Resistance of Human Liver Mesenchymal Stem Cells to FAS-Induced Cell Death.

Mesenchymal stem cells (MSCs) have a pronounced therapeutic potential in various pathological conditions. Though therapeutic effects of MSC transplantation have been studied for a long time, the underlying mechanisms are still not clear. It has been shown that transplanted MSCs are rapidly eliminated, presumably by apoptosis. As the mechanisms of MSC apoptosis are not fully understood, in the present work we analyzed MSC sensitivity to Fas-induced apoptosis using MSCs isolated from the biopsies of liver fibrosis patients (L-MSCs). The level of cell death was analyzed by flow cytometry in the propidium iodide test. The luminescent ATP assay was used to measure cellular ATP levels; and the mitochondrial membrane potential was assessed using the potential-dependent dye JC-1. We found that human L-MSCs were resistant to Fas-induced cell death over a wide range of FasL and anti-Fas mAb concentrations. At the same time, intrinsic death signal inducers CoCl2 and staurosporine caused apoptosis of L-MSCs in a dose-dependent manner. Despite the absence of Fas-induced cell death treatment of L-MSCs with low concentrations of FasL or anti-Fas mAb resulted in a cellular ATP level decrease, while high concentrations of the inducers caused a decline of the mitochondrial membrane potential. Pre-incubation of L-MSCs with the pro-inflammatory cytokine TNF-α did not promote L-MSC cell death. Our data indicate that human L-MSCs have increased resistance to receptor-mediated cell death even under inflammatory conditions.

Optimizing NK-92 serial killers: gamma irradiation, CD95/Fas-ligation, and NK or LAK attack limit cytotoxic efficacy.

BACKGROUND: The NK cell line NK-92 and its genetically modified variants are receiving attention as immunotherapies to treat a range of malignancies. However, since NK-92 cells are themselves tumors, they require irradiation prior to transfer and are potentially susceptible to attack by patients' immune systems. Here, we investigated NK-92 cell-mediated serial killing for the effects of gamma-irradiation and ligation of the death receptor Fas (CD95), and NK-92 cell susceptibility to attack by activated primary blood NK cells. METHODS: To evaluate serial killing, we used 51Cr-release assays with low NK-92 effector cell to target Raji, Daudi or K562 tumor cell (E:T) ratios to determine killing frequencies at 2-, 4-, 6-, and 8-h. RESULTS: NK-92 cells were able to kill up to 14 Raji cells per NK-92 cell in 8 h. NK-92 cells retained high cytotoxic activity immediately after irradiation with 10 Gy but the cells surviving irradiation lost > 50% activity 1 day after irradiation. Despite high expression of CD95, NK-92 cells maintained their viability following overnight Fas/CD95-ligation but lost some cytotoxic activity. However, 1 day after irradiation, NK-92 cells were more susceptible to Fas ligation, resulting in decreased cytotoxic activity of the cells surviving irradiation. Irradiated NK-92 cells were also susceptible to killing by both unstimulated and IL-2 activated primary NK cells (LAK). In contrast, non-irradiated NK-92 cells were more resistant to attack by NK and LAK cells. CONCLUSIONS: Irradiation is deleterious to both the survival and cytotoxicity mediated by NK-92 cells and renders the NK-92 cells susceptible to Fas-initiated death and death initiated by primary blood NK cells. Therefore, replacement of irradiation as an antiproliferative pretreatment and genetic deletion of Fas and/or NK activation ligands from adoptively transferred cell lines are indicated as new approaches to increase therapeutic efficacy.

Clusters of apoptotic signaling molecule-enriched rafts, CASMERs: membrane platforms for protein assembly in Fas/CD95 signaling and targets in cancer therapy.

