLT2 is a high-affinity receptor for 12-hydroxyheptadecatrienoic acid, which is involved in the maintenance of dermal and intestinal barrier function, and the acceleration of skin and corneal wound healing. Thus, BLT1 antagonists and BLT2 agonists are promising candidates in the treatment of inflammatory diseases.