Soluble FasL Research Articles

Exogenous binding immunoglobulin protein (BiP) enhance immune regulatory phenotype in ex-vivo Mtb infected PBMCs stratified based on QuantiFERON response.

Even though anti-tuberculosis (TB) treatment is readily available, Mycobacterium tuberculosis (Mtb) infection continues to be a global threat with a high death rate recorded from a single infectious agent. This highlights the significance of developing new strategies to curb the growing Mtb infection cases. Host-directed therapies (HDT) offer a promising approach that includes both drug discovery and drug repurposing, aimed at identifying host targets and promoting immune cell populations that can lead to better infection outcomes. In this context, we investigated the potential of exogenous Binding Immunoglobulin Protein (BiP) to induce such changes ex-vivo using PBMCs from healthy (QFN-) and Mtb exposed (QFN+) individuals. We analysed cell surface expression and cytokine profiles across eight different stimulation conditions including human full-length BiP protein (20μg/ml), TLR-9a (0.5μM), BiP/TLR-9a combination, isoniazid (1μM), H37Rv (MOI: 1: 10), and pooled bronchoalveolar lavage (BAL) samples collected at TB diagnosis (TBdx) and at month 6 (M6) of anti-TB treatment. Our results revealed that BiP-stimulated PBMCs showed a significant reduction of interleukin (IL)-10 secretion, along with increased IL-4, IL-5, IL-13, and soluble Fas-L (sFasL) secretion. We also observed that BiP stimulation enhanced the expression of membrane bound Fas-L (CD178) and IL5Ra (CD125) in B-cells isolated from both QFN- and QFN+ groups. Additionally, BiP exhibited a synergistic effect with TLR-9a, further boosting this co-expression. Moreover, we observed that BiP induced IL5Ra expression in both CD3+CD5lo and CD3+CD5hi T-cell populations. This study explores the effects of exogenous BiP on cell functionality and provides valuable insights into its potential to modulate host cell responses, which could be explored as a host-directed therapy for TB in the future.

PTPRZ1-Targeting RNA CAR T Cells Exert Antigen-Specific and Bystander Antitumor Activity in Glioblastoma.

The great success of chimeric antigen receptor (CAR) T-cell therapy in the treatment of patients with B-cell malignancies has prompted its translation to solid tumors. In the case of glioblastoma (GBM), clinical trials have shown modest efficacy, but efforts to develop more effective anti-GBM CAR T cells are ongoing. In this study, we selected protein tyrosine phosphatase receptor type Z (PTPRZ1) as a target for GBM treatment. We isolated six anti-human PTPRZ1 single-chain variable fragments from a human phage display library and produced second-generation CAR T cells in an RNA format. Patient-derived GBM PTPRZ1-knockin cell lines were used to select the CAR construct that showed high cytotoxicity while consistently displaying high CAR expression (471_28z). CAR T cells incorporating 471_28z were able to release IFNγ, IL2, TNFα, granzyme B, IL17A, IL6, and soluble FasL and displayed low tonic signaling. Additionally, they maintained an effector memory phenotype after in vitro killing. In addition, 471_28z CAR T cells displayed strong bystander killing against PTPRZ1-negative cell lines after preactivation by PTPRZ1-positive tumor cells but did not kill antigen-negative nontumor cells. In an orthotopic xenograft tumor model using NOD/SCIDγ mice, a single dose of anti-PTPRZ1 CAR T cells significantly delayed tumor growth. Taken together, these results validate PTPRZ1 as a GBM target and prompt the clinical translation of anti-PTPRZ1 CAR T cells.