Mammalian cells show the ability to commit suicide through the activation of death receptors at the cell surface. Death receptors, among which Fas/CD95 is one of their most representative members, lack enzymatic activity, and depend on protein-protein interactions to signal apoptosis. Fas/CD95 death receptor-mediated apoptosis requires the formation of the so-called death-inducing signaling complex (DISC), bringing together Fas/CD95, Fas-associated death domain-containing protein and procaspase-8. In the last two decades, cholesterol-rich lipid raft platforms have emerged as scaffolds where Fas/CD95 can be recruited and clustered. The co-clustering of Fas/CD95 and rafts facilitates DISC formation, bringing procaspase-8 molecules to be bunched together in a limited membrane region, and leading to their autoproteolytic activation by oligomerization. Lipid raft platforms serve as a specific region for the clustering of Fas/CD95 and DISC, as well as for the recruitment of additional downstream signaling molecules, thus forming the so-called cluster of apoptotic signaling molecule-enriched rafts, or CASMER. These raft/CASMER structures float in the membrane like icebergs, in which the larger portion lies inside the cell and communicates with other subcellular structures to facilitate apoptotic signal transmission. This allows an efficient spatiotemporal compartmentalization of apoptosis signaling machinery during the triggering of cell death. This concept of proapoptotic raft platforms as a basic chemical-biological structure in the regulation of cell death has wide-ranging implications in human biology and disease, as well as in cancer therapy. Here, we discuss how these raft-centered proapoptotic hubs operate as a major linchpin for apoptosis signaling and as a promising target in cancer therapy.

Lipid rafts as signaling hubs in cancer cell survival/death and invasion: implications in tumor progression and therapy: Thematic Review Series: Biology of Lipid Rafts.

Cholesterol/sphingolipid-rich membrane domains, known as lipid rafts or membrane rafts, play a critical role in the compartmentalization of signaling pathways. Physical segregation of proteins in lipid rafts may modulate the accessibility of proteins to regulatory or effector molecules. Thus, lipid rafts serve as sorting platforms and hubs for signal transduction proteins. Cancer cells contain higher levels of intracellular cholesterol and lipid rafts than their normal non-tumorigenic counterparts. Many signal transduction processes involved in cancer development (insulin-like growth factor system and phosphatidylinositol 3-kinase-AKT) and metastasis [cluster of differentiation (CD)44] are dependent on or modulated by lipid rafts. Additional proteins playing an important role in several malignant cancers (e.g., transmembrane glycoprotein mucin 1) are also being detected in association with lipid rafts, suggesting a major role of lipid rafts in tumor progression. Conversely, lipid rafts also serve as scaffolds for the recruitment and clustering of Fas/CD95 death receptors and downstream signaling molecules leading to cell death-promoting raft platforms. The partition of death receptors and downstream signaling molecules in aggregated lipid rafts has led to the formation of the so-called cluster of apoptotic signaling molecule-enriched rafts, or CASMER, which leads to apoptosis amplification and can be pharmacologically modulated. These death-promoting rafts can be viewed as a linchpin from which apoptotic signals are launched. In this review, we discuss the involvement of lipid rafts in major signaling processes in cancer cells, including cell survival, cell death, and metastasis, and we consider the potential of lipid raft modulation as a promising target in cancer therapy.

XIAP Interacts with and Regulates the Activity of FAF1.

Cell death depends on the balance between the activities of pro- and anti-apoptotic factors. X-linked inhibitor of apoptosis protein (XIAP) plays an important role in the cytoprotective process by inhibiting the caspase cascade and regulating pro-survival signaling pathways. While searching for novel interacting partners of XIAP, we identified Fas-associated factor 1 (FAF1). Contrary to XIAP, FAF1 is a pro-apoptotic factor that also regulates several signaling pathways in which XIAP is involved. However, the functional relationship between FAF1 and XIAP is unknown. Here, we describe a new interaction between XIAP and FAF1 and describe the functional implications of their opposing roles in cell death and NF-κB signaling. Our results clearly demonstrate the interaction of XIAP with FAF1 and define the specific region of the interaction. We observed that XIAP is able to block FAF1-mediated cell death by interfering with the caspase cascade and directly interferes in NF-κB pathway inhibition by FAF1. Furthermore, we show that XIAP promotes ubiquitination of FAF1. Conversely, FAF1 does not interfere with the anti-apoptotic activity of XIAP, despite binding to the BIR domains of XIAP; however, FAF1 does attenuate XIAP-mediated NF-κB activation. Altered expression of both factors has been implicated in degenerative and cancerous processes; therefore, studying the balance between XIAP and FAF1 in these pathologies will aid in the development of novel therapies.

