FasL Research Articles

Pleural fluid soluble Fas ligand and tuberculous pleural effusion: a prospective diagnostic test accuracy study.

The diagnosis of tuberculous pleural effusion (TPE) is challenging for pulmonologists. Adenosine deaminase (ADA), interferon-gamma (IFN-γ), and interleukin-27 (IL-27) have some limitations for diagnosing TPE. Soluble Fas ligand (sFasL) had a high diagnostic value for TPE. However, it remains unknown: (I) whether sFasL has an additional diagnostic value to the traditional markers (e.g., ADA); (II) whether sFasL provides a net benefit in patients with undiagnosed pleural effusion; (III) factors affecting the diagnostic accuracy of sFasL for TPE. This study aimed to evaluate the additional diagnostic value and benefit of pleural fluid sFasL for TPE. We prospectively enrolled 211 patients with undiagnosed pleural effusion. The concentration of sFasL in pleural fluid was measured by an enzyme-linked immunosorbent assay (ELISA). The diagnostic accuracy and net benefit of sFasL and ADA for TPE were analyzed by a receiver operating characteristic (ROC) curve, decision curve analysis (DCA), net reclassification improvement (NRI), and integrated discriminant improvement (IDI). The area under the ROC curves (AUCs) of sFasL and ADA were 0.74 (95% CI: 0.65-0.83) and 0.80 (95% CI: 0.71-0.90), respectively. The decision curve of sFasL revealed net benefit. The continuous NRI and IDI of sFasL were 0.36 (0.00-0.72, P=0.05) and 0.02 (-0.01-0.06, P=0.18), respectively. Pleural fluid sFasL has moderate diagnostic accuracy for TPE.

Inhibiting Endothelial Cell-Mediated T Lymphocyte Apoptosis with Integrin-Targeting Peptide-Drug Conjugate Filaments for Chemoimmunotherapy of Triple-Negative Breast Cancer.

Tumor-associated endothelial cells (TECs) limit antitumor immunity via inducing apoptosis of infiltrating T lymphocytes through a Fas ligand (FasL) mediated mechanism. Herein, this work creates a peptide-drug conjugate (PDC) by linking 7-ethyl-10-hydroxycamptothecin (SN38) to hydrophilic segments with either RGDR or HKD motif at their C-terminus through a glutathione-responsive linker. The PDCs spontaneously assemble into filaments in aqueous solution. The PDC filaments containing 1% of SN38-RGDR (SN38-HKD/RGDR) effectively target triple-negative breast cancer (TNBC) cells and TECs with upregulated expression of integrin, and induce immunogenic cell death (ICD) of tumor cells and FasL downregulation of TECs. SN38-HKD/RGDR increases infiltration, activity, and viability of CD8+ T cells, and thus inhibits the growth of primary tumors and pulmonary metastasis. This study highlights the synergistic modulation of cancerous cells and TECs with integrin-targeting PDC filaments as a promising strategy for TNBC chemoimmunotherapy.

Plasma Immune Proteins and Circulating Tumor DNA Predict the Clinical Outcome for Non-Small-Cell Lung Cancer Treated with an Immune Checkpoint Inhibitor

Immunotherapy has altered the therapeutic landscape for patients with non-small-cell lung cancer (NSCLC). The immune checkpoint inhibitor pembrolizumab targets the PD-1/PD-L1 signaling axis and produces durable clinical responses, but reliable biomarkers are lacking. Using 115 plasma samples from 42 pembrolizumab-treated patients with NSCLC, we were able to identify predictive biomarkers. In the plasma samples, we quantified the level of 92 proteins using the Olink proximity extension assay and circulating tumor DNA (ctDNA) using targeted next-generation sequencing. Patients with an above-median progression-free survival (PFS) had significantly higher expressions of Fas ligand (FASLG) and inducible T-cell co-stimulator ligand (ICOSLG) at baseline than patients with a PFS below the median. A Kaplan–Meier analysis demonstrated that high levels of FASLG and ICOSLG were predictive of longer PFS and overall survival (OS) (PFS: 10.83 vs. 4.49 months, OS: 27.13 vs. 18.0 months). Furthermore, we identified a subgroup with high expressions of FASLG and ICOSLG who also had no detectable ctDNA mutations after treatment initiation. This subgroup had significantly longer PFS and OS rates compared to the rest of the patients (PFS: 25.71 vs. 4.52 months, OS: 34.62 vs. 18.0 months). These findings suggest that the expressions of FASLG and ICOSLG at baseline and the absence of ctDNA mutations after the start of treatment have the potential to predict clinical outcomes.