Mechano-inhibition of endocytosis sensitizes cancer cells to Fas-induced Apoptosis

The transmembrane death receptor Fas transduces apoptotic signals upon binding its ligand, FasL. Although Fas is highly expressed in cancer cells, insufficient cell surface Fas expression desensitizes cancer cells to Fas-induced apoptosis. Here, we show that the increase in Fas microaggregate formation on the plasma membrane in response to the inhibition of endocytosis sensitizes cancer cells to Fas-induced apoptosis. We used a clinically accessible Rho-kinase inhibitor, fasudil, that reduces endocytosis dynamics by increasing plasma membrane tension. In combination with exogenous soluble FasL (sFasL), fasudil promoted cancer cell apoptosis, but this collaborative effect was substantially weaker in nonmalignant cells. The combination of sFasL and fasudil prevented glioblastoma cell growth in embryonic stem cell-derived brain organoids and induced tumor regression in a xenograft mouse model. Our results demonstrate that sFasL has strong potential for apoptosis-directed cancer therapy when Fas microaggregate formation is augmented by mechano-inhibition of endocytosis.

FasL impacts Tgfb signaling in osteoblastic cells

Fas ligand (FasL, CD178) belongs to classical apoptotic molecules, however, recent evidence expands the spectrum of FasL functions into non-apoptotic processes which also applies for the bone. Tgfb subfamily members (Tgfb1, Tgfb2, Tgfb3) represent major components in osteogenic pathways and extracellular matrix. Their possible association with FasL has not yet been investigated but can be postulated. To test such a hypothesis, FasL deficient (gld) calvaria-derived cells were examined with a focus on the expression of Tgfb receptor ligands. The qPCR analysis revealed significantly increased expression of Tgfb1, Tgfb2 and Tgfb3 in gld cells. To check the vice versa effect, the gld cells were stimulated by soluble FasL. As a consequence, a dramatic decrease in expression levels of all three ligands was observed. This phenomenon was also confirmed in IDG-SW3 (osteoblastic cells of endochondral origin).TFLink gateway identified Fosl2 as an exclusive candidate of FasL capable to impact expression of all three Tgfb ligands. However, Fosl2 siRNA did not cause any significant changes in expression of Tgfb ligands. Therefore, the upregulation of the three ligands is likely to occur separately. In this respect, we tested the only exclusive candidate transcription factor for Tgfb3, Prrx1. Additionally, an overlapping candidate for Tgfb1 and Tgfb2, Mef2c capable to modulate expression of sclerostin, was examined. Prrx1 as well as Mef2c were found upregulated in gld samples and their expression decreased after addition of FasL. The same effect of FasL treatment was observed in the IDG-SW3 model.Taken together, FasL deficiency causes an increase in the expression of Tgfb ligands and stimulation by FasL reduces Tgfb expression in osteoblastic cells. The candidates mediating the effect comprise Prrx1 for Tgfb3 and Mef2c for Tgfb1/2. These results indicate FasL as a novel cytokine interfering with Tgfb signaling and thus the complex osteogenic network. The emerging non-apoptotic functions of FasL in bone development and maintenance should also be considered in treatment strategies such as the anti-osteoporotic factor.

A connection between levels of soluble Fas and Fas ligand in the aqueous humor and the parameters of structural and functional damage of glaucoma patients

Introduction/Objective. Fas ligant (FasL) induces apoptosis when interacting with the Fas-receptor. The aim was to determine the concentration of soluble Fas (sFas) and FasL (sFasL) in the aqueous humor of open-angle glaucoma patients, and establish a connection between these markers of apoptosis and the parameters of structural and functional glaucoma damage. Methods. This study examined 88 aqueous humor samples; 35 primary open-angle glaucoma with elevated intraocular pressure (POAG-HTG) patients, 24 open angle pseudoexfoliative glaucoma patients (XFG) and 29 patients with senile cataract (CAT). The concentration of sFas and sFasL was determined by commercial ELISA tests in the aqueous humor. Results. The conducted study showed that aqueous humor sFas concentration was the highest in XFG (720.14 ? 167.39 pg/ml), and slightly lower in POAG-HTG (713.43 ? 162.69 pg/ml), than in cataract patients (632.46 ? 217.11 pg/ml), without statistical significance. There was a significant negative correlation of sFas concentration and RNFL Inf thickness in POAG-HTG (p < 0.05). The concentration of sFasL was the lowest in POAG-HTG (9.28 ? 0.551 pg/ml), higher in XFG (9.45 ? 0.61 pg/ml; p = 0.0566), and the highest in the cataract group (9.48 ? 0.73 pg/ml). A negative correlation of sFasL and MD in the POAG-HTG, and a negative correlation with RNFL Sup in the XFG were significant. Conclusion. sFasL has an active role in the regulation of the inflammatory process in glaucoma. sFas and sFasL, as markers of apoptosis, are associated with the parameters of structural, RNFL thinning, and functional glaucoma damage, namely visual field defects.

