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Background Achalasia subtypes on high-resolution manometry (HRM) prognosticate treatment response and help direct management plan. We aimed to utilize parameters of distension-induced contractility and pressurization on functional luminal imaging probe (FLIP) panometry and machine learning to predict HRM achalasia subtypes. Methods One hundred eighty adult patients with treatment-naive achalasia defined by HRM per Chicago Classification (40 type I, 99 type II, 41 type III achalasia) who underwent FLIP panometry were included: 140 patients were used as the training cohort and 40 patients as the test cohort. FLIP panometry studies performed with 16-cm FLIP assemblies were retrospectively analyzed to assess distensive pressure and distension-induced esophageal contractility. Correlation analysis, single tree, and random forest were adopted to develop classification trees to identify achalasia subtypes. Key results Intra-balloon pressure at 60 mL fill volume, and proportions of patients with absent contractile response, repetitive retrograde contractile pattern, occluding contractions, sustained occluding contractions (SOC), contraction-associated pressure changes >10 mm Hg all differed between HRM achalasia subtypes and were used to build the decision tree-based classification model. The model identified spastic (type III) vs non-spastic (types I and II) achalasia with 90% and 78% accuracy in the train and test cohorts, respectively. Achalasia subtypes I, II, and III were identified with 71% and 55% accuracy in the train and test cohorts, respectively. Conclusions and inferences Using a supervised machine learning process, a preliminary model was developed that distinguished type III achalasia from non-spastic achalasia with FLIP panometry. Further refinement of the measurements and more experience (data) may improve its ability for clinically relevant application.

BACKGROUND: The functional lumen imaging probe (FLIP) system is an FDA-approved tool for dynamic evaluation of the esophagogastric junction (EGJ). Even though commercially available since 2009, FLIP utilization remains low, partly due to lack of consensus in methodology and interpretation. Therefore, we aimed to analyze the influence of concurrent endoscopy on FLIP measurements. METHODS: In this single-center study, we reviewed data from 93 patients undergoing FLIP for symptomatic esophageal motility disorders between 2016 and 2018. During sedated endoscopy, we measured luminal values (distensibility, cross-sectional area (CSA), and balloon pressure) at the EGJ and distal esophagus using 30, 40, and 50 mL distension volumes, with and without concurrent endoscope presence. All recorded values were compared at the various distension volumes between the two measurements using a Wilcoxon rank sum test. KEY RESULTS: There was a significant difference in distensibility and CSA with index distension volume (40 mL) at the EGJ comparing the two measurements: Lower median distensibility was 2.1 mm$^{2}$ mm Hg$^{-1}$ in the group with concurrent inserted endoscope, respectively, 3.4 mm$^{2}$ mm Hg$^{-1}$ without endoscope (P

During assembly of the bacterial flagellum, protein subunits that form the exterior structures are exported through a specialized secretion apparatus energized by the proton gradient. This category of protein transport, together with the similar process that occurs in the injectisomes of gram-negative pathogens, is termed type-III secretion. The membrane-embedded part of the flagellar export apparatus contains five essential proteins: FlhA, FlhB, FliP, FliQ and FliR. Here, we have undertaken a variety of experiments that together support the proposal that the protein-conducting conduit is formed primarily, and possibly entirely, by FliP. Chemical modification experiments demonstrate that positions near the center of certain FliP trans-membrane (TM) segments are accessible to polar reagents. FliP expression sensitizes cells to a number of chemical agents, and mutations at predicted channel-facing positions modulate this effect. Multiple assays are used to show that FliP suffices to form a channel that can conduct a variety of medium-sized, polar molecules. Conductance properties are strongly modulated by mutations in a methionine-rich loop that is predicted to lie at the inner mouth of the channel, which might form a gasket around cargo molecules undergoing export. The results are discussed in the framework of an hypothesis for the architecture and action of the cargo-conducting part of the type-III secretion apparatus.