Platelet-Derived TGF-β1 Induces Functional Reprogramming of Myeloid-Derived Suppressor Cells in Immune Thrombocytopenia

Introduction: Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by isolated thrombocytopenia and varying degrees of bleeding symptoms. Platelets are anucleate cytoplasmic cell fragments that play dual physiological roles as both cellular mediators of thrombosis and immune modulatory cells. Platelets are the largest storage pool of TGF-β1, a crucial enforcer of immune homeostasis and tolerance. Studies have shown that TGF-β1 released by platelets contributes to the pathogenesis of pulmonary arterial hypertension and cervical carcinoma. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immature myeloid cells with potent immunosuppressive functions. Our previous studies have shown that MDSCs are impaired in number and function in patients with ITP. The objective of this study was to elucidate the underlying molecular mechanisms of MDSCs reprogramming by platelet-derived TGF-β1. Methods: A transwell assay was used to determine the impact of platelets on MDSCs induced from peripheral blood mononuclear cells (PBMCs) of ITP patients and healthy controls. The percentage of circulating MDSCs in ITP patients was detected before and after administration of thrombopoietin receptor agonists (TPO-RAs). Besides, anti-CD61 immune-sensitized splenocytes were transferred into SCID mice to establish an active murine model of ITP. Subsequently, MDSCs from TPO-RA-treated ITP mice were transplanted as cell therapy to rescue treatment-free ITP mice. In specific, adeno-associated viral (AAV) vectors efficiently infected MDSCs to enable cell-specific Smad2/3 knock-down. Finally, platelet specific TGF-β1-deficient mice were used to validate the effect of platelet-derived TGF-β1 on MDSCs. Results: TGF-β1 levels in the supernatant of MDSCs co-cultured with platelets of high concentration was significantly higher than that in MDSCs co-cultured with platelets of low concentration or alone. FACS analysis showed that platelet mass enhanced the inhibitory effects of MDSCs on the proliferation of CD4 + T cells, which could be offset by TGF-β1 inhibitor (TGF-βi). Besides, TPO-RAs treatment induced significantly increased percentage of circulating MDSCs in responders with chronic ITP. Likewise, TPO-RA significantly expanded MDSC population in active ITP murine models. Furthermore, after receiving donor MDSCs from TPO-RA-treated ITP mice, arrested differentiation of plasma cells, reduced Fas ligand expression on cytotoxic T cells, and re-balanced T cell subsets were found in the recipient mice, indicating augmented inhibitory functions of MDSCs. Mechanistically, transcriptome analysis confirmed the participation of TGF-β/Smad pathway in donor MDSCs from TPO-RA-treated ITP mice, which was offset by Smad2/3 knockdown. Finally, by constructing ITP models using Pf4 cre-Tgfb1 fl/fl and Pf4 cre+Tgfb1fl/fl mice, we found that TGF-β1 inhibition and platelet TGF-β1-deficence, both, could counteract the elevated platelet count and MDSCs percentage, as well as improved immunosuppressive capacity of MDSCs trigged by TPO-RA modulation. Conclusion: To sum up, it was demonstrated that platelet TGF-β1 induced the expansion and functional reprogramming of MDSCs via TGF-β/Smad pathway. These data indicate that platelet recovery not only serves as an endpoint of remission, but also paves the way for tolerance induction in immune-mediated thrombocytopenia. Our exploratory study deciphered immunological changes induced by TPO-RAs and helped to identify molecular mechanisms responsible for long-term remission after TPO-RA discontinuation.