Combining Apoptotic Resistance and Cytokine Signaling to Improve Persistence and Anti-Tumor Activity of CAR Vδ2 T Cells In Vivo

Vγ9Vδ2 T cells (Vδ2 T cells hereafter) are an attractive platform for allogeneic chimeric antigen receptor (CAR)-modified cell therapy, as their invariant TCR minimizes their risk of graft-versus-host disease. However, the susceptibility of Vδ2 T cells to activation-induced cell death (AICD) and dependency on exogenous cytokine supplementation for prolonged persistence limit their in vivo efficacy. Addressing these limitations in would increase therapeutic potential of engineered Vδ2 T cells. Fas signaling is critical for AICD induction in Vδ2 T cells. To attenuate AICD, we generated a truncated Fas receptor lacking a signaling endodomain (tFas), to prevent downstream activation of apoptosis. Incubation with soluble FasL for 24h induced apoptosis in unmodified Vδ2 T cells and CD19.CAR-expressing Vδ2 T cells, while tFas-armed CD19.CAR-Vδ2 T cells resisted FasL-induced cell death. In extended (9-day) coculture experiments with CD19(+) NALM6 target cells at a 1:2 E:T ratio without exogenous cytokines, tFas-armed CD19.CAR-Vδ2 T cells produced enhanced anti-tumor control at early time points, compared to unarmed CD19.CAR-Vδ2 T cells which exhibited only transient activity, resulting in tumor regrowth. However, the lack of cytokine support limited long-term expansion of tFas/CD19.CAR-Vδ2 T cells resulting in tumor regrowth after 6 days of coculture. IL-18 signaling via MyD88 stimulates Vδ2 T cells, promoting their growth. Hence, we hypothesized combining IL-18/MyD88 signaling with AICD suppression would synergize to improve anti-tumor function of Vδ2 T cells. We generated a chimeric receptor consisting of the extracellular domain of Fas linked with intracellular MyD88 (Fas88) aiming to both prevent AICD and transmit MyD88 signaling upon engagement with FasL. Fas88-armed CD19.CAR-Vδ2 T cells resisted FasL-induced apoptosis similar to those armed with a tFas construct. Furthermore, in cocultures with NALM6 cells, Fas88/CD19.CAR-Vδ2 T cells resisted AICD and, in contrast to tFas/CD19.CAR-Vδ2 T cells, expanded over 9 days in the absence of exogenous cytokines and mediated a significantly superior tumor control. Next, we investigated the activity of armed CD19.CAR-Vδ2 T cells in a xenograft mouse model, where NALM6 cells (0.5 x 10 6 cells/mouse) were intravenously infused into NSG mice followed 5 days later by a single injection of Vδ2 T cells (4 x 10 6 cells/mouse). Both tumor and T cell populations were tracked via dual bioluminescent imaging. Unmodified CD19.CAR-Vδ2 T cells extended median survival from 18 days (control group) to 30 days but demonstrated limited persistence. tFas-armed CD19.CAR-Vδ2 T cells produced comparable tumor control, with a median survival of 31.5 days, suggesting that AICD suppression alone is insufficient for the long-term activity of CD19.CAR-Vδ2 T cells in vivo. In contrast, Fas88/CD19.CAR-Vδ2 T cells persisted and expanded in tumor-bearing mice improving the median survival to 47 days (p = 0.0478), indicating the ability of Fas88 to sustain functional persistence of CD19.CAR-Vδ2 T cells. This data shows that combining AICD suppression with MyD88 signaling improves the therapeutic potency of CAR-engineered Vδ2 T cells, demonstrating a means for exploiting Vδ2 T cells for allogeneic adoptive immunotherapy. Current work is focused on evaluating this approach for clinical translation.