Objective: Macrophages are critical in the pathogenesis of rheumatoid arthritis (RA). We recently demonstrated that FLIP (FLICE-like inhibitory protein) is necessary for the differentiation and/or survival of macrophages. We also identified that FLIP is highly expressed in RA synovial macrophages. This study was performed to determine if the reduction of Flip in macrophages would reduce synovial tissue macrophages and ameliorate serum transfer induced arthritis (STIA). Methods: Mice with Flip deleted in myeloid cells (Flipf/fLysMc/+ mice) and littermate controls were employed. STIA was induced by intraperitoneal injection of K/BxN serum. Arthritis was evaluated by clinical score and change in thickness of the ankles. Joints were examined by histology and immunohistochemistry. Cells were isolated from the ankles and bone marrow and examined by flow cytometry, qRT-PCR or Western blot. Results: In contrast to expectations, Flipf/fLysMc/+ mice developed more severe arthritis early in the clinical course, however the peak arthritis was attenuated and the resolution phase more complete. Prior to the induction of STIA, tissue resident macrophages were reduced. Further at day 9 post arthritis induction the number of F4/80hi macrophages in the joints of the Flipf/fLysMc/+ mice was not decreased, but increased. Flip was reduced in the F4/80hi macrophages in the ankles of the Flipf/fLysMc/+ mice, while F4/80hi population expressed an anti-inflammatory phenotype in both the Flipf/fLysMc/+ and control mice. Conclusions: These observations suggest that reduction of FLIP in macrophages, by increasing the number of anti-inflammatory macrophages, may be an effective therapeutic approach to suppress inflammation, depending upon the disease stage. This article is protected by copyright. All rights reserved.

Background Distensibility evaluation of the esophageal body using the functional lumen imaging probe (FLIP) offers an objective measure to characterize patients with eosinophilic esophagitis (EoE), though this analysis may be limited by unrecognized catheter movement and esophageal contractility. The aims of this study were to report novel FLIP analytic methods of esophageal distensibility measurement in EoE and to assess the effect of contractility. Methods Nine healthy controls (six female; ages 20–49) and 20 EoE patients (four female; ages 19–64; grouped by degree of distension-mediated contractility identified on FLIP) were evaluated with a 16-cm FLIP device during step-wise balloon distension during upper endoscopy. A distensibility plateau (DP) was generated using multiple methods to identify the narrowest esophageal body diameter: (i) wavelet decomposition (WD), (ii) maximal diameter (MD), and (iii) FLIP Analytics software. Key Results Distensibility was reduced in EoE patients compared with controls using the WD (p = 0.002) and MD (p = 0.001) methods; a trend was detected using the FLIP Analytics method (p = 0.055). Significant intra-subject differences were detected between methods among both patients and controls (p-values

Purpose To evaluate the effects of increasing the flip angle during the hepatocyte phase of gadobenate dimeglumine-enhanced magnetic resonance imaging (MRI) in cirrhotic patients with hepatocellular carcinoma (HCC). Materials and Methods Sixty-three patients with liver cirrhosis underwent gadobenate dimeglumine-enhanced 1.5T MRI with 90-minute delayed hepatocyte phase with flip angles of 10°, 20°, 30°, consecutively. Relative enhancement and signal-to-noise ratio (SNR) of liver parenchyma at hepatocyte phase according to flip angle were calculated. The liver-to-lesion (low signal intensity HCCs, n = 63; ≥1 cm) and contrast-to-noise ratio (CNR) at the hepatocyte phase according to flip angle were calculated. Two radiologists independently assessed the presence of HCCs using a 5-point scale, and detection sensitivity of HCCs was calculated according to flip angle. Results The relative enhancement of hepatic parenchyma differed significantly according to flip angle (10°, mean relative enhancement = 0.69 ± 0.46; 20°, mean relative enhancement = 0.63 ± 0.47; 30°, mean relative enhancement = 0.49 ± 0.45; P = 0.043). The SNR of hepatic parenchyma was significantly different according to flip angle (10°, mean SNR = 26.2 ± 5.6; 20°, mean SNR = 25.3 ± 5.7; 30°, mean SNR = 22.8 ± 6.1; P = 0.004). The CNR of lesion was not significantly different according to flip angle (10°, mean CNR = 7.5 ± 6.6; 20°, mean CNR = 10.2 ± 6.9; 30°, mean CNR = 10.1 ± 7.1; P = 0.051). The sensitivities with 10° and 20° for HCCs were significantly higher than those with 30° for one reader (P < 0.05). Conclusion In patients with cirrhosis, hepatocyte phase gadobenate dimeglumine-enhanced 1.5T MRI with 20° flip angle should be recommended rather than 10° and 30° flip angle. J. Magn. Reson. Imaging 2015.