1530. Reactivation of Latent HIV in CD4 T Cells Procured from Patients on ART by Autologous CD8 T cells or Fas Ligand

Abstract Background HIV persists in a long-lived infected CD4 T cell reservoir, which harbors an integrated transcriptionally latent virus. We reported that cytotoxic CD8 T cells conjugate with, and kill, autologous HIV-infected CD4 T cells isolated from all stages of HIV infection including reservoir cells of patients under antiretroviral therapy (ART) (image 1). This killing is attenuated by HIV Nef protein. We also previously reported that FasL presented by the cytotoxic T cells interacts with Fas on the surface of the target cells, resulting in the apoptosis of the target cells. Wajant demonstrated that Fas/FasL interaction can result in the target cell activation through NFkB, whereas NFkB binding site is present in the Long Terminal Repeat of HIV. Therefore, we hypothesize that the interaction of FasL and autologous CD8 T cells with CD4 T cells can result in the reactivation of latent HIV. Conjugation and apoptosis of CD4 T cells by autologous CD8 T cells procured from patients on ART and analyzed by ImageStream. The CD4 T cells in apoptosis are positive for activated caspase 3 (yellow) and DNA fragmentation/ TUNEL assay (orange). CD4 T cells (green), CD8 T cells (red), DAPI (blue). Methods CD4 T cells and CD8 T cells were procured from the PBMC of 20 HIV-infected patients on ART with undetectable viral load and 10 healthy volunteers. The cells were isolated using magnetic beads. Resting memory CD4 T cells (CD25-, CD69- and HLA-DR-) were isolated using a two-step bead depletion purification procedure. These cells were then incubated with soluble FasL (sFasL) and autologous CD8 T cells, for 2 and 16 hours. P24 was looked for in the supernatant by ELISA, and the expression of viral proteins was looked for inside CD4 T cells using immunohistochemistry and confocal microscopy Results CD4 T cells and resting memory CD4 T cells, procured from PBMC of HIV-infected patients on ART, showed HIV reactivation after incubation with sFasL and autologous CD8 T cells. This reactivation was demonstrated by the appearance of P24 in the supernatant (figure 1). HIV proteins p24, gp120, and Nef appeared inside the CD4 T cells (Image 2). HIV reactivation was not demonstrated in the CD4 T cells procured from HIV-infected patients that were incubated without sFasL or autologous CD8 T cells for 18 hours (figure 1). Immunohistochemistry and confocal microscopy of CD4 T cells incubated with sFasL for 18 hours. The cells were then incubated with a primary fluorescent antibody against HIV proteins (P24, Nef, GP120) and a secondary antibody ALEXA594 (red). The cells were also marked with DAPI (blue). We can see the expression of HIV proteins inside the CD4 T cells (red arrows). ELISA for the expression of P24 in the supernatant of CD4 T cells procured from HIV-infected patients, following different manipulations On the left, we note positive ELISA for P24 in the supernatant of the CD4 T cells of patients 11 to 16 (pg/ml). On the right, we note positive ELISA for P24 in the supernatant of the latent CD4 T cells of patients 17-20 (pg/ml). Conclusion HIV manipulates the cellular immune system in two ways; First, HIV attenuates CD8 T cells killing of HIV-infected CD4 T cells by the HIV-Nef protein. Second, reactivation of latent HIV by CD8 T cells and sFasL, that results in further infection of additional CD4 T cells. Disclosures All Authors: No reported disclosures

Variant rs4986790 of toll-like receptor 4 affects the signaling and induces cell dysfunction in patients with severe COVID-19

ObjectivesWe investigated the expression of toll-like receptor (TLR)-4 on the cell surface of innate and adaptive cells from patients with COVID-19 carrying the rs4986790 GG genotype in the TLR4 gene and the functional profile of these cells. MethodsWe included 1169 hospitalized patients with COVID-19. The rs4986790 in TLR4 was identified by real-time polymerase chain reaction. Peripheral blood mononuclear cells were isolated and cultured to evaluate TLR-4 expression on immune cells. Supernatants recovered culture assays were stored, and we measured cytokines and cytotoxic molecules. ResultsWe showed that the rs4986790 (GG) was significantly associated (P = 0.0310) with severe COVID-19. Cells of patients with COVID-19 carrying the GG genotype have increased the frequency of monocytes and activated naïve and non-switched B cells positive to TLR-4 when cells are stimulated with lipopolysaccharide and with spike protein of SARS-CoV-2. Also, cells from patients with GG COVID-19 cannot produce pro-inflammatory cytokines after lipopolysaccharide stimulus, but they are high producers of cytotoxic molecules at baseline. ConclusionsThe rs4986790 GG genotype of the TLR4 is associated with the risk of COVID-19 and acute respiratory distress syndrome. Peripheral blood mononuclear cells of patients carrying the rs4986790 (TLR4) GG genotype had a limited delivery of pro-inflammatory cytokines compared to the AA and AG genotypes in which TLR-4 stimulation induces IL-10, IL-6, tumor necrosis factor-α, and Fas ligand production.

Abstract 17813: Markers of Cellular Damage, Inflammatory Response, Coagulation and Platelet Activity, and Apoptosis During Catheter Ablation for Atrial Fibrillation Using Radiofrequency and Pulsed-Field Energy

Introduction: Pulsed-field ablation (PFA) presents a new technology for the ablation of AF using non-thermal ablation energy. Ablation energies producing thermal injury are associated with an inflammatory response, platelet activation, and coagulation cascade activation. The study aimed to compare markers of cell damage and platelet and coagulation activation in patients undergoing PVI using PF and radiofrequency (RF) energy. Hypothesis: PFA will be associated will more pronounced myocardial damage, but less pronounced activation of platelets and inflammation. Methods: Patients with AF indicated for PVI were enrolled and randomly assigned to undergo PVI using RF (CARTO Smart Touch, Biosense Webster, USA) or PF (Farapulse, Boston-Scientific, USA) energy. Markers of myocardial damage (high-sensitivity troponin I), apoptosis (Fas ligand, caspase-3), inflammation (interleukin-6), coagulation (D-dimers, Fibrin monomers, von Willebrand antigen and factor activity), platelet activation (P-selectin, activated Gp IIb/IIIa antigen and CD42b), and neuron axon damage (NGF-β) were measured before the procedure (T1), after transseptal puncture, (T2), after completing the ablation in the left atrium (T3), and one day after the procedure (T4). Results: Sixty-five patients were enrolled in the PFA (n=33) and RFA (n=32) groups. Both groups were similar in baseline characteristics (age 60.5±12.7 vs. 64.0±10.7 and 20 (60.6%) non-paroxysmal vs. 20 (62.5%) paroxysmal AF). Procedural and left atrial dwelling times were substantially shorter in the PFA group (55:09±11:57 vs. 151:19±41:25 min, p< 0.001; 36:00±8:05 vs. 115:58±36:49 min, p<0.001). Troponin release (1 d after the procedure) was substantially higher in the PFA group (10,102, IQR 8,272-14,207 vs. 1,006, IQR 603-1,433) and was ten times higher in PFA patients. However, during the procedure, markers of platelet activation, coagulation activation, and inflammation were similar between groups. Conclusions: PF ablation was associated with substantially more myocardial damage compared to radiofrequency ablation. However, coagulation activation, platelet activation, and inflammatory responses were similar.