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

Prognostic value of humoral markers in patients with anthracycline-related cardiac dysfunction

Aim. To carry out a 12-month study on the prognostic role of humoral markers responsible for the main mechanisms of initiation of cardiotoxic myocardial damage (endothelin-1, soluble Fas-L, N-terminal pro-brain natriuretic peptide (NT-proBNP), tumor necrosis factor-α, interleukin (IL)-1β, matrix metalloproteinase (MMP)-2 and MMP9, soluble form of the ST2 protein (sST2), a tissue inhibitor of metalloproteinase-1, and tetranectin) in assessing the risk of progression of anthracycline-related left ventricular dysfunction.Materials and methods. The study included a total of 114 women aged 48.0 (46.0; 52.0) years without concomitant cardiovascular diseases and risk factors who received chemotherapy with anthracyclines in the past. The levels of serum biomarkers were determined using the enzyme immunoassay. Transthoracic echocardiography was performed at baseline and at 12 months of follow-up.Results. After 12 months of follow-up, all patients were retrospectively divided into 2 groups: group 1 (n = 54) included patients with an unfavorable course of anthracycline-related cardiac dysfunction (ARCD), group 2 (n = 60) encompassed patients with a favorable course of the disease. According to the ROC analysis, MMP-2 ≥ ≥ 338.8 pg / ml (sensitivity 57%, specificity 78%; AUC = 0.629; p = 0.025), MMP-9 ≥ 22.18 pg / ml (sensitivity 89%, specificity 87%; AUC = 0.886; p < 0.001), sST2 ≥ 32.4 ng / ml (sensitivity 64%, specificity 70.5%; AUC = 0.691; p = 0.002), and tetranectin ≤ 15.4 pg / ml (sensitivity 69%, specificity 72%; AUC = 0.764; p < 0.001) were identified as predictors of an adverse course of ARCD. When comparing ROC curves, it was found that the concentration of MMP-9 (p = 0.002) was the most significant predictor of the progression of ARCD.Conclusion. MMP-2 and -9, soluble ST2, and tetranectin can be considered as non-invasive markers for assessing the risk of ARCD progression. At the same time, an increased level of MMP-9 is the most significant predictor of ARCD progression.

Blocking soluble Fas Ligand ameliorates pemphigus: PC111 efficacy in ex-vivo human pemphigus models.

Pemphigus is a life-threatening, chronic, autoimmune bullous disease affecting both the skin and the mucous membranes. Based on the mainstream concept that blister formation occurs upon binding of autoantibodies to their antigen proteins (desmoglein1, DSG1 and desmoglein3, DSG3), current therapies mostly aim to suppress the immune system. To avoid the severe side effects associated with the chronic use of immunosuppressive treatments, we have developed PC111, a fully human monoclonal antibody targeting human Fas ligand (FasL). We have provided a number of in vitro and in vivo evidences showing that soluble FasL induces keratinocyte apoptosis followed by acantholysis. An anti-murine FasL prevents blister formation in the pemphigus neonatal mouse model. To confirm the mechanism of action (MoA) and the efficacy of PC111 in a human pemphigus context, we used the keratinocyte dissociation assay and two independent Human Skin Organ Cultures (HSOC) pemphigus models. PC111 reduced acantholysis in vitro, as shown by the dose-dependent reduction of fragments in the monolayer cultures. In the first HSOC model, normal human skin was subcutaneously injected with a scFv antibody fragment directed against DSG1 and DSG3, resulting in a severe acantholysis (70-100%) after 24 hours. PC111 inhibited blister formation to around 50% of control. In the second model, normal human skin was injected with a mixture of pemphigus patients' autoantibodies resulting in a less severe acantholysis (20-30%). PC111 significantly suppressed blister formation to more than 75% up to 72 hours. These results confirm PC111 MoA and demonstrates the efficacy of the anti-FasL antibody also in a pemphigus setting.

