Induction Of Cytokines Research Articles

EXTH-54. CO-CULTURE OF PATIENT-DERIVED GLIOBLASTOMA CELL LINES WITH AUTOLOGOUS PBMCS FOR THE INVESTIGATION OF ONCOLYTIC VIRUS RESPONSE

Abstract BACKGROUND The dismal prognosis of glioblastoma (GBM) patients, with a median survival of less than 15 months despite maximal therapy urgently warrants new therapeutic approaches. Clinical trials employing oncolytic viruses (OVs) have shown encouraging results, however, in each OV clinical trial only a small subset of patients responded to treatment. As inter-tumoral heterogeneity has been the key challenge in treating GBM, we hypothesized that development of an in vitro co-culture model for assessment of viral replication and subsequent immune response might ultimately predict in vivo OV efficacy for individual GBM patients. METHODS 20 patient-derived GBM cell cultures were tested with dose-ranges of two clinically relevant OVs (DNX2401 and rQnestin34.5 V1) to determine the EC50 values (half-maximal effective concentration). To discriminate between responders and non-responders six of these cultures were infected using MOI 10 for DNX2401 and 0.25 for rQnestin34.5V1, and co-cultured with autologous PBMCs. OV-induced changes in gene and protein expression of immune associated genes were assessed using targeted gene expression (NanoString Technology) and ELISA. RESULTS DNX2401 EC50 values ranged from 0.35 to > 600 and from 0.04 to 1.77 for rQnestin34.5V1. Induction of pro-inflammatory cytokines and chemokines differed per virus and per patient. Enhanced OV infection or oncolysis efficiency did not lead to increased levels of IFNγ production. Importantly, longer exposure to dexamethasone prior to PBMC isolation correlated with suppressed IFNγ production. CONCLUSION We established an autologous GBM cells/PBMCs co-culture model which reflects inter-tumoral heterogeneity in terms of cytokine induction and virus-specific changes in gene and protein expression upon OV infection. Immune activation is not directly related to degree of infection or oncolysis and can be hampered by prolonged prior dexamethasone use. These first results support our hypothesis that improved prediction of response rates in oncolytic virotherapy for GBM may require a personalized approach with an in vitro test system.

Anti-inflammatory effects of PRIMA-1MET (mutant p53 reactivator) induced by inhibition of nuclear factor-κB on rheumatoid arthritis fibroblast-like synoviocytes.

Fibroblast-like synoviocytes (FLSs), the main pathological cells in rheumatoid arthritis (RA), display tumor-like phenotype, including hyper-proliferation, apoptosis resistance, and aggressive phenotype. Excessive proliferation and insufficient apoptosis of RA-FLSs can lead to hyperplastic synovial pannus tissue, excess production of inflammatory mediators, and destruction of joints. In this article, we investigate the effect of PRIMA-1MET on the apoptosis induction and inhibition of pro-inflammatory cytokines in RA-FLSs. Synovial tissue samples were obtained from 10 patients with RA. The FLSs were treated with different concentrations of PRIMA-1MET. The rate of apoptosis and cell survival was assessed by flow cytometry and MTT assay and Real-time quantitative PCR was performed to evaluate the transcription of p53, IL-6, IL-1β, TNF-α, Noxa, p21, PUMA, Bax, Survivin, and XIAP in treated RA-FLSs. The protein level of p53, IκBα, and phospho-IκBα were measured using Western blotting. The results showed that PRIMA-1MET induced apoptosis in RA-FLSs and increased significantly the expression of Noxa, and decreased significantly IL-6, IL-1β, p53, and phospho-IκBα expression. PRIMA-1MET can induce apoptosis in RA-FLSs through induction of Noxa expression while p53 was downregulated. Furthermore, PRIMA-1MET treatment results in the suppression of pro-inflammatory cytokine production and NF-κB inhibition. Given the role of p53 and NF-κB in RA-FLSs, PRIMA-1MET can be considered as a new therapeutic strategy for rheumatoid arthritis.