Loss of BID Delays FASL-Induced Cell Death of Mouse Neutrophils and Aggravates DSS-Induced Weight Loss.

Neutrophils are key players in the early defense against invading pathogens. Due to their potent effector functions, programmed cell death of activated neutrophils has to be tightly controlled; however, its underlying mechanisms remain unclear. Fas ligand (FASL/CD95L) has been shown to induce neutrophil apoptosis, which is accelerated by the processing of the BH3-only protein BH3 interacting domain death agonist (BID) to trigger mitochondrial apoptotic events, and been attributed a regulatory role during viral and bacterial infections. Here, we show that, in accordance with previous works, mouse neutrophils underwent caspase-dependent apoptosis in response to FASL, and that this cell death was significantly delayed upon loss of BID. However, pan-caspase inhibition failed to protect mouse neutrophils from FASL-induced apoptosis and caused a switch to RIPK3-dependent necroptotic cell death. Intriguingly, such a switch was less evident in the absence of BID, particularly under inflammatory conditions. Delayed neutrophil apoptosis has been implicated in several auto-inflammatory diseases, including inflammatory bowel disease. We show that neutrophil and macrophage driven acute dextran sulfate sodium (DSS) induced colitis was slightly more aggravated in BID-deficient mice, based on significantly increased weight loss compared to wild-type controls. Taken together, our data support a central role for FASL > FAS and BID in mouse neutrophil cell death and further underline the anti-inflammatory role of BID.

The molecular signature of murine T cell homeostatic proliferation reveals both inflammatory and immune inhibition patterns.

T lymphocyte homeostatic proliferation, driven by the engagement of T cell antigen receptor with self-peptide/major histocompatibility complexes, and signaling through the common γ-chain-containing cytokine receptors, is critical for the maintenance of the T cell compartment and is regulated by the Fas death receptor (Fas, CD95). In the absence of Fas, Fas-deficient lymphoproliferation spontaneous mutation (lpr) mice accumulate homeostatically expanded T cells. The functional consequences of sequential rounds of homeostatic expansion are not well defined. We thus examined the gene expression profiles of murine wild-type and Fas-deficient lpr CD8+ T cell subsets that have undergone different amounts of homeostatic proliferation as defined by their level of CD44 expression, and the CD4-CD8-TCRαß+ T cell subset that results from extensive homeostatic expansion of CD8+ T cells. Our studies show that recurrent T cell homeostatic proliferation results in global gene expression changes, including the progressive upregulation of both cytolytic proteins such as Fas-Ligand and granzyme B as well as inhibitory proteins such as programmed cell death protein 1 (PD-1) and lymphocyte activating 3 (Lag3). These findings provide an explanation for how augmented T cell homeostatic expansion could lead to the frequently observed clinical paradox of simultaneous autoinflammatory and immunodeficiency syndromes and provide further insight into the regulatory programs that control chronically stimulated T cells.

Epstein-Barr virus Latent Membrane Protein 2A (LMP2A)-mediated changes in Fas expression and Fas-dependent apoptosis: Role of Lyn/Syk activation.

Epstein-Barr virus Latent Membrane Protein 2A (LMP2A) is expressed in EBV-infected B cells in the germinal center, a site of significant apoptosis induced by engagement of Fas on activated B cells. Signals from the B cell receptor (BCR) protect germinal center B cells from Fas-mediated apoptosis, and since LMP2A is a BCR mimic, we hypothesized that LMP2A would also protect B cells from Fas-mediated apoptosis. Surprisingly, latently-infected human and murine B cell lines expressing LMP2A were more sensitive to Fas-mediated apoptosis, as determined by increases in Annexin-V staining, and cleavage of caspase-8, -3 and PARP. Additional studies show that LMP2A-expressing B cell lines demonstrate a Lyn- and Syk-dependent increase in sensitivity to Fas-mediated apoptosis, due to an LMP2A-dependent enhancement in Fas expression. These findings demonstrate the ability for LMP2A to directly increase a pro-apoptotic molecule and have implications for EBV latency as well as the treatment of EBV-associated malignancies.

Quantitative gene analysis of methylation and expression (Q-GAME) in fresh or fixed cells and tissues.

