CD4 Receptor Research Articles

Inactivation of cell-free HIV-1 by designing potent peptides based on mutations in the CD4 binding site.

Human immunodeficiency virus type 1 (HIV-1) is a major global health problem, with over 38 million people infected worldwide. Current anti-HIV-1 drugs are limited in their ability to prevent the virus from replicating inside host cells, making them less effective as preventive measures. In contrast, viral inhibitors that inactivate the virus before it can bind to a host cell have great potential as drugs. In this study, we aimed to design mutant peptides that could block the interaction between gp120 and the CD4 receptor on host cells, thus preventing HIV-1 infection. We designed a 20-amino-acid peptide that mimicked the amino acids of the CD4 binding site and docked it to gp120. Molecular dynamics simulations were performed to calculate the energy of MMPBSA (Poisson-Boltzmann Surface Area) for each residue of the peptide, and unfavorable energy residues were identified as potential mutation points. Using MAESTRO (Multi AgEnt STability pRedictiOn), we measured ΔΔG (change in the change in Gibbs free energy) for mutations and generated a library of 240 mutated peptides using OSPREY software. The peptides were then screened for allergenicity and binding affinity. Finally, molecular dynamics simulations (via GROMACS 2020.2) and control docking (via HADDOCK 2.4) were used to evaluate the ability of four selected peptides to inhibit HIV-1 infection. Three peptides, P3 (AHRQIRQWFLTRGPNRSLWQ), P4 (VHRQIRQWFLTRGPNRSLWQ), and P9 (AHRQIRQMFLTRGPNRSLWQ), showed practical and potential as HIV inhibitors, based on their binding affinity and ability to inhibit infection. These peptides have the ability to inactivate the virus before it can bind to a host cell, thus representing a promising approach to HIV-1 prevention. Our findings suggest that mutant peptides designed to block the interaction between gp120 and the CD4 receptor have potential as HIV-1 inhibitors. These peptides could be used as preventive measures against HIV-1 transmission, and further research is needed to evaluate their safety and efficacy in clinical settings.

Soluble CD4 and low molecular weight CD4-mimetic compounds sensitize cells to be killed by anti-HIV cytotoxic immunoconjugates.

HIV infection can be effectively treated to prevent the development of AIDS, but it cannot be cured. We have attached poisons to anti-HIV antibodies to kill the infected cells that persist even after years of effective antiviral therapy. Here we show that the killing of infected cells can be markedly enhanced by the addition of soluble forms of the HIV receptor CD4 or by mimics of CD4.

Structural and functional characterization of Aedes aegypti pupal cuticle protein that controls dengue virus infection.

The pupal cuticle protein from Aedes aegypti (AaPC) inhibits dengue virus (DENV) infection; however, the underlying mechanism of this inhibition remains unknown. Here, we report that AaPC is an intrinsically disordered protein and interacts with domain I/II of the DENV envelope protein via residues Asp59, Asp61, Glu71, Asp73, Ser75, and Asp80. AaPC can directly bind to and cause the aggregation of DENV, which in turn blocks virus infection during the virus-cell fusion stage. AaPC may also influence viral recognition and attachment by interacting with human immune receptors DC-SIGN and CD4. These findings enhance our understanding of the role of AaPC in mitigating viral infection and suggest that AaPC is a potential target for developing inhibitors or antibodies to control dengue virus infection.

HIV Preintegration Transcription and Host Antagonism.

Retrovirus integration is an obligatory step for the viral life cycle, but large amounts of unintegrated DNA (uDNA) accumulate during retroviral infection. For simple retroviruses, in the absence of integration, viral genomes are epigenetically silenced in host cells. For complex retroviruses such as HIV, preintegration transcription has been found to occur at low levels from a large population of uDNA even in the presence of host epigenetic silencing mechanisms. HIV preintegration transcription has been suggested to be a normal early process of HIV infection that leads to the syntheses of all three classes of viral transcripts: multiply-spliced, singly-spliced, and unspliced genomic RNA; only viral early proteins such as Nef are selectively translated at low levels in blood CD4 T cells and macrophages, the primary targets of HIV. The initiation and persistence of HIV preintegration transcription have been suggested to rely on viral accessory proteins, particularly virion Vpr and de novo Tat generated from uDNA; both proteins have been shown to antagonize host epigenetic silencing of uDNA. In addition, stimulation of latently infected resting T cells and macrophages with cytokines, PKC activator, or histone deacetylase inhibitors has been found to greatly upregulate preintegration transcription, leading to low-level viral production or even replication from uDNA. Functionally, Nef synthesized from preintegration transcription is biologically active in modulating host immune functions, lowering the threshold of T cell activation, and downregulating surface CD4, CXCR4/CCR5, and HMC receptors. The early Tat activity from preintegration transcription antagonizes repressive minichromatin assembled onto uDNA. The study of HIV preintegration transcription is important to understanding virus-host interaction and antagonism, viral persistence, and the mechanism of integrase drug resistance. The application of unintegrated lentiviral vectors for gene therapy also offers a safety advantage for minimizing retroviral vector-mediated insertional mutagenesis.