Neurovascular injury associated non-apoptotic endothelial caspase-9 and astroglial caspase-9 mediate inflammation and contrast sensitivity decline

Retinal neurovascular injuries are a leading cause of vision loss in young adults presenting unmet therapeutic needs. Neurovascular injuries damage homeostatic communication between endothelial, pericyte, glial, and neuronal cells through signaling pathways that remain to be established. To understand the mechanisms that contribute to neuronal death, we use a mouse model of retinal vein occlusion (RVO). Using this model, we previously discovered that after vascular damage, there was non-apoptotic activation of endothelial caspase-9 (EC Casp9); knock-out of EC Casp9 led to a decrease in retinal edema, capillary ischemia, and neuronal death. In this study, we aimed to explore the role of EC Casp9 in vision loss and inflammation. We found that EC Casp9 is implicated in contrast sensitivity decline, induction of inflammatory cytokines, and glial reactivity. One of the noted glial changes was increased levels of astroglial cl-caspase-6, which we found to be activated cell intrinsically by astroglial caspase-9 (Astro Casp9). Lastly, we discovered that Astro Casp9 contributes to capillary ischemia and contrast sensitivity decline after RVO (P-RVO). These findings reveal specific endothelial and astroglial non-apoptotic caspase-9 roles in inflammation and neurovascular injury respectively; and concomitant relevancy to contrast sensitivity decline.

Carum carvi Modulates Acetaminophen-Induced Hepatotoxicity: Effects on TNF-α, NF-κB, and Caspases

Carum carvi is a well-known herb traditionally used as a spice in Asian countries. Acetaminophen is a known marketed drug mainly used as an analgesic. It has been scientifically proven that consumption of acetaminophen (paracetamol) is associated with liver toxicity if taken in high doses without medical supervision. The present study evaluated the in vivo antioxidant and hepatoprotective efficacy of Carum carvi against acetaminophen-induced hepatotoxicity in Wistar rats. Our results demonstrate that Carum carvi, at doses (mg/kg) of 100 (D1) and 200 (D2), showed inhibitory properties for DNA-sugar damage, lipid peroxidation, DPPH scavenging, and increased reducing potential in a concentration-dependent manner. Our results also confirm that liver toxicity associated with paracetamol, such as depletion of reduced glutathione and antioxidant enzyme levels, as well as induction of cytochrome P450, oxidative stress, apoptosis, and inflammatory cytokines, was efficiently restored by Carum carvi treatment in rats. Moreover, the expression of redox-sensitive transcription factors, namely, NF-κB and TNF-α levels, was also modulated by Carum carvi in the rats. In summary, our study confirms that Carum carvi inhibits inflammation and oxidative stress, thereby protecting liver cells from paracetamol prompted hepatotoxicity.

Surface layer protein A from hypervirulent Clostridioides difficile ribotypes induce significant changes in the gene expression of tight junctions and inflammatory response in human intestinal epithelial cells

BackgroundSurface layer protein A (SlpA), the primary outermost structure of Clostridioides difficile, plays an essential role in C. difficile pathogenesis, although its interaction with host intestinal cells are yet to be understood. The aim of this study was to investigate the effects of SlpA extracted from C. difficile on tight junction (TJ) proteins expression and induction of pro-inflammatory cytokines in human colon carcinoma cell line HT-29. SlpA was extracted from three toxigenic C. difficile clinical strains including RT126, RT001, RT084 as well as C. difficile ATCC 700057 as non-toxigenic strain. Cell viability was performed by MTT assay, and the mRNA expression of TJ proteins and inflammation-associated genes was determined using quantitative RT-PCR. Additionally, the secretion of IL-8, IL-1β and TNF-α cytokines was measured by ELISA.ResultsC. difficile SlpA from selected RTs variably downregulated the expression level of TJs-assassinated genes and increased the expression level of TLR-4 and pro-inflammatory cytokines in HT-29 treated cells. SlpA from RT126 significantly (padj<0.05) decreased the gene expression level of claudins family and JAM-A and increased the secretion of IL-8, TNF-α and IL1-β as compared to untreated cells. Moreover, only SlpA from RT001 could significantly induce the expression of IL-6 (padj<0.05).ConclusionThe results of the present study highlighted the importance of SlpA in the pathogenesis of CDI and C. difficile-induced inflammatory response in the gut. Further studies are required to unravel the significance of the observed results in promoting the intestinal inflammation and immune response induced by C. difficile SlpA from different RTs.