Epigenetic regulation of gene expression by DNA methylation is a central mechanism governing the silencing of tumor suppressor genes in many forms of cancer. Current methods have not proven optimal for the quantitative analysis of DNA methylation and corresponding in situ protein expression within cells in small specimens like skin biopsies. We have overcome this limitation by combining and modifying several techniques: target cell enrichment, DNA micro-isolation, one-step denaturation/bisulphite conversion/in-column desulphonation, specially designed PCR amplification, pyrosequencing and multispectral image analysis. Using this approach optimized for small samples, we can quantify minor alterations in gene methylation and protein expression using minimal amounts of tissue. Comparative studies of fresh and processed cells showed that our method is valid for DNA in both fresh and formalin-fixed, paraffin-embedded specimens. We can measure the effects of DNA methylation inhibitors, administered in vitro or in vivo, on the promoter methylation and protein expression of selected genes in specific cells. This novel approach should prove useful for a wide variety of investigative and clinical applications in dermatology and other specialties where the collection of small, routinely processed biopsy specimens is common. We refer to this method as Q-GAME (quantitative gene analysis of methylation and expression).

CD74-AKT Axis Is a Potential Therapeutic Target in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) cells are often resistant to FAS (CD95)-mediated apoptosis, but the underlying molecular mechanism(s) is not fully understood yet. Notably, the expression of the type II transmembrane protein, CD74, is correlated with chemotherapy-resistant and more invasive forms of cancers via unknown mechanisms. Here, we analyzed gene expression pattern of cancer patients and/or patient-derived xenograft (PDX) models and found that mRNA and protein levels of CD74 are highly expressed in TNBC and correlated with cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) properties. Mechanistically, we found that AKT activation is likely critical for maintaining CD74 expression and protein stability to favor its oncogenic functions. Physiologically, epidermal growth factor (EGF) along with CD74 could activate AKT signaling, likely through binding of phosphorylated AKT (S473) to CD74, whereas inhibition of AKT could impair stability of CD74. We also revealed that CD74 binds to FAS and interferes with the intrinsic signaling of FAS-mediated apoptosis. As such, selective targeting of the CD74/FAS complex using the AKT inhibitor along with the CD74-derived peptide could synergistically restore and activate FAS-mediated apoptosis. Therefore, our approach of mobilizing apoptosis pathways likely provides a rationale for TNBC treatment by targeting the CD74/FAS and CD74-AKT axes.

Prognostic Value of Fas/Fas Ligand Expression on Circulating Tumor Cells (CTCs) and Immune Cells in the Peripheral Blood of Patients with Metastatic Breast Cancer.

The Fas/Fas ligand (FasL) system is a major apoptosis-regulating pathway with a key role in tumor immune surveillance and metastasis. The expression of Fas/FasL on mammary tumor tissues holds prognostic value for breast cancer (BC) patients. We herein assessed Fas/FasL expression on circulating tumor cells (CTCs) and matched peripheral blood mononuclear cells (PBMCs) from 98 patients with metastatic BC receiving first-line treatment. Fas+, FasL+, and Fas+/FasL+ CTCs were identified in 88.5%, 92.3%, and 84.6% of CTC-positive patients, respectively. In addition, Fas+/FasL+, Fas-/FasL+, and Fas-/FasL- PBMCs were identified in 70.3%, 24.2%, and 5.5% of patients, respectively. A reduced progression-free survival (PFS) was revealed among CTC-positive patients (median PFS: 9.5 versus 13.4 months; p = 0.004), and specifically among those harboring Fas+/FasL+ CTCs (median PFS: 9.5 vs. 13.4 months; p = 0.009). On the other hand, an increased overall survival (OS) was demonstrated among patients with Fas+/FasL+ PBMCs rather than those with Fas-/FasL+ and Fas-/FasL- PBMCs (median OS: 35.7 vs. 25.9 vs. 14.4 months, respectively; p = 0.008). These data provide for the first time evidence on Fas/FasL expression on CTCs and PBMCs with significant prognostic value for patients with metastatic BC, thus highlighting the role of the Fas/FasL system in the peripheral immune response and metastatic progression of BC.