LONG-TERM COVID-19 EFFECT TO ENDOTHELIAL DAMAGE TROUGH EXTRINSIC APOPTOSIS LED TO CARDIOVASCULAR DISEASE PROGRESSION: AN UPDATE REVIEW

COVID-19 can involve persistence, sequelae, and other medical complications that last weeks to months after initial recovery; these prolonged symptoms called as long-term covid-19 effect. Symptoms, signs, or abnormal clinical parameters persisting two or more weeks after COVID-19 onset that do not return to a healthy baseline can potentially be long-term effects of the disease. SARS-CoV-2 affects the cardiovascular system and causes conditions such as myocarditis, arrhythmias, and myocardial injury. Vascular damage from COVID-19 has been affected directly by the SARS-CoV-2 virus infection and indirectly by systemic inflammatory cytokine storm. This damage can be long-lasting and lead to various cardiovascular complications. Fas ligand (FasL)-Fas complex is a death factor that induces cell apoptosis. Fas and FasL have been detected in the endothelial wall, and it has been proposed that Fas-mediated apoptosis has a role in physiological and pathological cell turnover in the endothelial wall. High concentrations of inflammatory cytokines, such as cytokines storm induced by SARS-CoV-2 infection, are thought to increase the expression of FasL, which leads to an increase in the regulation of extrinsic apoptosis in endothelial cells leading to endothelial damage. This article summarises the current understanding of the long-term covid-19 effect on endothelial damage through extrinsic apoptosis Fas-FasL complex.

Immune Mediators Profiles in the Aqueous Humor of Patients with Simple Diabetic Retinopathy.

Various immune mediators identified to date are associated with the development of advanced forms of diabetic retinopathy (DR), such as proliferative DR and diabetic macular edema, although the exact pathophysiological mechanisms of early stages of DR such as simple DR remain unclear. We determined the immune mediator profile in the aqueous humor of eyes with simple DR. Fifteen eyes of fifteen patients with simple DR were studied. Twenty-two eyes of twenty-two patients with cataracts and no DR served as controls. Undiluted aqueous humor samples were collected, and a cytometric bead array was used to determine the aqueous humor concentrations of 32 immune mediators comprising 13 interleukins (IL), interferon-γ, interferon-γ-inducible protein-10 (IP-10), monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, regulated on activation, normal T cell expressed and secreted (RANTES), monokine induced by interferon-γ, basic fibroblast growth factor (bFGF), Fas ligand, granzyme A, granzyme B, interferon-inducible T-cell alpha chemoattractant (ITAC), fractalkine, granulocyte macrophage colony-stimulating factor, granulocyte colony-stimulating factor (G-CSF), vascular endothelial growth factor (VEGF), angiogenin, tumor necrosis factor-α, and CD40 ligand. Among the 32 immune mediators, 10 immune mediators, including bFGF, CD40 ligand, fractalkine, G-CSF, IL-6, IL-8, MIP-α, MIP-1β, and VEGF, showed significantly higher aqueous humor concentrations and the Fas ligand had significantly lower concentration (p < 0.05) in eyes with simple DR compared with control eyes. Of these 10 cytokines with significant concentration alteration, protein-protein interaction analysis revealed that 8 established an intricate interaction network. Various immune mediators may contribute to the pathogenesis of simple DR. Attention should be given to the concentrations of immune mediators in ocular fluids even in simple DR. Large-scale studies are warranted to assess whether altered aqueous humor concentrations of these 10 immune mediators are associated with an increased risk of progression to advanced stages of DR.

Effect of safranal or candesartan on 3-nitropropionicacid-induced biochemical, behavioral and histological alterations in a rat model of Huntington's disease.