Edible exosome-like nanoparticles from portulaca oleracea L mitigate DSS-induced colitis via facilitating double-positive CD4+CD8+T cells expansion

Plant-derived exosome-like nanoparticles (PDENs) have been paid great attention in the treatment of ulcerative colitis (UC). As a proof of concept, we isolated and identified Portulaca oleracea L-derived exosome-like nanoparticles (PELNs) from edible Portulaca oleracea L, which exhibited desirable nano-size (~ 160 nm) and a negative zeta potential value (-31.4 mV). Oral administration of PELNs effectively suppressed the expressions of pro-inflammatory cytokines (TNF-α, IL-6, IL-12, and IL-1β) and myeloperoxidase (MPO), increased levels of the anti-inflammatory cytokine (IL-10), and alleviated acute colitis in dextran sulfate sodium (DSS)-induced C57 mice and IL-10−/− mice. Notably, PELNs exhibited excellent stability and safety within the gastrointestinal tract and displayed specific targeting to inflamed sites in the colons of mice. Mechanistically, oral administration of PELNs played a crucial role in maintaining the diversity and balance of gut microbiota. Furthermore, PELNs treatment enhanced Lactobacillus reuteri growth and elevated indole derivative levels, which might activate the aryl-hydrocarbon receptor (AhR) in conventional CD4+ T cells. This activation downregulated Zbtb7b expression, leading to the reprogramming of conventional CD4+ T cells into double-positive CD4+CD8+T cells (DP CD4+CD8+ T cells). In conclusion, our findings highlighted the potential of orally administered PELNs as a novel, natural, and colon-targeted agent, offering a promising therapeutic approach for managing UC.Graphic abstractSchematic illustration of therapeutic effects of oral Portulaca oleracea L -derived natural exosome-like nanoparticles (PELNs) on UC. PELNs treatment enhanced Lactobacillus reuteri growth and elevated indole derivative levels, which activate the aryl-hydrocarbon receptor (AhR) in conventional CD4+ T cells leading to downregulate the expression of Zbtb7b, reprogram of conventional CD4+ T cells into double-positive CD4+CD8+T cells (DP CD4+CD8+ T cells), and decrease the levels of pro-inflammatory cytokines.

Human A2-CAR T Cells Reject HLA-A2 + Human Islets Transplanted Into Mice Without Inducing Graft-versus-host Disease.

Type 1 diabetes is an autoimmune disease characterized by T-cell-mediated destruction of pancreatic beta-cells. Islet transplantation is an effective therapy, but its success is limited by islet quality and availability along with the need for immunosuppression. New approaches include the use of stem cell-derived insulin-producing cells and immunomodulatory therapies, but a limitation is the paucity of reproducible animal models in which interactions between human immune cells and insulin-producing cells can be studied without the complication of xenogeneic graft-versus-host disease (xGVHD). We expressed an HLA-A2-specific chimeric antigen receptor (A2-CAR) in human CD4 + and CD8 + T cells and tested their ability to reject HLA-A2 + islets transplanted under the kidney capsule or anterior chamber of the eye of immunodeficient mice. T-cell engraftment, islet function, and xGVHD were assessed longitudinally. The speed and consistency of A2-CAR T-cell-mediated islet rejection varied depending on the number of A2-CAR T cells and the absence/presence of coinjected peripheral blood mononuclear cells (PBMCs). When <3 million A2-CAR T cells were injected, coinjection of PBMCs accelerated islet rejection but also induced xGVHD. In the absence of PBMCs, injection of 3 million A2-CAR T cells caused synchronous rejection of A2 + human islets within 1 wk and without xGVHD for 12 wk. Injection of A2-CAR T cells can be used to study rejection of human insulin-producing cells without the complication of xGVHD. The rapidity and synchrony of rejection will facilitate in vivo screening of new therapies designed to improve the success of islet-replacement therapies.