Patients with hepatocellular carcinoma that die during the first year of liver transplantation have high blood sFasL concentrations.

Fas ligand (FasL) is one ligand that activates extrinsic apoptosis pathway. High expression in lymphocytes of FasL have been found in patients with acute rejection of liver transplantation (LT). No high blood concentrations of soluble FasL (sFasL) have been found in patients with acute LT rejection; however, the samples size of those studies was small. To determine whether patients with hepatocellular carcinoma (HCC) that dead during the first year of LT have higher blood sFasL concentrations previously to LT that those who that remain alive in a study of higher sample size. Patients underwent LT due to HCC were included in this retrospective study. Serum sFasL levels prior to LT were measured and one-year LT mortality was registered. Non-surviving patients (n = 14) showed higher serum sFasL levels [477 (269-496) vs 85 (44-382) pg/mL; P < 0.001] than surviving patients (n = 113). Serum sFasL levels (pg/mL) were associated with mortality (OR = 1.006; 95%CI = 1.003-1.010; P = 0.001) independently of age of LT donor in the logistic regression analysis. We report for the first time that HCC patients who die within the first year of HT have higher blood sFasL concentrations prior to HT than those who remain alive.

Revisiting autoimmune lymphoproliferative syndrome caused by Fas ligand mutations

Fas ligand (FasL) is expressed by activated T cells and induces death in target cells upon binding to Fas. Loss-of-function FAS or FASLG mutations cause autoimmune-lymphoproliferative syndrome (ALPS) characterized by expanded double-negative T cells (DNT) and elevated serum biomarkers. While most ALPS patients carry heterozygous FAS mutations, FASLG mutations are rare and usually biallelic. Only 2 heterozygous variants were reported, associated with an atypical clinical phenotype. We revisited the significance of heterozygous FASLG mutations as a cause of ALPS. Clinical features and biomarkers were analyzed in 24 individuals with homozygous or heterozygous FASLG variants predicted to be deleterious. Cytotoxicity assays were performed with patient T cells and biochemical assays with recombinant FasL. Homozygous FASLG variants abrogated cytotoxicity and resulted in early-onset severe ALPS with elevated DNT, raised vitamin B12, and usually no soluble FasL. In contrast, heterozygous variants affected FasL function by reducing expression, impairing trimerization, or preventing Fas binding. However, they were not associated with elevated DNT and vitamin B12, and they did not affect FasL-mediated cytotoxicity. The dominant-negative effects of previously published variants could not be confirmed. Even Y166C, causing loss of Fas binding with a dominant-negative effect in biochemical assays, did not impair cellular cytotoxicity or cause vitamin B12 and DNT elevation. Heterozygous loss-of-function mutations are better tolerated for FASLG than for FAS, which may explain the low frequency of ALPS-FASLG.

Assessment Of Soluble Fas Ligand In Patients With Chronic Myeloid Leukemia

Background: Soluble FasL (sFasL) generating from Membrane-bound FasL (mFasL) cleaved by matrix metalloproteinases (MMPs). sFasL inhibits the apoptotic and inflammatory activity of mFasL because of sFasL competes with the mFasL and binds to Fas. To evaluate serum soluble Fas ligand (sFasL) level in patients with chronic myeloid leukemia.&#x0D; Materials and Method: Serum levels of sFasL were measured by ELISA method after venous blood was collected from 56 CML patients (newly diagnosed and optimally treated) and 28 healthy subjects as control group.&#x0D; Results: There were no significant increases in serum sFasL patient compared to healthy control with P=0.07. When the mean sFasL concentration was obviously highest in newly diagnosed (216.9pg/ml) followed by healthy control (152.5pg/ml) and lowest in optimally treated (147.7 pg/ml).&#x0D; Conclusion: Production of sFasL in tumor patients may be a key mechanism to inhibit Fas-mediated apoptosis.&#x0D;

Fas and Fas ligand are highly expressed in lymphocytes from cervical intraepithelial neoplasia and cervical cancer patients: A possible role for immune escaping.