3-nitropropionic acid (3-NP) is a potent mitochondrial inhibitor mycotoxin. Systemic administration of 3-NP can induce Huntington's disease (HD)-like symptoms in experimental animals. Safranal (Safr) that is found in saffron essential oil has antioxidant, anti-inflammatory and anti-apoptotic actions. Candesartan (Cands) is an angiotensin receptor blocker that has the potential to prevent cognitive deficits. The present study aims to investigate the potential neuroprotective efficacy of Safr or Cands in 3-NP-induced rat model of HD. The experiments continued for nine consecutive days. Rats were randomly assigned into seven groups. The first group (Safr-control) was daily intraperitoneally injected with paraffin oil. The second group (Cands- and 3-NP-control) daily received an oral dose of 0.5% carboxymethylcellulose followed by an intraperitoneal injection of 0.9% saline. The third and fourth groups received a single daily dose of 50 mg/kg Safr (intraperitoneal) and 1 mg/kg Cands (oral), respectively. The sixth group was daily treated with 50 mg Safr kg/day (intraperitoneal) and was intraperitoneally injected with 20 mg 3-NP/ kg, from the 3rd till the 9th day. The seventh group was daily treated with 1 mg Cands /kg/day (oral) and was intraperitoneally injected with 20 mg 3-NP/ kg, from the 3rd till the 9th day. The present results revealed that 3-NP injection induced a considerable body weight loss, impaired memory and locomotor activity, reduced striatal monoamine levels. Furthermore, 3-NP administration remarkably increased striatal malondialdehyde and nitric oxide levels, whereas markedly decreased the total antioxidant capacity. Moreover, 3-NP significantly upregulated the activities of inducible nitric oxide synthase and caspase-3 as well as the Fas ligand, in striatum. On the contrary, Safr and Cands remarkably alleviated the above-mentioned 3-NP-induced alterations. In conclusion, Safr and Cands may prevent or delay the progression of HD and its associated impairments through their antioxidant, anti-inflammatory, anti-apoptotic and neuromodulator effects.

Combined thermal ablation and liposomal granulocyte-macrophage colony stimulation factor increases immune cell trafficking in a small animal tumor model.

To characterize intratumoral immune cell trafficking in ablated and synchronous tumors following combined radiofrequency ablation (RFA) and systemic liposomal granulocyte-macrophage colony stimulation factor (lip-GM-CSF). Phase I, 72 rats with single subcutaneous R3230 adenocarcinoma were randomized to 6 groups: a) sham; b&c) free or liposomal GM-CSF alone; d) RFA alone; or e&f) combined with blank liposomes or lip-GM-CSF. Animals were sacrificed 3 and 7 days post-RFA. Outcomes included immunohistochemistry of dendritic cells (DCs), M1 and M2 macrophages, T-helper cells (Th1) (CD4+), cytotoxic T- lymphocytes (CTL) (CD8+), T-regulator cells (T-reg) (FoxP3+) and Fas Ligand activated CTLs (Fas-L+) in the periablational rim and untreated index tumor. M1/M2, CD4+/CD8+ and CD8+/FoxP3+ ratios were calculated. Phase II, 40 rats with double tumors were randomized to 4 groups: a) sham, b) RFA, c) RFA-BL and d) RFA-lip-GM-CSF. Synchronous untreated tumors collected at 7d were analyzed similarly. RFA-lip-GMCSF increased periablational M1, CTL and CD8+/FoxP3+ ratio at 3 and 7d, and activated CTLs 7d post-RFA (p<0.05). RFA-lip-GMSCF also increased M2, T-reg, and reduced CD4+/CD8+ 3 and 7d post-RFA respectively (p<0.05). In untreated index tumor, RFA-lip-GMCSF improved DCs, M1, CTLs and activated CTL 7d post-RFA (p<0.05). Furthermore, RFA-lip-GMSCF increased M2 at 3 and 7d, and T-reg 7d post-RFA (p<0.05). In synchronous tumors, RFA-BL and RFA-lip-GM-CSF improved DC, Th1 and CTL infiltration 7d post-RFA. Systemic liposomal GM-CSF combined with RFA improves intratumoral immune cell trafficking, specifically populations initiating (DC, M1) and executing (CTL, FasL+) anti-tumor immunity. Moreover, liposomes influence synchronous untreated metastases increasing Th1, CTL and DCs infiltration.

CD138 promotes the accumulation and activation of autoreactive T cells in autoimmune MRL/lpr mice.