HIV infection.

The AIDS epidemic has been a global public health issue for more than 40 years and has resulted in ~40 million deaths. AIDS is caused by the retrovirus, HIV-1, which is transmitted via body fluids and secretions. After infection, the virus invades host cells by attaching to CD4 receptors and thereafter one of two major chemokine coreceptors, CCR5 or CXCR4, destroying the host cell, most often a T lymphocyte, as it replicates. If unchecked this can lead to an immune-deficient state and demise over a period of ~2-10 years. The discovery and global roll-out of rapid diagnostics and effective antiretroviral therapy led to a large reduction in mortality and morbidity and toan expanding group of individuals requiring lifelong viral suppressive therapy. Viral suppression eliminates sexual transmission of the virus and greatly improves health outcomes. HIV infection, although still stigmatized, is now a chronic and manageable condition. Ultimate epidemic control will require prevention and treatment to be made available, affordable and accessible for all. Furthermore, the focus should be heavily oriented towards long-term well-being, care for multimorbidity and good quality of life. Intense research efforts continue for therapeutic and/or preventive vaccines, novel immunotherapies and a cure.

The truncated IFITM3 facilitates the humoral immune response in inactivated influenza vaccine-vaccinated mice via interaction with CD81

ABSTRACT A single-nucleotide polymorphism (SNP) rs12252-C of interferon-induced transmembrane protein 3 (IFITM3), resulting in a truncated IFITM3 protein lacking 21 N-terminus amino acids, is associated with severe influenza infection in the Chinese population. However, the effect of IFITM3 rs12252-C on influenza vaccination and the underlying mechanism is poorly understood. Here, we constructed a mouse model with a deletion of 21 amino acids at the N-terminus (NΔ21) of IFITM3 and then compared the antibody response between Quadrivalent influenza vaccine (QIV) immunized wild-type (WT) mice and NΔ21 mice. Significantly higher levels of haemagglutination inhibition (HI) titre, neutralizing antibodies (NAb), and immunoglobulin G (IgG) to H1N1, H3N2, B/Victory, and B/Yamagata viruses were observed in NΔ21 mice compared to WT mice. Correspondingly, the numbers of splenic germinal centre (GC) B cells, plasma cells, memory B cells, QIV-specific IgG+ antibody-secreting cells (ASC), and T follicular helper cells (TFH) in NΔ21 mice were higher compared with WT mice. Moreover, the 21-amino-acid deletion caused IFITM3 translocation from the endocytosis compartment to the periphery of cells, which also prevented the degradation of a co-stimulatory molecule of B cell receptor (BCR) CD81 on the cell surface. More importantly, a more interaction was observed between NΔ21 protein and CD81 compared to the interaction between IFITM3 and CD81. Overall, our study revealed a potential mechanism of NΔ21 protein enhancing humoral immune response by relocation to prevent the degradation of CD81, providing insight into SNP affecting influenza vaccination.

The mRNA expression and secretion of granzyme B are up-regulated via the histamine H2 receptor in human CD4+ T cells

IntroductionGranzyme B (GZMB), a serine protease with cytotoxic and immunomodulatory functions, shows elevated levels in blood plasma of patients with atopic dermatitis (AD). It has been observed that GZMB expression in CD4+ and CD8+ T cells is higher in lesional skin in AD than in healthy skin. Since histamine is present in high concentration in the skin of AD patients, we investigated the regulation of GZMB in human CD4+ T cells by histamine.MethodsNaïve CD4+ T cells polarized into Th2 cells, total CD4+ T cells treated with IL-4 for 72 h and CD4+ T cells isolated from healthy donors and AD patients were investigated. The cells were stimulated with histamine or with different histamine-receptor agonists. Gene expression was evaluated by RNA-Seq. GZMB mRNA expression was detected by quantitative real time PCR, whereas GZMB secretion was measured by ELISpot and ELISA. T cell degranulation was evaluated by flow cytometry using CD107a surface expression as a degranulation marker.ResultsBy RNA-Seq, we identified the up-regulation of various genes of the cytotoxic pathway, in particular of GZMB, by histamine in Th2-polarized CD4+ T cells. In Th2-polarized CD4+ T cells and in CD4+ T cells activated by IL-4 the mRNA expression of GZMB was significantly up-regulated by histamine and by histamine H2 receptor (H2R) agonists. The induction of GZMB secretion by histamine was significantly higher in CD4+ T cells from AD patients than in those from healthy donors. CD107a surface expression was up-regulated by trend in response to histamine in Th2-polarized CD4+ T cells.ConclusionOur findings may help to elucidate novel mechanisms of the H2R and to achieve a better understanding of the role of GZMB in the pathogenesis of AD.