NGAL Mediates Anaplastic Thyroid Carcinoma Cells Survival Through FAS/CD95 Inhibition.

Neutrophil gelatinase-associated lipocalin (NGAL), a siderophore-mediated iron binding protein, is highly expressed in human anaplastic thyroid carcinomas (ATCs) where it plays pleiotropic protumorigenic roles including that of a prosurvival protein. Here we show that NGAL inhibits FAS/CD95 death receptor to control ATC cell survival. FAS/CD95 expression in human specimens from patients with ATC and in ATC-derived cell lines negatively correlate with NGAL expression. Silencing of NGAL in ATC cells leads to FAS/CD95 upregulation, whereas NGAL overexpression determines the opposite effect. As a result, an agonist anti-FAS/CD95 antibody induces cell death in NGAL-silenced cells while it is ineffective on NGAL-overexpressing cells. Interestingly, the inhibitory activity of NGAL on FAS/CD95 is due to its iron carrier property given that perturbing iron homeostasis of NGAL-proficient and -deficient ATC cells directly influences FAS/CD95 expression. Accordingly, conditioned media containing a mutant form of NGAL unable to bind siderophores cannot rescue cells from FAS/CD95-dependent death, whereas NGAL wild type-containing conditioned media abolish the effects of the agonist antibody. We also find that downregulation of FAS/CD95 expression is mediated by iron-dependent NGAL suppression of p53 transcriptional activity. Our results indicate that NGAL contributes to ATC cell survival by iron-mediated inhibition of p53-dependent FAS/CD95 expression and suggest that restoring FAS/CD95 by NGAL suppression could be a helpful strategy to kill ATC cells.

Bifidobacterium animalis sup F1-7 Acts as an Effective Activator to Regulate Immune Response Via Casepase-3 and Bak of FAS/CD95 Pathway.

Intestinal microecology was closely related to immune regulation, but the related mechanism was still unclear. This study aimed to reveal how microorganisms improved immune response via casepase-3 and Bak of FAS/CD95 pathway. Bifidobacterium animalis F1-7 inhibited the melanoma B16-F10 cells in vitro effectively; had a potent anticancer effect of lung cancer mice; effectively improved the spleen immune index and CD3+ (75.8%) and CD8+ (19.8%) expression level; strengthened the phagocytosis of macrophages; inhibited the overexpression of inflammatory factors IL-6 (319.10 ± 2.46 pg/mL), IL-8 (383.05 ± 9.87 pg/mL), and TNF-α (2003.40 ± 11.42 pg/mL); and promoted the expression of anti-inflammatory factor IL-10 (406.00 ± 3.59 pg/mL). This process was achieved by promoting caspase-8/3 and BH3-interacting domain death agonist (Bid), Bak genes, and protein expression. This study confirmed the B. animalis F1-7 could act as an effective activator to regulate immune response by promoting the expression of caspase-8/3, Bid and Bak genes, and proteins and by activating the FAS/CD95 pathway. Our study provided a data support for the application of potentially beneficial microorganisms of B. animalis F1-7 as an effective activator to improve immunity.

Lipid raft involvement in signal transduction in cancer cell survival, cell death and metastasis.

Lipid rafts are cholesterol- and sphingolipid-enriched specialized membrane domains within the plasma membrane. Lipid rafts regulate the density and activity of signal receptors by compartmentalizing them, promoting signalling cascades that play important roles in the survival, death and metastasis of cancer cells. In this review, we emphasize the current concept initially postulated by F. Mollinedo and C. Gajate on the importance of lipid rafts in cancer survival, death and metastasis by describing representative signalling pathways, including the IGF system and the PI3K/AKT, Fas/CD95, VEGF/VEGFR2 and CD44 signalling pathways, and we also discuss the concept of CASMER (cluster of apoptotic signalling molecule-enriched rafts), coined, originally introduced and further advanced by F. Mollinedo and C. Gajate in the period 2005-2010. Then, we summarize relevant research progress and suggest that lipid rafts play important roles in the survival, death and metastasis of cancer cells, making them promising targets for cancer therapy.

Blunted sFasL signalling exacerbates TNF-driven neutrophil necroptosis in critically ill COVID-19 patients.