Single-cell RNA sequencing reveals different signatures of mesenchymal stromal cell pluripotent-like and multipotent populations

Somatic stem cells are advantageous research targets for understanding the properties required to maintain stemness. Human bone marrow-mesenchymal stromal cells (BM-MSCs) were separated into pluripotent-like SSEA-3(+) Muse cells (Muse-MSCs) and multipotent SSEA-3(-) MSCs (MSCs) and were subjected to single-cell RNA sequencing analysis. Compared with MSCs, Muse-MSCs exhibited higher expression levels of the p53 repressor MDM2; signal acceptance-related genes EGF, VEGF, PDGF, WNT, TGFB, INHB, and CSF; ribosomal protein; and glycolysis and oxidative phosphorylation. Conversely, MSCs had higher expression levels of FGF and ANGPT; Rho family and caveola-related genes; amino acid and cofactor metabolism; MHC class I/II, and lysosomal enzyme genes than Muse-MSCs. Unsupervised clustering further divided Muse-MSCs into two clusters stratified by the expression of cell cycle-related genes, and MSCs into three clusters stratified by the expression of cell cycle-, cytoskeleton-, and extracellular matrix-related genes. This study evaluating the differentiation ability of BM-MSC subpopulations provides intriguing insights for understanding stemness.

Endogenous reparative pluripotent Muse cells with a unique immune privilege system: Hint at a new strategy for controlling acute and chronic inflammation.

Multilineage-differentiating stress enduring (Muse) cells, non-tumorigenic endogenous pluripotent stem cells, reside in the bone marrow (BM), peripheral blood, and connective tissue as pluripotent surface marker SSEA-3(+) cells. They express other pluripotent markers, including Nanog, Oct3/4, and Sox2 at moderate levels, differentiate into triploblastic lineages, self-renew at a single cell level, and exhibit anti-inflammatory effects. Cultured mesenchymal stromal cells (MSCs) and fibroblasts contain several percent of SSEA-3(+)-Muse cells. Circulating Muse cells, either endogenous or administered exogenously, selectively accumulate at the damaged site by sensing sphingosine-1-phosphate (S1P), a key mediator of inflammation, produced by damaged cells and replace apoptotic and damaged cells by spontaneously differentiating into multiple cells types that comprise the tissue and repair the tissue. Thus, intravenous injection is the main route for Muse cell treatment, and surgical operation is not necessary. Furthermore, gene introduction or cytokine induction are not required for generating pluripotent or differentiated states prior to treatment. Notably, allogenic and xenogenic Muse cells escape host immune rejection after intravenous injection and survive in the tissue as functioning cells over 6 and ∼2months, respectively, without immunosuppressant treatment. Since Muse cells survive in the host tissue for extended periods of time, therefore their anti-inflammatory, anti-fibrotic, and trophic effects are long-lasting. These unique characteristics have led to the administration of Muse cells via intravenous drip in clinical trials for stroke, acute myocardial infarction, epidermolysis bullosa, spinal cord injury, neonatal hypoxic ischemic encephalopathy, amyotrophic lateral sclerosis, and COVID-19 acute respiratory distress syndrome without HLA-matching or immunosuppressive treatment.

Levels of Influenza A Virus Defective Viral Genomes Determine Pathogenesis in the BALB/c Mouse Model.