HIV-1 virological synapse formation enhances infection spread by dysregulating Aurora Kinase B.

HIV-1 spreads efficiently through direct cell-to-cell transmission at virological synapses (VSs) formed by interactions between HIV-1 envelope proteins (Env) on the surface of infected cells and CD4 receptors on uninfected target cells. Env-CD4 interactions bring the infected and uninfected cellular membranes into close proximity and induce transport of viral and cellular factors to the VS for efficient virion assembly and HIV-1 transmission. Using novel, cell-specific stable isotope labeling and quantitative mass spectrometric proteomics, we identified extensive changes in the levels and phosphorylation states of proteins in HIV-1 infected producer cells upon mixing with CD4+ target cells under conditions inducing VS formation. These coculture-induced alterations involved multiple cellular pathways including transcription, TCR signaling and, unexpectedly, cell cycle regulation, and were dominated by Env-dependent responses. We confirmed the proteomic results using inhibitors targeting regulatory kinases and phosphatases in selected pathways identified by our proteomic analysis. Strikingly, inhibiting the key mitotic regulator Aurora kinase B (AURKB) in HIV-1 infected cells significantly increased HIV activity in cell-to-cell fusion and transmission but had little effect on cell-free infection. Consistent with this, we found that AURKB regulates the fusogenic activity of HIV-1 Env. In the Jurkat T cell line and primary T cells, HIV-1 Env:CD4 interaction also dramatically induced cell cycle-independent AURKB relocalization to the centromere, and this signaling required the long (150 aa) cytoplasmic C-terminal domain (CTD) of Env. These results imply that cytoplasmic/plasma membrane AURKB restricts HIV-1 envelope fusion, and that this restriction is overcome by Env CTD-induced AURKB relocalization. Taken together, our data reveal a new signaling pathway regulating HIV-1 cell-to-cell transmission and potential new avenues for therapeutic intervention through targeting the Env CTD and AURKB activity.

Signatures of VH1-69-derived hepatitis C virus neutralizing antibody precursors defined by binding to envelope glycoproteins

An effective preventive vaccine for hepatitis C virus (HCV) remains a major unmet need. Antigenic region 3 (AR3) on the E1E2 envelope glycoprotein complex overlaps with the CD81 receptor binding site and represents an important epitope for broadly neutralizing antibodies (bNAbs) and is therefore important for HCV vaccine design. Most AR3 bNAbs utilize the VH1-69 gene and share structural features that define the AR3C-class of HCV bNAbs. In this work, we identify recombinant HCV glycoproteins based on a permuted E2E1 trimer design that bind to the inferred VH1-69 germline precursors of AR3C-class bNAbs. When presented on nanoparticles, these recombinant E2E1 glycoproteins efficiently activate B cells expressing inferred germline AR3C-class bNAb precursors as B cell receptors. Furthermore, we identify critical signatures in three AR3C-class bNAbs that represent two subclasses of AR3C-class bNAbs that will allow refined protein design. These results provide a framework for germline-targeting vaccine design strategies against HCV.

Small but Highly Versatile: The Viral Accessory Protein Vpu.

Human and simian immunodeficiency viruses (HIVs and SIVs, respectively) encode several small proteins (Vif, Vpr, Nef, Vpu, and Vpx) that are called accessory because they are not generally required for viral replication in cell culture. However, they play complex and important roles for viral immune evasion and spread in vivo. Here, we discuss the diverse functions and the relevance of the viral protein U (Vpu) that is expressed from a bicistronic RNA during the late stage of the viral replication cycle and found only in HIV-1 and closely related SIVs. It is well established that Vpu counteracts the restriction factor tetherin, mediates degradation of the primary viral CD4 receptors, and inhibits activation of the transcription factor nuclear factor kappa B. Recent studies identified additional activities and provided new insights into the sophisticated mechanisms by which Vpu enhances and prolongs the release of fully infectious viral particles. In addition, it has been shown that Vpu prevents superinfection not only by degrading CD4 but also by modulating DNA repair mechanisms to promote degradation of nuclear viral complementary DNA in cells that are already productively infected.