Objective(s):Infection with high-risk human papillomavirus is required to develop cervical cancer. Some viruses modulate the Fas/FasL signaling to evade the immune response; the role of these molecules in cervical cancer is not clear. In this study, we measured the expression levels of Fas and FasL mRNA, soluble proteins, and cell surface proteins in peripheral blood mononuclear cells from patients with low- and high-grade squamous intraepithelial lesions and cervical cancer in relation to healthy women, to gain new insights into the role of Fas/FasL in cervical cancer development. Materials and Methods:Fas/FasL mRNA expression was measured in cervical tissues and peripheral blood mononuclear cells from patients and healthy subjects; serum soluble proteins Fas/FasL were measured by ELISA, and cell-surface protein expression was detected by flow cytometry. Results:Varying expression levels were found for both molecules. Cervical Fas and FasL mRNA expression was decreased in low- and high-grade lesions, but it was increased in cervical cancer cases. While, systemic Fas mRNA expression increased as malignity progressed; systemic FasL mRNA expression was increased in low- and high-grade lesions, but it was decreased in cancer patients. Soluble FasL levels decreased as lesions progressed, while soluble Fas levels increased. Finally, overexpression of Fas/FasL on the surface of peripheral blood mononuclear cells was found in patients with low-grade lesion with respect to healthy donors. Conclusion:Fas and FasL act as negative modulators of the immune response, probably by removing specific cytotoxic T lymphocytes against papillomavirus -infected cells and tumor cells.

T cell apoptosis characterizes severe Covid-19 disease

Severe SARS-CoV-2 infections are characterized by lymphopenia, but the mechanisms involved are still elusive. Based on our knowledge of HIV pathophysiology, we hypothesized that SARS-CoV-2 infection-mediated lymphopenia could also be related to T cell apoptosis. By comparing intensive care unit (ICU) and non-ICU COVID-19 patients with age-matched healthy donors, we found a strong positive correlation between plasma levels of soluble FasL (sFasL) and T cell surface expression of Fas/CD95 with the propensity of T cells to die and CD4 T cell counts. Plasma levels of sFasL and T cell death are correlated with CXCL10 which is part of the signature of 4 biomarkers of disease severity (ROC, 0.98). We also found that members of the Bcl-2 family had modulated in the T cells of COVID-19 patients. More importantly, we demonstrated that the pan-caspase inhibitor, Q-VD, prevents T cell death by apoptosis and enhances Th1 transcripts. Altogether, our results are compatible with a model in which T-cell apoptosis accounts for T lymphopenia in individuals with severe COVID-19. Therefore, a strategy aimed at blocking caspase activation could be beneficial for preventing immunodeficiency in COVID-19 patients.

Anthracycline-induced cardiotoxicity in women without cardiovascular diseases: molecular and genetic predictors

Objective To evaluate role of molecular (endothelin-1, soluble Fas-L, NT-proBNP, TNF-α, interleukin-1β,) and genetic factors (NOS3 (rs1799983), EDNRA (C + 70G, rs5335), NADPH oxidase (C242T, rs4673), p53 protein (polymorphic marker-Arg72Pro exon 4, rs1042522), NOS3 (Glu298Asp, rs1799983), Caspase 8 (CASP8, rs3834129 and rs1045485), interleukin-1β gene (Il-1β, rs1143634), TNF-α gene (rs1800629), SOD2 (rs4880), GPX1 (rs1050450) in development of anthracycline-induced cardiotoxicity (AIC) in women without cardiovascular diseases. Methods A total of 176 women with breast cancer and without cardiovascular diseases who received anthracyclines were enrolled in the study. After the 12 months of chemotherapy (CT), all patients were divided into two groups: group 1 (n = 52) comprised patients with AIC, group 2 (n = 124) comprised those without it. Results Based on ROC-analysis, levels of endothelin-1 of ≥9.0 pg/mL (AUC of 0.699), sFas-L of ≥98.3 ng/mL (AUC of 0.990), and NT-proBNP of ≥71.5 pg/mL (AUC of 0.994;) were identified as a cut-off values predicting AIC during 12 months after CT. Whereas, NT-proBNP and sFas-L were more significant predictors than endothelin-1 (p < 0.001). The development of AIC was significantly related to Arg/Arg of p53 protein gene (OR = 2.972; p = 0.001), T/T of NOS3 gene (OR = 3.059, p = 0.018), T/T of NADPH oxidase gene (OR = 2.753, p = 0.008), and C/C of GPX1 (OR = 2.345; p = 0.007). Conclusion Evaluation of polymorphisms genes of p53 (rs1042522), NOS3 (rs1799983), GPX1 (rs1050450), and NADPH oxidase (rs4673) can be recommended before CT for the risk assessment of AIC development. The serum levels of NT-proBNP and soluble Fas-L after CT may be considered as non-invasive biomarkers for prediction of AIC development during the 12 months.