Autoreactive T cells, specifically CD138+ (syndecan-1) T cells produced in Fas-deficient systemic lupus erythematosus (SLE) mouse models, were shown to significantly promote the generation of autoantibodies. In the present study, Murphy Roths Large lymphoproliferative (MRL/lpr) lupus mice were used to investigate the role of CD138 protein expression in T cells in the progression of SLE. Measurement of flow cytometry, immunofluorescence and Luminex were performed to determine the effect of CD138 on T cells in MRL/lpr mice. The results demonstrate that CD138+ T cells induce apoptosis via a Fas-dependent pathway. CD138 protein expression in T cells of MRL/lpr mice significantly reduced T cell apoptosis and contributed to the accumulation of T cells and double negative (DN) T cells, whilst simultaneously promoting T cell activation in Fas-deficient lupus mice. CD138 protein expression in DN T cells also significantly increased the protein expression of Fas ligand to enhance the cytotoxicity of DN T cells. Furthermore, phorbol 12-myristate 13-acetate and ionomycin (PI) stimulation reduced CD138 protein expression in CD3+ T cells and prevented CD138+ T cell accumulation by inducing specific apoptosis. PI stimulation also activated T cells in MRL/lpr mice to increase CD69 protein expression. CD69 protein expression in CD138+ T cells significantly increased the frequency of apoptotic CD138+ T cells. In addition, results from the present study demonstrated that CD138- T cells of MRL/lpr lupus mice had an activation defect. CD138 protein expression in T cells significantly reversed the defective activation and activating T cells could significantly reduce CD138 protein expression in CD3+ T cells of MRL/lpr mice. This suggests that CD138 protein expression in CD3+CD138- T cells of MRL/lpr mice may be a consequence of the impaired activation in autoreactive T cells prior to exposure to self-antigens by the immune system. CD138 expression in autoreactive T cells has a central role in promoting the progression and development of autoimmune response in MRL/lpr mice.

TCR-Engineered Lymphocytes Targeting NY-ESO-1: In Vitro Assessment of Cytotoxicity against Tumors.

Adoptive T-cell therapies tailored for the treatment of solid tumors encounter intricate challenges, necessitating the meticulous selection of specific target antigens and the engineering of highly specific T-cell receptors (TCRs). This study delves into the cytotoxicity and functional characteristics of in vitro-cultured T-lymphocytes, equipped with a TCR designed to precisely target the cancer-testis antigen NY-ESO-1. Flow cytometry analysis unveiled a notable increase in the population of cells expressing activation markers upon encountering the NY-ESO-1-positive tumor cell line, SK-Mel-37. Employing the NanoString platform, immune transcriptome profiling revealed the upregulation of genes enriched in Gene Ontology Biological Processes associated with the IFN-γ signaling pathway, regulation of T-cell activation, and proliferation. Furthermore, the modified T cells exhibited robust cytotoxicity in an antigen-dependent manner, as confirmed by the LDH assay results. Multiplex immunoassays, including LEGENDplex™, additionally demonstrated the elevated production of cytotoxicity-associated cytokines driven by granzymes and soluble Fas ligand (sFasL). Our findings underscore the specific targeting potential of engineered TCR T cells against NY-ESO-1-positive tumors. Further comprehensive in vivo investigations are essential to thoroughly validate these results and effectively harness the intrinsic potential of genetically engineered T cells for combating cancer.

Study of anticancer effects of platinum levetiracetam and levetiracetam via cancer biomarkers genes expression on HepG2 cell line.

High expression of some anticancer biomarkers such as telomerase and B cell lymphoma-2(Bcl-2), microRNA-21(miRNA-21), and low expression FAS ligand (FASLG) are reported in many cancers. Some anticancer drugs such as Levetiracetam(Lev) produce their effects via the change of expression of these biomarkers. The present study aimed to evaluate the anti-cancer effects of a new compound, Platinum Levetiracetam(Pt-Lev), gene expression of mentioned biomarkers on hepatocyte G2 (HepG2) cells compared to Lev. In this study, Human Dermal fibroblast cells (HDF) were used as the negative control group (group A) HepG2 cells were divided into three groups: untreated cancer cells as positive group (group B), groups C and D were treated with, Lev and Pt-Lev, respectively. After evaluating lethal concentration 50% (LC50) for the examined drugs using the MTT test, biomarker gene expression was evaluated by real-time PCR. No Apoptotic cell was found in groups C or D before drug treatment, but it was present using different concentrations of the drugs. Results indicated that telomerase and miRNA-21 genes expression was significantly lower and FASLG was higher in group D compared with group C but there was no significant difference for Bcl-2 expression between these two groups. For the first time, it was indicated that Pt-Lev has anticancer effects by inhibiting telomerase and Bcl-2 and miRNA-21 and increasing FASLG gene expression and its effects were more than Lev. It effectively exerted its anticancer effects by extending apoptosis on HepG2 cells.

Exploring TCR-like CAR-Engineered Lymphocyte Cytotoxicity against MAGE-A4.