Lysis and phenotypic modulation of mesenchymal stromal cells upon blood contact triggers anti-inflammatory skewing of the peripheral innate immune repertoire

Background aimsMesenchymal stromal cells (MSCs) are used to treat immune-related disorders, including graft-versus-host disease. Upon intravenous infusion, MSCs trigger the instant blood-mediated inflammatory response, resulting in activation of both complement and coagulation cascades, and are rapidly cleared from circulation. Despite no/minimal engraftment, long-term immunoregulatory properties are evident. The aim of this study was to establish the effects of blood exposure on MSC viability and immunomodulatory functions. MethodsHuman, bone marrow derived MSCs were exposed to human plasma +/– heat inactivation or whole blood. MSC number, viability and cellular damage was assessed using the JC-1 mitochondrial depolarization assay and annexin V staining. C3c binding and expression of the inhibitory receptors CD46, CD55 and CD59 and complement receptors C3aR and C5aR were evaluated by flow cytometry. MSCs pre-exposed to plasma were cultured with peripheral blood mononuclear cells (PBMCs) and monocyte subsets characterized by flow cytometry. The PBMC and MSC secretome was assessed using enzyme-linked immunosorbent assays against tumor necrosis factor alpha, interleukin (IL)-6 and IL-10. Monocyte recruitment towards the MSC secretome was evaluated using Boyden chambers and screened for chemotactic factors including monocyte chemoattractant protein (MCP)-1. MSC effects on the peripheral immune repertoire was also evaluated in whole blood by flow cytometry. ResultsPlasma induced rapid lysis of 57% of MSCs, which reduced to 1% lysis with heat inactivation plasma. Of those cells that were not lysed, C3c could be seen bound to the surface of the cells, with a significant swelling of the MSCs and induction of cell death. The MSC secretome reduced monocyte recruitment, in part due to a reduction in MCP-1, and downregulated PBMC tumor necrosis factor alpha secretion while increasing IL-6 levels in the co-culture supernatant. A significant decrease in CD14+ monocytes was evident after MSC addition to whole blood alongside a significant increase in IL-6 levels, with those remaining monocytes demonstrating an increase in classical and decrease in non-classical subsets. This was accompanied by a significant increase in both mononuclear and polymorphonuclear myeloid-derived suppressor cells. ConclusionsThis study demonstrates that a significant number of MSCs are rapidly lysed upon contact with blood, with those surviving demonstrating a shift in their phenotype, including a reduction in the secretion of monocyte recruitment factors and an enhanced ability to skew the phenotype of monocytes. Shifts in the innate immune repertoire, towards an immunosuppressive profile, were also evident within whole blood after MSC addition. These findings suggest that exposure to blood components can promote peripheral immunomodulation via multiple mechanisms that persists within the system long after the infused MSCs have been cleared.

Mapping the neutralizing specificity of human anti-HIV serum by deep mutational scanning

Understanding the specificities of human serum antibodies that broadly neutralize HIV can inform prevention and treatment strategies. Here, we describe a deep mutational scanning system that can measure the effects of combinations of mutations to HIV envelope (Env) on neutralization by antibodies and polyclonal serum. We first show that this system can accurately map how all functionally tolerated mutations to Env affect neutralization by monoclonal antibodies. We then comprehensively map Env mutations that affect neutralization by a set of human polyclonal sera that neutralize diverse strains of HIV and target the site engaging the host receptor CD4. The neutralizing activities of these sera target different epitopes, with most sera having specificities reminiscent of individual characterized monoclonal antibodies, but one serum targeting two epitopes within the CD4-binding site. Mapping the specificity of the neutralizing activity in polyclonal human serum will aid in assessing anti-HIV immune responses to inform prevention strategies.

Asymmetric conformations of cleaved HIV-1 envelope glycoprotein trimers in styrene-maleic acid lipid nanoparticles

During virus entry, the pretriggered human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer initially transits into a default intermediate state (DIS) that remains structurally uncharacterized. Here, we present cryo-EM structures at near-atomic resolution of two cleaved full-length HIV-1 Env trimers purified from cell membranes in styrene-maleic acid lipid nanoparticles without antibodies or receptors. The cleaved Env trimers exhibited tighter subunit packing than uncleaved trimers. Cleaved and uncleaved Env trimers assumed remarkably consistent yet distinct asymmetric conformations, with one smaller and two larger opening angles. Breaking conformational symmetry is allosterically coupled with dynamic helical transformations of the gp41 N-terminal heptad repeat (HR1N) regions in two protomers and with trimer tilting in the membrane. The broken symmetry of the DIS potentially assists Env binding to two CD4 receptors—while resisting antibody binding—and promotes extension of the gp41 HR1 helical coiled-coil, which relocates the fusion peptide closer to the target cell membrane.