Possible protective effect of pantoprazole against cisplatin-induced nephrotoxicity in head and neck cancer patients: a randomized controlled trial.

Cisplatin is used to treat solid malignancies including head and neck cancer. However, nephrotoxicity limits its use. In this study, we looked for a possible protective effect of pantoprazole against cisplatin-induced nephrotoxicity. We used novel biomarkers for early detection of nephrotoxicity. Sixty chemotherapy naïve head and neck cancer patients completed the study. Following complete history taking and thorough clinical examination, patients were randomly divided into three groups: 20 patients in each. Group I (control group) received cisplatin without pantoprazole, groups II and III received pantoprazole 80mg and 40mg, respectively, concurrently with cisplatin. Blood and urine samples were collected at baseline, and 48h after the first and third cycles of cisplatin administration. Assessment of serum creatinine and soluble FasL (sFasL), as well as urinary neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) was performed. Nephrotoxicity was detected in 6 patients in group I, none in group II and 3 patients in group III. Serum creatinine significantly increased at the end of treatment in group I compared to groups II and III. Group I also had significantly higher urinary KIM-1 and NGAL and serum sFasL compared to groups II and III after the first and third cycles. On the contrary, there was no significant difference between groups II and III. Pantoprazole prevented the increase in acute kidney injury biomarkers in cisplatin-treated patients. Therefore, it is a promising agent in reducing cisplatin-induced nephrotoxicity.Trial registration Clinical Trials.gov identifier:NCT04217512, registered in January 2020 " retrospectively registered".

Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2.

To date, no study has demonstrated that soluble Fas ligand (sFasL)-mediated inflammation is regulated via interaction with Fas in vivo. We found that FasL interacts specifically with tumor necrosis factor receptor superfamily (TNFRSF)10B, also known as death receptor (DR)5. Autoantibody-induced arthritis (AIA) was attenuated in FasL (Faslgld/gld)- and soluble FasL (FaslΔs/Δs)-deficient mice, but not in Fas (Faslpr/lpr and Fas-/-)- or membrane FasL (FaslΔm/Δm)-deficient mice, suggesting sFasL promotes inflammation by binding to a Fas-independent receptor. Affinity purification mass spectrometry analysis using human (h) fibroblast-like synovial cells (FLSCs) identified DR5 as one of several proteins that could be the elusive Fas-independent FasL receptor. Subsequent cellular and biochemical analyses revealed that DR5 interacted specifically with recombinant FasL-Fc protein, although the strength of this interaction was approximately 60-fold lower than the affinity between TRAIL and DR5. A microarray assay using joint tissues from mice with arthritis implied that the chemokine CX3CL1 may play an important downstream role of the interaction. The interaction enhanced Cx3cl1 transcription and increased sCX3CL1 production in FLSCs, possibly in an NF-κB-dependent manner. Moreover, the sFasL-DR5 interaction-mediated CX3CL1-CX3CR1 axis initiated and amplified inflammation by enhancing inflammatory cell influx and aggravating inflammation via secondary chemokine production. Blockade of FasL or CX3CR1 attenuated AIA. Therefore, the sFasL-DR5 interaction promotes inflammation and is a potential therapeutic target.