TCR-like chimeric antigen receptor (CAR-T) cell therapy has emerged as a game-changing strategy in cancer immunotherapy, offering a broad spectrum of potential antigen targets, particularly in solid tumors containing intracellular antigens. In this study, we investigated the cytotoxicity and functional attributes of in vitro-generated T-lymphocytes, engineered with a TCR-like CAR receptor precisely targeting the cancer testis antigen MAGE-A4. Through viral transduction, T-cells were genetically modified to express the TCR-like CAR receptor and co-cultured with MAGE-A4-expressing tumor cells. Flow cytometry analysis revealed a significant surge in cells expressing activation markers CD69, CD107a, and FasL upon encountering tumor cells, indicating robust T-cell activation and cytotoxicity. Moreover, immune transcriptome profiling unveiled heightened expression of pivotal T-effector genes involved in immune response and cell proliferation regulation. Additionally, multiplex assays also revealed increased cytokine production and cytotoxicity driven by granzymes and soluble Fas ligand (sFasL), suggesting enhanced anti-tumor immune responses. Preliminary in vivo investigations revealed a significant deceleration in tumor growth, highlighting the therapeutic potential of these TCR-like CAR-T cells. Further investigations are warranted to validate these revelations fully and harness the complete potential of TCR-like CAR-T cells in overcoming cancer's resilient defenses.

The Protective Effect of Chronic Intermittent Hypobaric Hypoxia on Preventing the Destruction of CD34+ Haematopoietic Stem Cells in Aplastic Anaemia by Modulating the Th1/Th2 Balance.

Aplastic anaemia (AA) is a haematopoietic disorder caused by immune-mediated attack on haematopoietic stem cells (HSCs). Stem cell transplantation and immunosuppressive therapy remain the major treatment choice for AA patients but have limited benefits and undesired side effects. The aim of our study was to clarify the protective role of immunity of chronic intermittent hypobaric hypoxia (CIHH) and the underlying mechanism in AA. Our integrative analysis demonstrated that CIHH pre-treatment significantly improved haematopoiesis and survival in an AA rat model. We further confirmed that CIHH pre-treatment was closely associated with the Th1/Th2 balance and a large number of negative regulatory haematopoietic factors, such as TNF-α and IFN-γ, produced by hyperactive Th1 lymphocytes released in AA rats, which induced the death program in a large number of CD34+ HSCs by activating the Fas/FasL apoptosis pathway, while CIHH pre-treatment effectively downregulated the expression of TNF-α and IFN-γ, resulting in a reduction in Fas antigen expression in CD34+ HSCs. In summary, this study provides evidence that CIHH has good protective effect against AA by modulating immune balance in Th1/Th2 cells and may provide a new therapeutic strategy.

A novel Fas ligand plays an important role in cell apoptosis of Crassostrea hongkongensis: molecular cloning, expression profiles and functional identification of ChFasL.

Apoptosis regulates normal development, homeostasis, immune tolerance and response to environmental stress by eliminating unwanted or diseased cells, and plays a key role in non-specific immunity of invertebrates. The exogenous pathway mediated by death receptors and death ligands is a very important pathway for cell apoptosis. Death ligands are mainly members of the tumour necrosis factor (TNF) family, of which FasL is an important member. The deep involvement of FasL in vertebrates cell apoptosis and immunity has been reported many times, but there is limited research on the FasL gene in shellfish, and its functional importance in oyster cell apoptosis and immunity remains unclear. The full length of ChFasL was identified and cloned based on the genome of Crassostrea hongkongensis. Quantitative PCR was used to detect the relative expression of ChFasL in different developmental stages and tissues, as well as the changes of relative expression in hemocytes after bacterial infection. The expression position of ChFasL in HEK293T cells was also located by subcellular localization, and the effect of increased recombinant protein content on the activity of reporter genes p53 and p21 was studied by dual-fluorescence reporter gene. Finally, the changes of apoptosis rate in hemocytes after ChFasL silencing was identified by RNA interference technology. We identified a novel FasL gene from C. hongkongensis and named it ChFasL. We found that ChFasL has potential N-linked glycosylation site, a transmembrane domain and a TNF region, which was a typical characteristics of TNF family. ChFasL was expressed in all developmental stages of larvae and in all tissues of oysters. After stimulation by V. alginolyticus or S. haemolyticus, its relative expression in hemocytes increased significantly, suggesting that ChFasL was deeply engaged in the immune response process of C. hongkongensis to external microbial stimulation. The results of subcellular localization showed that ChFasL was mainly distributed in the cytoplasm of HEK293T cells. With the overexpression of the recombinant protein pcDNA3 1- ChFasL, the activity of p53 and p21 significantly increased, showing a positive regulatory effect. Moreover, after dsRNA successfully reduced the relative expression of ChFasL, the apoptosis rate of hemocytes was significantly lower than that the dsGFP group. These results comprehensively confirmed the important role of ChFasL in the apoptosis process of C. hongkongensis, which provided the basis and premise for the in-depth understanding of the immune function of apoptosis in molluscs, and also contributed to the research on the pathogenic death mechanism and disease resistance breeding of marine bivalves.

Activation and Denitrosylation of Procaspase-3 in KA-induced Excitotoxicity.