Temsavir Modulates HIV-1 Envelope Conformation by Decreasing Its Proteolytic Cleavage.

HIV-1 envelope glycoproteins (Envs) mediate viral entry and represent a target of choice for small molecule inhibitors. One of them, temsavir (BMS-626529) prevents the interaction of the host cell receptor CD4 with Env by binding the pocket under the β20-β21 loop of the Env subunit gp120. Along with its capacity to prevent viral entry, temsavir stabilizes Env in its "closed" conformation. We recently reported that temsavir affects glycosylation, proteolytic processing, and overall conformation of Env. Here, we extend these results to a panel of primary Envs and infectious molecular clones (IMCs), where we observe a heterogeneous impact on Env cleavage and conformation. Our results suggest that the effect of temsavir on Env conformation is associated with its capacity to decrease Env processing. Indeed, we found that the effect of temsavir on Env processing affects the recognition of HIV-1-infected cells by broadly neutralizing antibodies and correlates with their capacity to mediate antibody-dependent cellular cytotoxicity (ADCC).

Combined Drug with Antibacterial Effect Supports the Normal Intestinal Microflora.

Widespread use of antibiotics leads to an imbalance of normal intestinal microflora and to the development of multidrug resistance. The problem can be solved by administration of the antibiotics in combination with the drugs that have an immunotropic effect. We studied the effect of the drug containing technologically processed affinity purified antibodies to IFNγ, CD4 receptor, β2-microglobulin of MHC class I, and β2-domain of MHC II combined with antibiotics on the composition of intestinal microflora of pigs and the total number of microbiome resistance genes. Using the methods of NGS sequencing and quantitative PCR, we found that the drug contributes to the maintenance of normal microflora and, consequently, to the symbiotic relationship of the host with microflora, and prevents the reproduction of pathogenic bacterial species. Analysis for the presence of the resistance genes of gastrointestinal microorganisms showed that the drug does not affect the qualitative and quantitative composition of these genes of the intestinal microbiome.

Abstract 6793: In vivo effects and molecular mechanisms of Minnelide mediated enhancement of measles virus oncolysis in human colorectal cancer

Abstract Background: The development of novel biotherapies for metastatic colorectal cancer (CRC), a fatal disease when advanced, is an urgent medical need. Oncolytic viruses (OVs) are novel cancer biotherapies, and the oncolytic measles virus (MV) has demonstrated safety and antitumor activity in early clinical studies. Triptolide (TRP), a diterpenoid epoxide extracted from the thunder god vine, and Minnelide (MN), its water-soluble compound, are novel agents associated with potent antitumor, anti-proliferative, pro-apoptotic, antiangiogenic, and ER stress inducing effects. The objectives of this study are to characterize the in vitro and in vivo effects and molecular mechanisms of measles virotherapy in combination with TRP and MN in CRC. Methods: The in vitro effects of TRP, MV-GFP (Edmonston strain of Measles virus expressing eGFP), MV-CD46-muPA (dual targeted MV against the murine uPA receptor and human CD46), or virus-triptolide combinations on tumor cell cytotoxicity were assessed on HT-29, HCT116 and SW620 CRC cells. Molecular and mechanistic characterization of TRP’s effects alone and combination with MV in HT29 cells was analyzed by the Reverse Phase Protein Array (RPPA) and validated by western blot analysis. In vivo effects (tumor progression and survival) of MN alone and in combination with MV vectors using different treatment schemes were assessed in HT-29 tumor bearing NSG mice, and tumor correlative studies were performed. Results: MV and TRP had single agent antitumor activity against human CRC cell lines. MV-TRP combinations were associated with increased in vitro cytotoxicity compared to single agent treatments. Functional proteomics analysis showed that TRP-MV combinations were associated with inhibition of survival and proliferation pathways, such as AKT, ERK, inhibition of HSP70 as well as induction of autophagy, at a higher level than each agent alone. In vivo, both a 7-days MN treatment and a 21-days MN treatment significantly improved long term tumor control and significantly improved survival, compared to each agent alone. Tumor studies showed that MN increased MV tumor deposition, as well as enhancement in apoptosis and inhibition of proliferation. Molecular characterization of oncogenic pathways modulated by MV-Minnelide combinations (Nanostring) are underway and will be presented at the meeting. Conclusions: Our results demonstrated that, in human CRC xenografts, Minnelide safely and significantly enhances measles virus oncolysis in vivo, leading to improved antitumor effects and prolonged survival. These effects were associated with enhanced antiproliferative and pro-apoptotic effects with the MV-Minnelide combination, suggesting in vivo synergy. Studies characterizing the molecular mechanisms of in vivo synergy and assessing this promising combination in immunocompetent CRC models are underway. Citation Format: Valery A. Chavez, Floritza Bustamante, Carolina Merchan-Mendes, Ashok Saluja, Jaime Merchan. In vivo effects and molecular mechanisms of Minnelide mediated enhancement of measles virus oncolysis in human colorectal cancer [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 6793.