Purpose To develop and validate clinically a single-shot fast spin echo (SSFSE) sequence utilizing variable flip angle refocusing pulses to shorten acquisition times via reductions in specific absorption rate (SAR) and improve image quality. Materials and methods A variable refocusing flip angle SSFSE sequence (vrfSSFSE) was designed and implemented, with simulations and volunteer scans performed to determine suitable flip angle modulation parameters. With Institutional Review Board (IRB) approval/informed consent, patients referred for 3T abdominal magnetic resonance imaging (MRI) were scanned with conventional SSFSE and either half-Fourier (n = 25) or full-Fourier vrfSSFSE (n = 50). Two blinded radiologists semiquantitatively scored images on a scale from -2 to 2 for contrast, noise, sharpness, artifacts, cardiac motion-related signal loss, and the ability to evaluate the pancreas and kidneys. Results vrfSSFSE demonstrated significantly increased speed (∼2-fold, P Conclusion vrfSSFSE increases speed at 3T over conventional SSFSE via reduced SAR, and when combined with full-Fourier acquisition can improve image quality, although with some increased sensitivity to cardiac motion-related signal loss.

Novel agents are desperately needed for improving the quality of life and 5-year survival to more than 30% for metastatic castrate-resistant prostate cancer. Previously we showed that Nexrutine, Phellodendron amurense bark extract, inhibits prostate tumor growth in vitro and in vivo. Subsequently using biochemical fractionation we identified butanol fraction contributes to the observed biological activities. We report here that palmatine, which is present in the butanol fraction, selectively inhibits growth of prostate cancer cells without significant effect on non-tumorigenic prostate epithelial cells. By screening receptor tyrosine kinases in a protein kinase array, we identified ribosomal protein S6, a downstream target of p70S6K and the Akt/mTOR signaling cascade as a potential target. We further show that palmatine treatment is associated with decreased activation of NFκB and its downstream target gene FLIP. These events led to inhibition of invasion. Similar results were obtained using parent extract Nexrutine (Nx) suggesting that palmatine either in the purified form or as one of the components in Nx is a potent cytotoxic agent with tumor invasion inhibitory properties. Synergistic inhibition of rpS6/NFκB/FLIP axis with palmatine may have therapeutic potential for the treatment of prostate cancer and possibly other malignancies with their constitutive activation. These data support a biological link between rpS6/NFκB/FLIP in mediating palmatine-induced inhibitory effects and warrants additional preclinical studies to test its therapeutic efficacy.

In this study, we show that atraxia telangiectasia mutated kinase (ATM) activity is generally upregulated by different apoptotic stimuli, i.e. TNF-α, TRAIL, paclitaxel, or UV. Apoptotic progression is markedly attenuated by siATM-RNA through down regulation of caspase-8 and caspase-9 in parallel with decreases in FLIP-S (short form of cellular FLICE inhibitory protein) protein levels and Bid cleavage. In addition, ATM activity is upregulated through t-Cdc6 while caspase-8 and caspase-9 activities increase. Taken together, we suggest that ATM regulates caspase-8 activation by influencing levels of FLIP-S, ATM kinase activity is upregulated by t-Cdc6, and increased ATM activity plays an essential role in the amplification of apoptosis in TNF-α-stimulated HeLa cells.