Defective viral genomes (DVGs), which are generated by the viral polymerase in error during RNA replication, can trigger innate immunity and are implicated in altering the clinical outcome of infection. Here, we investigated the impact of DVGs on innate immunity and pathogenicity in a BALB/c mouse model of influenza virus infection. We generated stocks of influenza viruses containing the internal genes of an H5N1 virus that contained different levels of DVGs (indicated by different genome-to-PFU ratios). In lung epithelial cells, the high-DVG stock was immunostimulatory at early time points postinfection. DVGs were amplified during virus replication in myeloid immune cells and triggered proinflammatory cytokine production. In the mouse model, infection with the different virus stocks produced divergent outcomes. The high-DVG stock induced an early type I interferon (IFN) response that limited viral replication in the lungs, resulting in minimal weight loss. In contrast, the virus stock with low levels of DVGs replicated to high titers and amplified DVGs over time, resulting in elevated levels of proinflammatory cytokines accompanied by rapid weight loss and increased morbidity and mortality. Our results suggest that the timing and levels of immunostimulatory DVGs generated during infection contribute to H5N1 pathogenesis. IMPORTANCE Mammalian infections with highly pathogenic avian influenza viruses (HPAIVs) cause severe disease associated with excessive proinflammatory cytokine production. Aberrant replication products, such as defective viral genomes (DVGs), can stimulate the antiviral response, and cytokine induction is associated with their emergence in vivo. We show that stocks of a recombinant virus containing HPAIV internal genes that differ in their amounts of DVGs have vastly diverse outcomes in a mouse model. The high-DVG stock resulted in extremely mild disease due to suppression of viral replication. Conversely, the stock that contained low DVGs but rapidly accumulated DVGs over the course of infection led to severe disease. Therefore, the timing of DVG amplification and proinflammatory cytokine production impact disease outcome, and these findings demonstrate that not all DVG generation reduces viral virulence. This study also emphasizes the crucial requirement to examine the quality of virus preparations regarding DVG content to ensure reproducible research.

Schisandrin B protects against LPS-induced inflammatory lung injury by targeting MyD88

BackgroundAcute lung injury (ALI) is a challenging clinical syndrome that manifests as an acute inflammatory response. Schisandrin B (Sch B), a bioactive lignan from Schisandra genus plants, has been shown to suppress inflammatory responses and oxidative stress. However, the underlying molecular mechanisms have remained elusive. Hypothesis/purposeThis study performed an in-depth investigation of the anti-inflammatory mechanism of Sch B in macrophages and in an animal model of ALI. MethodsqPCR array was used to probe the differential effects and potential target of Sch B. ALI was induced by intratracheal administration of LPS in experimental mice with or without Sch B treatment. ResultsOur studies show that Sch B differentially modulates inflammatory factor induction by LPS in macrophages by directly binding myeloid differentiation response factor-88 (MyD88), an essential adaptor protein in the toll-like receptor-4 (TLR4) pathway. Sch B spares non-MyD88-pathways downstream of TLR4. Such inhibition suppressed key signaling mediators such as TAK1, MAPKs, and NF-κB, and pro-inflammatory factor induction. Pull down assay using biotinylated-Sch B validate the direct interaction between Sch B and MyD88 in macrophages. Treatment of mice with Sch B prior to LPS challenge reduced inflammatory cell infiltration in lungs, induction of MyD88-pathway signaling proteins, and prevented inflammatory cytokine induction. ConclusionIn summary, our studies have identified MyD88 as a direct target of Sch B for its anti-inflammatory activity, and suggest that Sch B may have therapeutic value for acute lung injury and other MyD88-dependent inflammatory diseases.

SARS-CoV-2 Variants Show a Gradual Declining Pathogenicity and Pro-Inflammatory Cytokine Stimulation, an Increasing Antigenic and Anti-Inflammatory Cytokine Induction, and Rising Structural Protein Instability: A Minimal Number Genome-Based Approach.