Target-specificity of different amyrin subunits in impeding HCV influx mechanism inside the human cells considering the quantum tunnel profiles and molecular strings of the CD81 receptor: a combined in silico and in vivo study.

HCV is a hepatotropic RNA virus recognized for its frequent virulence and fatality worldwide. Despite many vaccine development programs underway, researchers are on a quest for natural bioactive compounds due to their multivalent efficiencies against viral infections, considering which the current research aimed to figure out the target-specificity and therapeutic potentiality of α, β, and δ subunits of amyrin, as novel bioactive components against the HCV influx mechanism. Initially, the novelty of amyrin subunits was conducted from 203 pharmacophores, comparing their in-silico pharmacokinetic and pharmacodynamic profiles. Besides, the best active site of CD81 was determined following the quantum tunneling algorithm. The molecular dynamic simulation was conducted (100ns) following the molecular docking steps to reveal the parameters- RMSD (Å); Cα; RMSF (Å); MolSA (Å2); Rg (nm); PSA (Å); SASA (Å2), and the MM-GBSA dG binding scores. Besides, molecular strings of CD81, along with the co-expressed genes, were classified, as responsible for encoding CD81-mediated protein clusters during HCV infection, resulting in the potentiality of amyrins as targeted prophylactics in HCV infection. Finally, in vivo profiling of the oxidative stress marker, liver-specific enzymes, and antioxidant markers was conducted in the DMN-induced mice model, where β-amyrin scored the most significant values in all aspects.

Deficient leptin receptor signaling in T cells of human SLE.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease mainly mediated by IgG autoantibody. While follicular helper T (Tfh) cells are crucial for supporting IgG autoantibody generation in human SLE, underlying mechanisms for Tfh cell mal-differentiation remain unclear. In total, 129 SLE patients and 37 healthy donors were recruited for this study. Circulating leptin was determined by ELISA from patients with SLE and healthy individuals. CD4 T cells isolated from SLE patients and healthy donors were activated with anti-CD3/CD28 beads under cytokine-unbiased conditions in the presence or absence of recombinant leptin protein, followed by detection for Tfh cell differentiation by quantifying intracellular transcription factor Bcl-6 and cytokine IL-21. AMPK activation was assessed by analyzing phosphor-AMPK using phosflow cytometry and immunoblots. Leptin receptor expression was determined using flow cytometry and its overexpression was achieved by transfection with an expression vector. Humanized SLE chimeras were induced by injecting patients' immune cells into immune-deficient NSG mice and used for translational studies. Circulating leptin was elevated in patients with SLE, inversely associated with disease activity. In healthy individuals, leptin efficiently inhibited Tfh cell differentiation through inducing AMPK activation. Meanwhile, leptin receptor deficiency was a feature of CD4 T cells in SLE patients, impairing the inhibitory effect of leptin on the differentiation of Tfh cells. As a result, we observed the coexistence of high circulating leptin and increased Tfh cell frequencies in SLE patients. Accordingly, overexpression of leptin receptor in SLE CD4 T cells abrogated Tfh cell mal-differentiation and IgG anti-dsDNA generation in humanized lupus chimeras. Leptin receptor deficiency blocks the inhibitory effect of leptin on SLE Tfh cell differentiation, serving as a promising therapeutic target for lupus management.