OBJECTIVES: Critically ill coronavirus disease 2019 (COVID-19) patients are characterised by a severely dysregulated cytokine profile and elevated neutrophil counts, impacting disease severity. However, it remains unclear how neutrophils contribute to pathophysiology during COVID-19. Here, we assessed the impact of the dysregulated cytokine profile on the regulated cell death (RCD) programme of neutrophils. METHODS: Regulated cell death phenotype of neutrophils isolated from critically ill COVID-19 patients or healthy donors and stimulated with COVID-19 or healthy plasma ex vivo was assessed by flow cytometry, time-lapse microscopy and cytokine multiplex analysis. Immunohistochemistry of COVID-19 patients and control biopsies were performed to assess the in situ neutrophil RCD phenotype. Plasma cytokine levels of COVID-19 patients and healthy donors were measured by multiplex analysis. Clinical parameters were correlated to cytokine levels of COVID-19 patients. RESULTS: COVID-19 plasma induced a necroptosis-sensitive neutrophil phenotype, characterised by cell lysis, elevated release of damage-associated molecular patterns (DAMPs), increased receptor-interacting serine/threonine-protein kinase (RIPK) 1 levels and mixed lineage kinase domain-like pseudokinase (MLKL) involvement. The occurrence of neutrophil necroptosis MLKL axis was further confirmed in COVID-19 thrombus and lung biopsies. Necroptosis was induced by the tumor necrosis factor receptor 1 (TNFRI)/TNF-α axis. Moreover, reduction of soluble Fas ligand (sFasL) levels in COVID-19 patients and hence decreased signalling to Fas directly increased RIPK1 levels, exacerbated TNF-driven necroptosis and correlated with disease severity, which was abolished in patients treated with glucocorticoids. CONCLUSION: Our results suggest a novel role for sFasL signalling in the TNF-α-induced RCD programme in neutrophils during COVID-19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome.

Lipid Raft Isolation by Sucrose Gradient Centrifugation and Visualization of Raft-Located Proteins by Fluorescence Microscopy: The Use of Combined Techniques to Assess Fas/CD95 Location in Rafts During Apoptosis Triggering.

Lipid rafts are heterogeneous membrane domains enriched in cholesterol, sphingolipids, and gangliosides that serve as sorting platforms to compartmentalize and modulate signaling pathways. Death receptors and downstream signaling molecules have been reported to be recruited into these raft domains during the triggering of apoptosis. Here, we provide two protocols that support the presence of Fas/CD95 in lipid rafts during apoptosis, involving lipid raft isolation and confocal microscopy techniques. A detailed protocol is provided for the isolation of lipid rafts, by taking advantage of their resistance to Triton X-100 solubilization at 4 °C, followed by subsequent sucrose gradient centrifugation and analysis of the protein composition of the different gradient fractions by Western blotting. In addition, we also provide a detailed protocol for the visualization of the coclustering of Fas/CD95 death receptor and lipid rafts, as assessed by using anti-Fas/CD95 antibodies and fluorescent dye-conjugated cholera toxin B subunit that binds to ganglioside GM1, a main component of lipid rafts, by immunofluorescence and confocal microscopy. These protocols can be extended to any protein of interest to be analyzed for its association to lipid rafts.

Diagnostic accuracy of hepatocyte growth factor, Fas/CD95 and Endostatin for non-invasive assessment of hepatic fibrosis in biopsy-proven hepatitis C virus patients.