Hyper-transmissibility with decreased disease severity is a typical characteristic of the SARS-CoV-2 Omicron variant. To understand this phenomenon, we used various bioinformatics approaches to analyze randomly selected genome sequences (one each) of the Gamma, Delta, and Omicron variants submitted to NCBI from December 15 to 31, 2021. We report that the pathogenicity of SARS-CoV-2 variants decreases in the order of Wuhan > Gamma > Delta > Omicron; however, the antigenic property follows the order of Omicron > Gamma > Wuhan > Delta. The Omicron spike RBD shows lower pathogenicity but higher antigenicity than other variants. The reported decreased disease severity by the Omicron variant may be due to its decreased pro-inflammatory and IL-6 stimulation and increased IFN-γ and IL-4 induction efficacy. The mutations in the N protein are probably associated with this decreased IL-6 induction and human DDX21-mediated increased IL-4 production for Omicron. Due to the mutations, the stability of S, M, N, and E proteins decreases in the order of Omicron > Gamma > Delta > Wuhan. Although a stronger spike RBD-hACE2 binding of Omicron increases its transmissibility, the lowest stability of its spike protein makes spike RBD-hACE2 interaction weak for systemic infection and for causing severe disease. Finally, the highest instability of the Omicron E protein may also be associated with decreased viral maturation and low viral load, leading to less severe disease and faster recovery. Our findings will contribute to the understanding of the dynamics of SARS-CoV-2 variants and the management of emerging variants. This minimal genome-based method may be used for other similar viruses avoiding robust analysis.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10753-022-01734-w.

Design, Synthesis, and Biochemical and Biological Evaluation of Novel 7-Deazapurine Cyclic Dinucleotide Analogues as STING Receptor Agonists.

Cyclic dinucleotides (CDNs) are second messengers that activate stimulator of interferon genes (STING). The cGAS-STING pathway plays a promising role in cancer immunotherapy. Here, we describe the synthesis of CDNs containing 7-substituted 7-deazapurine moiety. We used mouse cyclic GMP-AMP synthase and bacterial dinucleotide synthases for the enzymatic synthesis of CDNs. Alternatively, 7-(het)aryl 7-deazapurine CDNs were prepared by Suzuki-Miyaura cross-couplings. New CDNs were tested in biochemical and cell-based assays for their affinity to human STING. Eight CDNs showed better activity than 2'3'-cGAMP, the natural ligand of STING. The effect on cytokine and chemokine induction was also evaluated. The best activities were observed for CDNs bearing large aromatic substituents that point above the CDN molecule. We solved four X-ray structures of complexes of new CDNs with human STING. We observed π-π stacking interactions between the aromatic substituents and Tyr240 that are involved in the stabilization of CDN-STING complexes.

Activation of cytokine responses by Candida africana

Candida africana is a fungal pathogen that rarely causes invasive infections, but is mainly isolated from patients with vaginal infections. Vulvovaginal candidiasis is associated with dysregulated inflammatory responses of the host, however, the innate immune responses against C. africana are currently unknown. In this study, we explored the cytokine production of human peripheral blood mononuclear cells (PBMCs) in response to different C. africana isolates (intra-species diversity), and compared it with that induced by other yeasts belonging to the C. albicans species complex such as C. dubliniensis and C. albicans. Candida africana isolates induced both pro- and anti-inflammatory cytokines broadly similar to other Candida species. Candida africana-stimulated PBMCs tended to produce lower Interleukin (IL)-17 and IL-22 levels in comparison with C. albicans, whereas the induction of trained immunity was similar between C. africana and other Candida species. Overall, our results demonstrate that C. africana induces similar innate immune responses as the other Candida species. Therefore, its propensity to cause vulvovaginal infections is not due to an increased capacity to induce cytokine-related immune pathology. Nor is the infrequent occurrence of invasive infection by C. africana explained by a quantitatively different cytokine induction.