Nanoscale FasL Organization on DNA Origami to Decipher Apoptosis Signal Activation in Cells.

Cell signaling is initiated by characteristic protein patterns in the plasma membrane, but tools to decipher their molecular organization and activation are hitherto lacking. Among the well-known signaling pattern is the death inducing signaling complex with a predicted hexagonal receptor architecture. To probe this architecture, DNA origami-based nanoagents with nanometer precise arrangements of the death receptor ligand FasL are introduced and presented to cells. Mimicking different receptor geometries, these nanoagents act as signaling platforms inducing fastest time-to-death kinetics for hexagonal FasL arrangements with 10nm inter-molecular spacing. Compared to naturally occurring soluble FasL, this trigger is faster and 100× more efficient. Nanoagents with different spacing, lower FasL number or higher coupling flexibility impede signaling. The results present DNA origami as versatile signaling scaffolds exhibiting unprecedented control over molecular number and geometry. They define molecular benchmarks in apoptosis signal initiation and constitute a new strategy to drive particular cell responses.

Cord blood soluble Fas ligand linked to allergic rhinitis and lung function in seven-year-old children.

Serum or cord blood soluble Fas ligand (FasL) has been related to asthma, allergic rhinitis, and atopic dermatitis in cross-sectional and short-term follow-up studies. However, the association of cord blood soluble FasL with long-term allergic outcomes has seldom been investigated. The Prediction of Allergies in Taiwanese Children birth cohort study recruited healthy newborns upon delivery. At birth, blood was collected from the umbilical cords of these children, and the cord blood soluble Fas ligand levels were measured. At the age of seven years, the allergic outcome of each child was diagnosed by pediatric allergists and pulmonologists. Tests were conducted to measure the specific immunoglobulin E, fractional exhaled nitric oxide (FeNO), and pulmonary function levels of each child. Cord blood soluble FasL levels were higher in seven-year-old children with allergic rhinitis (Odds ratio [OR]=2.41, p=0.012) and expiratory airway obstruction (the highest forced expiratory volume in 1second/forced vital capacity<90%, OR=2.11, p=0.022). The FeNO and Dermatophagoides pteronyssinus-specific immunoglobulin E levels of seven-year-old children were positively correlated with cord blood soluble FasL levels (p=0.006 and 0.02, respectively). In this birth cohort, the cord blood soluble FasL levels were associated with allergic rhinitis, obstructive-type lung function, FeNO, and house dust mite sensitization in 7-year-old children. The cord blood soluble FasL level might be used as a predictor for allergic diseases in children who are 7 years old.

The FasLane to ocular pathology-metalloproteinase cleavage of membrane-bound FasL determines FasL function.

Fas ligand (FasL) is best known for its ability to induce cell death in a wide range of Fas-expressing targets and to limit inflammation in immunoprivileged sites such as the eye. In addition, the ability of FasL to induce a much more extensive list of outcomes is being increasingly explored and accepted. These outcomes include the induction of proinflammatory cytokine production, T cell activation, and cell motility. However, the distinct and opposing functions of membrane-associated FasL (mFasL) and the C-terminal soluble FasL fragment (sFasL) released by metalloproteinase cleavage is less well documented and understood. Both mFasL and sFasL can form trimers that engage the trimeric Fas receptor, but only mFasL can form a multimeric complex in lipid rafts to trigger apoptosis and inflammation. By contrast, a number of reports have now documented the anti-apoptotic and anti-inflammatory activity of sFasL, pointing to a critical regulatory function of the soluble molecule. The immunomodulatory activity of FasL is particularly evident in ocular pathology where elimination of the metalloproteinase cleavage site and the ensuing increased expression of mFasL can severely exacerbate the extent of inflammation and cell death. By contrast, both homeostatic and increased expression of sFasL can limit inflammation and cell death. The mechanism(s) responsible for the protective activity of sFasL are discussed but remain controversial. Nevertheless, it will be important to consider therapeutic applications of sFasL for the treatment of ocular diseases such as glaucoma.