BACKGROUND/AIM: Evaluation of liver fibrosis in chronic hepatitis C patients (CHC) provides a high value, not only for the diagnosis of the disease, but also for the therapeutic decision. The aim of the current study is the construction of simple non-invasive and more accurate score for liver fibrosis staging in CHC patients and estimating its performance against three published non-invasive indexes. MATERIAL AND METHODS: CHC patients were divided into two groups: an estimated group (n = 75) and validated group (n = 50). Liver fibrosis was tested in biopsies by Metavair score system. Fas/CD95, hepatocyte growth factor (HGF) and endostatin were assayed by enzyme linked immunosorbent assay (ELISA). Statistical analysis was performed by stepwise linear discriminate analysis and area under-receiver operating curves (AUCs). RESULTS: The multivariate discriminate analysis (MDA) selects a function based on absolute values of five biochemical markers; FHEPA (Fas/CD95, HGF, Endostatin, Platelets&Albumin)-Test score = 1.2 × Fas/CD95 (ng/mL) + 0.006 × HGF (pg/mL) + 0.03 × Endostatin (ng/mL) - 0.007 × platelets count(109/L)-3.6 × Albumin (g/dL) - 8.6.FHEPA-Test producesAUCs 0.99, 0.877 and 0.847 to discriminate patients with significant fibrosis (F2-F4), advanced fibrosis (F3-F4) and cirrhosis (F4), respectively. CONCLUSION: FHEPA-Test is considered a novel non-invasive test which could be applied in assessment of liver fibrosis in HCV infected patients. Our novel score was more efficient than Immune Fibrosis Index, Fibrosis Index and FibroQ and thus it could be more applicable, feasible & economic for Egyptian HCV patients. Our Novel Scoring system could be globalized to other populations to confirm its advantageous use in early diagnosis of liver fibrosis.

Apoptosis-related Proteins Are Altered by Selective Tyrosine Kinase Inhibitors and Everolimus in HPV-dependent SCC.

BACKGROUND: Head and neck squamous cell cancer (HNSCC) affects the oral cavity and the pharynx. The aim of the study was to investigate the effects of selective tyrosine kinase inhibitors (TKIs) erlotinib, gefitinib, nilotinib and dasatinib and the mammalian target of rapamycin (mTOR) inhibitor everolimus on the expression of apoptosis-related proteins caspase-3, FAS cluster of differentiation (CD)-95 and FAS ligand in human papilloma virus (HPV)-dependent squamous cancer. MATERIALS AND METHODS: Two HPV-negative cell lines (UMSCC-11A/-14C) and one HPV-positive cell line (CERV196) were incubated with TKIs or everolimus and protein concentrations of target proteins were analyzed with enzyme-linked immunosorbent assay (ELISA). RESULTS: Caspase-3 was affected by the tested TKIs in HPV-positive SCC, whereas FAS CD95 and FAS ligand were influenced in HPV-negative SCC. DISCUSSION: This is the first study to analyze the influence of TKIs and everolimus on key proteins of apoptosis. Our results provide novel information contributing to a better understanding of the cell biology of HPV-dependent HNSCC and might contribute to the discovery of novel pharmaceutical treatment strategies for HNSCC.

Coarse Grained Molecular Dynamic Simulations for the Study of TNF Receptor Family Members' Transmembrane Organization.

The Tumor Necrosis Factor (TNF) and the TNF receptor (TNFR) superfamilies are composed of 19 ligands and 30 receptors, respectively. The oligomeric properties of ligands, both membrane bound and soluble, has been studied most. However, less is known about the oligomeric properties of TNFRs. Earlier reports identified the extracellular, membrane-distal, cysteine-rich domain as a pre-ligand assembly domain which stabilizes receptor dimers and/or trimers in the absence of ligand. Nevertheless, recent reports based on structural nuclear magnetic resonance (NMR) highlight the intrinsic role of the transmembrane domains to form dimers (p75NTR), trimers (Fas), or dimers of trimers (DR5). Thus, understanding the structural basis of transmembrane oligomerization may shed light on the mechanism for signal transduction and the impact of disease-associated mutations in this region. To this end, here we used an in silico coarse grained molecular dynamics approach with Martini force field to study TNFR transmembrane homotypic interactions. We have first validated this approach studying the three TNFR described by NMR (p75NTR, Fas, and DR5). We have simulated membrane patches composed of 36 helices of the same receptor equidistantly distributed in order to get unbiassed information on spontaneous proteins assemblies. Good agreement was found in the specific residues involved in homotypic interactions and we were able to observe dimers, trimers, and higher-order oligomers corresponding to those reported in NMR experiments. We have, applied this approach to study the assembly of disease-related mutations being able to assess their impact on oligomerization stability. In conclusion, our results showed the usefulness of coarse grained simulations with Martini force field to study in an unbiased manner higher order transmembrane oligomerization.