Four organosulfur compounds from the seeds of Capsella bursa-pastoris and their anti-inflammatory activities

Phytochemical investigation of the seeds of Capsella bursa-pastoris led to the isolation of four organosulfur compounds. There were two new compounds, 10-methylsulfinyl-decanamide (1) and 11-methylsulfinyl-undecanamide (2), along with two known compounds (3 − 4), which all have a sulfoxide group and an amide or a nitrile group. Their chemical structures were elucidated by analysing UV, IR, ESI-MS and NMR spectroscopy. In addition, compounds 1 − 4 were evaluated for their anti-inflammatory activities by using LPS-induced RAW 264.7 cells. Compounds 1 − 4 exhibited potential anti-inflammatory activities on NO release characterised by decreasing the mRNA expression levels of inducible NO synthase (iNOS), cytokines cyclooxygenase-2 (COX-2) and interleukin 6 (IL-6).

Melatonin ameliorates lipopolysaccharide induced brain inflammation through modulation of oxidative status and diminution of cytokine rush in Danio rerio

Lipopolysaccharide (LPS) is known to induce inflammation and immunonomodulation in a piscine model of Danio rerio. Present study aimed to explore the ability of melatonin in attenuating LPS-induced oxidative damages using this model. In LPS-exposed fish, activation of stress marker MDA was observed in brain with corresponding augmentation of multiple pro-inflammatory cytokines (IL1β, IL6, IL10 and TNFα). In addition, it also showed marked increase in the levels of heat shock factor (HSF) and heat shock proteins (HSPs) in association with transcription factors (NF-kB and NRF2) and mitogen-activated protein kinases (MAPKs). The changes in the levels of these mediators are highly correlated with the induction of pro-inflammatory cytokines. In melatonin-treated fishes, significant amelioration of oxidative stress was observed with reduced levels of MDA and pro-inflammatory cytokines. Melatonin also modulated expression of HSPs that facilitated the brain to overcome inflammation-induced stress by directly initiating NFkB/NRF2 translocation. In summary, melatonin effectively functions to reduce stress induced inflammatory signalling through modulation of oxidative stress and protects the brain from the neuropathological insult.

Leptin treatment has vasculo-protective effects in lipodystrophic mice

Lipodystrophy syndromes (LDs) are characterized by loss of adipose tissue, metabolic complications such as dyslipidemia, insulin resistance, and fatty liver disease, as well as accelerated atherosclerosis. As a result of adipose tissue deficiency, the systemic concentration of the adipokine leptin is reduced. A current promising therapeutic option for patients with LD is treatment with recombinant leptin (metreleptin), resulting in reduced risk of mortality. Here, we investigate the effects of leptin on endothelial to mesenchymal transition (EndMT), which impair the functional properties of endothelial cells and promotes atherogenesis in LD. Leptin treatment reduced inflammation and TGF-β2-induced expression of mesenchymal genes and prevented impairment of endothelial barrier function. Treatment of lipodystrophic- and atherosclerosis-prone animals (Ldlr-/-; aP2-nSrebp1c-Tg) with leptin reduced macrophage accumulation in atherosclerotic lesions, vascular plaque protrusion, and the number of endothelial cells with mesenchymal gene expression, confirming a reduction in EndMT in LD after leptin treatment. Treatment with leptin inhibited LD-mediated induction of the proatherosclerotic cytokine growth/differentiation factor 15 (GDF15). Inhibition of GDF15 reduced EndMT induction triggered by plasma from patients with LD. Our study reveals that in addition to the effects on adipose tissue function, leptin treatment exerts beneficial effects protecting endothelial function and identity in LD by reducing GDF15.

P126 Effects of Histoplasma capsulatum infection on activation and proliferation of hematopoietic stem cells