It has been reported that activation of glutamate kainate receptor subunit 2 (GluK2) subunit-containing glutamate receptors and the following Fas ligand(FasL) up-regulation, caspase-3 activation, subsequently results in delayed apoptosis-like neuronal death in hippocampus CA1 subfield after cerebral ischemia. Nitric oxide-mediated S-nitrosylation might inhibit the procaspase activation, whereas denitrosylation might contribute to cleavage and activation of procaspases. The study aimed to elucidate the molecular mechanisms underlying procaspase-3 denitrosylation and activation following kainic acid (KA)-induced excitotoxicity in rat hippocampus. S-nitrosylation of procaspase-3 was detected by biotin-switch method. Activation of procaspase-3 was shown as cleavage of procaspase-3 detected by immunoblotting. FasL expression was detected by immunoblotting. Cresyl violets and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining were used to detect apoptosis-like neuronal death in rat hippocampal CA1 and CA3 subfields. KA led to the activation of procaspase-3 in a dose- and time-dependent manner, and the activation was inhibited by KA receptor antagonist NS102. Procaspase-3 was denitrosylated at 3 h after kainic acid administration, and the denitrosylation was reversed by SNP and GSNO. FasL ASODNs inhibited the procaspase-3 denitrosylation and activation. Moreover, thioredoxin reductase (TrxR) inhibitor auranofin prevented the denitrosylation and activation of procaspase-3 in rat hippocampal CA1 and CA3 subfields. NS102, FasL AS-ODNs, and auranofin reversed the KAinduced apoptosis and cell death in hippocampal CA1 and CA3 subfields. KA led to denitrosylation and activation of procaspase-3 via FasL and TrxR. Inhibition of procaspase-3 denitrosylation by auranofin, SNP, and GSNO played protective effects against KA-induced apoptosis-like neuronal death in rat hippocampal CA1 and CA3 subfields. These investigations revealed that the procaspase-3 undergoes an initial denitrosylation process before becoming activated, providing valuable insights into the underlying mechanisms and possible treatment of excitotoxicity.

Immunological Role of Megakaryocytes and Platelets during Influenza A Virus Infection

Influenza viruses pose a serious threat to public health, with severe cases often characterized by lung damage and inflammation. However, the underlying mechanisms of these processes remain poorly understood. This study aimed to investigate the essential role of megakaryocytes (MKs) and platelets (PLTs) in influenza A virus (IAV) infections. Conducted at the Department of Rare Respiratory Diseases, Cystic Fibrosis, and Pulmonology, Nord University Hospital, Marseille, France, the study collected seventy blood samples between October 2018 and March 2019. Samples were obtained from healthy individuals and patients diagnosed with IAV. Messenger RNA was extracted from isolated PLTs and subjected to quantitative real-time-polymerase chain reaction using sets of primers targeting immune marker genes. Western blot analysis was also performed for confirmation, focusing on Fas Ligand (FasL). Results showed that PLTs from IAV-infected individuals expressed the FasL, tumor necrosis factor-related apoptosis-inducing ligand, and Granulysin (GNLY) receptors when activated. Furthermore, an in vitro assay revealed the presence of FasL receptors on infected CMK cell lines. In vivo investigations demonstrated that activated MKs and PLTs in mice also expressed FasL. Interestingly, none of the immune receptors under investigation were found in both MKs and PLTs in mouse model studies. In conclusion, MKs and PLTs play a significant role in influencing immune responses that may help prevent viral spread during infection. However, further examination of their mechanisms of action is warranted. Understanding the involvement of these cells in influenza pathogenesis could offer valuable insights for developing potential therapeutic strategies.

Correlations between histopathologic findings, serum biomarker levels, and clinical outcomes in Stevens–Johnson syndrome/toxic epidermal necrolysis (SJS/TEN)

Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe dermatological emergencies. The role of cytokines and chemokines in the pathogenesis, progression of the disease, and histopathologic features is not fully elucidated. To address this gap, we conducted a retrospective study examining the associations between 42 serum biomarkers, histopathologic findings, and clinical outcomes in SJS/TEN patients. We reviewed the medical records of 23 patients diagnosed with SJS/TEN. Regarding histopathology, our study did not reveal any significant associations between the degree of epidermal necrosis, dermal mononuclear cell infiltration, and clinical outcomes. However, an intriguing observation was made regarding the degree of dermal infiltration of CD8 + cells, which showed a negative correlation with the severity of acute ocular complications. Notably, serum levels of IFN-γ positively correlated with the number of CD8 + cells in dermal infiltration. Additionally, higher serum levels of myeloperoxidase were associated with greater degrees of epidermal necrosis, while serum Fas ligand and stem cell factor levels were elevated in individuals with increased dermal mononuclear cell infiltration. Furthermore, the levels of S100A8/A9 were significantly correlated with the SCORTEN and mortality rate. These findings provide insights into the intricate pathogenesis of the disease.