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMBackgroundHematopoietic stem cells (HSCs) are considered a multipotent population with high proliferative potential, and are widely used in the treatment of leukemias, multiple myeloma, and some lymphomas. In the context of infectious diseases, some microorganisms have been reported to induce changes in the expression of surface markers in HSCs by a direct effect or through the induction of cytokines. Systemic infections are characterized by inducing stress on the bone marrow, which is reflected in an increase or decrease in leukocytes and platelets in peripheral blood, a process known as ‘emergency hematopoiesis’. Histoplasmosis is a systemic mycosis caused by Histoplasma spp., which occurs mainly in immunosuppressed individuals; this mycosis can present a severe clinical picture with dissemination to various organs, including the bone marrow, and is associated with anemia and pancytopenia. So far, the effect of a possible interaction of Histoplasma with HSCs is unknown.ObjectivesTo evaluate, in vitro, the effects of Histoplasma capsulatum infection on activation and proliferation of HSCs.MethodsHSCs were obtained from bone marrow of C57BL/6 male mice; after isolation and purification, they were characterized by flow cytometry. Later, the basal expression of toll-like receptor (TLR)-2, TLR4, and Dectin-1 was determined using flow cytometry. HSCs were infected with H. capsulatum yeasts in a multiplicity of infection (MOI) of 5 and incubated for 24 h. In addition, some of the co-cultures were previously treated with specific blocking antibodies for TLR2 and TLR4 or with a blocking peptide specific for Dectin-1 (CLEC7A). Furthermore, phagocytosis, microbicidal, and cell proliferation assays were done, and the expression of the genes encoding the cytokines IL-1β, IL-6, IL-10, IL-17, TNF-α, and TGF-β as well as arginase-1 and iNOS were assessed.ResultsWe observed that H. capsulatum has the capability to adhere and internalize within these HSCs; nonetheless, this process did not affect the survival of the fungus. The interaction of H. capsulatum with HSCs induced a significantly increased expression of TLR2 and Dectin-1 but not TLR4. In addition, this fungal interaction significantly induced an augmented expression of IL-6, IL-1β, IL-10, IL-17, TNF-α, TGF-β, as well as the immune mediators Arg-1 and iNOS. Interestingly, blockade of these receptors significantly decreased the phagocytosis process as well the expression of all inflammatory mediators evaluated, especially when blocking TLR4 and Dectin-1. Of note, H. capsulatum induced apoptosis but did not inhibit the proliferation of these stem cells.ConclusionsThese results indicate that HSCs are capable of phagocytosing H. capsulatum but do not affect its survival; moreover, this fungal pathogen could induce changes in the expression of pattern-recognition receptors (PRRs), especially TLR2 and Dectin-1, and could subsequently activate the HSCs leading to the expression of inflammatory mediators as well as affecting the viability of these stem cells. Altogether, these findings indicate that H. capsulatum could affect the hematopoiesis process as reflected in an increase or decrease in leukocytes, erythrocytes, and platelets as observed in patients with severe and disseminated disease, especially in those with dissemination to bone marrow.

Intratumoral delivery of TransCon™ TLR7/8 Agonist promotes sustained anti-tumor activity and local immune cell activation while minimizing systemic cytokine induction

BackgroundIntratumoral (IT) delivery of toll-like receptor (TLR) agonists has shown encouraging anti-tumor benefit in preclinical and early clinical studies. However, IT delivery of TLR agonists may lead to rapid effusion from the tumor microenvironment (TME), potentially limiting the duration of local inflammation and increasing the risk of systemic adverse events.MethodsTo address these limitations, TransCon™ TLR7/8 Agonist—an investigational sustained-release prodrug of resiquimod that uses a TransCon linker and hydrogel technology to achieve sustained and predictable IT release of resiquimod—was developed. TransCon TLR7/8 Agonist was characterized for resiquimod release in vitro and in vivo, in mice and rats, and was assessed for anti-tumor efficacy and pharmacodynamic activity in mice.ResultsFollowing a single IT dose, TransCon TLR7/8 Agonist mediated potent tumor growth inhibition which was associated with sustained resiquimod release over several weeks with minimal induction of systemic cytokines. TransCon TLR7/8 Agonist monotherapy promoted activation of antigen-presenting cells in the TME and tumor-draining lymph nodes, with evidence of activation and expansion of CD8+ T cells in the tumor-draining lymph node and TME. Combination of TransCon TLR7/8 Agonist with systemic immunotherapy further promoted anti-tumor activity in TransCon TLR7/8 Agonist-treated tumors. In a bilateral tumor setting, combination of TransCon TLR7/8 Agonist with systemic IL-2 potentiated tumor growth inhibition in both injected and non-injected tumors and conferred protection against tumor rechallenge following complete regressions.ConclusionsOur findings show that a single dose of TransCon TLR7/8 Agonist can mediate sustained local release of resiquimod in the TME and promote potent anti-tumor effects as monotherapy and in combination with systemic immunotherapy, supporting TransCon TLR7/8 Agonist as a novel intratumoral TLR agonist for cancer therapy. A clinical trial to evaluate the safety and efficacy of TransCon TLR7/8 Agonist, as monotherapy and in combination with pembrolizumab, in cancer patients is currently ongoing (transcendIT-101; NCT04799054).

SARS-CoV-2 Nsp14 protein associates with IMPDH2 and activates NF-κB signaling.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to NF-κB activation and induction of pro-inflammatory cytokines, though the underlying mechanism for this activation is not fully understood. Our results reveal that the SARS-CoV-2 Nsp14 protein contributes to the viral activation of NF-κB signaling. Nsp14 caused the nuclear translocation of NF-κB p65. Nsp14 induced the upregulation of IL-6 and IL-8, which also occurred in SARS-CoV-2 infected cells. IL-8 upregulation was further confirmed in lung tissue samples from COVID-19 patients. A previous proteomic screen identified the putative interaction of Nsp14 with host Inosine-5’-monophosphate dehydrogenase 2 (IMPDH2), which is known to regulate NF-κB signaling. We confirmed the Nsp14-IMPDH2 protein interaction and identified that IMPDH2 knockdown or chemical inhibition using ribavirin (RIB) and mycophenolic acid (MPA) abolishes Nsp14- mediated NF-κB activation and cytokine induction. Furthermore, IMPDH2 inhibitors (RIB, MPA) or NF-κB inhibitors (bortezomib, BAY 11-7082) restricted SARS-CoV-2 infection, indicating that IMPDH2-mediated activation of NF-κB signaling is beneficial to viral replication. Overall, our results identify a novel role of SARS-CoV-2 Nsp14 in inducing NF-κB activation through IMPDH2 to promote viral infection.

Lymphangiogenesis and Prostanoids as Regulators in Disease States

The lymphatic vessels have crucial roles in the regulation of interstitial fluids, immune surveillance and the absorption of dietary fat in the intestine. Lymphatic functions are also closely related to the pathogenesis of various disease states such as inflammatory responses, lymphedema, and metastasis of tumors. Lymphangiogenesis, the formation of new lymphatic vessels from pre-existing lymphatics is critical determinant of the above pathological conditions. Prostanoids including prostaglandins and thromboxanes are the metabolites of arachidonic acid, C-20 unsaturated fatty acid, and they exhibit a variety of actions via specific receptors. Although some kinds of growth factors are well-characterized in the lymphangiogenesis, there is accumulating evidence that prostaglandinsand thromboxanesare important regulators of lymphangiogenesis. Previously prostanoids are reported to have immediate actions on the smooth muscles and vasculatures, however, they work as inducers of growth factors or cytokines that regulate lymphangiogenesis, and modulate the prolymphangiogenic microenvironment. This review discusses our current understanding of prostanoids as regulators of lymphangiogenesis, and the emerging importance of the lymphangiogenesis as a therapeutic target.

Not all T cell epitopes are equally desired: a review of in silico tools for the prediction of cytokine-inducing potential of T-cell epitopes.

Assessment of protective or harmful T cell response induced by any antigenic epitope is important in designing any immunotherapeutic molecule. The understanding of cytokine induction potential also helps us to monitor antigen-specific cellular immune responses and rational vaccine design. The classical immunoinformatics tools served well for prediction of B cell and T cell epitopes. However, in the last decade, the prediction algorithms for T cell epitope inducing specific cytokines have also been developed and appreciated in the scientific community. This review summarizes the current status of such tools, their applications, background algorithms, their use in experimental setup and functionalities available in the tools/web servers.