HIV-1 infection and replication are affected by host factors. Recent studies demonstrate that molecules from apoptotic pathways regulate HIV-1 replication. Therefore, studies on effects of host factors that maintain host cell survival and influence HIV-1 replication are critical to understanding the mechanisms of HIV-1 replicative cycle. Using the susceptible Jurkat cell line, CD4(+) T cells, and peripheral blood mononuclear cells (PBMCs), we studied the role of FLIP, an inhibitor of caspase-8, in HIV-1 production. Full length cellular FLIP (cFLIP) inhibited HIV-1 replication in these cells. cFLIP upregulated the expression of viral restriction factors, such as TRIM5, Apobec3G, and Bst2/tetherin, decreased nuclear factor 1C expression and inactivated ERK and p38 induced by HIV-1 in Jurkat cells. cFLIP blocked the trafficking of gp120 and Gag p24 capsid protein into lipid rafts with inhibition of Tsg101 and Alix in ESCRT signaling pathway. cFLIP also promoted Bst2/tetherin trafficking into lipid rafts. These results indicate that cFLIP may inhibit the HIV-1 replication cycle at multiple steps, including viral RNA release, transcription, traffic and assembly. We also found that cFLIP expression downregulated Fas expression and inactivated FADD in the Fas-mediated apoptotic pathway. The inactivated FADD also inhibited HIV-1 replication.

Cancerous growth is one of the most difficult diseases to target as there is no one clear cause, and targeting only one pathway does not generally produce quantifiable improvement. For a truly effective cancer therapy, multiple pathways must be targeted at the same time. One way to do this is to find a gene that is associated with several pathways; this approach expands the possibilities for disease targeting and enables multiple points of attack rather than one fixed point, which does not allow treatment to evolve over time as cancer does. Inducing programmed cell death (PCD) is a promising method to prevent or inhibit the progression of tumor cells. Intricate cross talk among various programmed cell death pathways including cell death by apoptosis, necroptosis or autophagy plays a critical role in the regulation of PCD. In addition, the complex and overlapping patterns of signaling and lack of understanding of such networks between these pathways generate hurdles for developing effective therapeutic approaches. This review article focuses on targeting FLIP (Fas-associated death domain-like interleukin-1-converting enzyme-like inhibitory protein) signaling as a bridge between various PCD processes as an effective approach for cancer management.

The multikinase inhibitor sorafenib is highly effective against certain types of cancer in the clinic and prevents colon cancer cell proliferation in vitro. Non-steroidal anti-inflammatory drugs, such as acetylsalicylic acid (aspirin), have shown activity against colon cancer cells. The aims of this study were to determine whether the combination of aspirin with sorafenib has enhanced anti-proliferative effects and increases recombinant human tumour necrosis factor-related apoptosis-inducing ligand (rhTRAIL)-induced apoptosis in the human SW948, Lovo, Colo205, Colo320, Caco-2 and HCT116 colon cancer cell lines. In four cell lines, aspirin strongly stimulated the anti-proliferative effects of sorafenib (similar to four-fold enhancement) by inducing cell cycle arrest. Furthermore, combining low doses of aspirin (

Objective(s) We have previously shown that Fas expression inversely correlates with the metastatic potential of osteosarcoma (OS) to the lung. FasL is constitutively expressed in the lung microenvironment and eliminates Fas+ osteosarcoma cells leaving Fas− cells to form metastases. Absence of FasL in the lung epithelium or blocking the Fas-signaling pathway interfered with this clearance mechanism allowing Fas+ cells to remain and form lung metastases. We also demonstrated that while the majority of patient OS lung metastases were Fas−, 10-20% of the lesions contain Fas+ cells, suggesting that these cells were not sensitive to FasL-induced apoptosis. The expression of c-FLIP, an inhibitor of the Fas pathway, has been associated with tumor development, progression, and resistance to chemotherapy. We therefore evaluated the expression of c-FLIP in OS patient tumor specimens and human xenograft lung metastases.

Various molecular mechanisms are involved in the efficacy of arsenic trioxide (ATO) against malignant hematologic and some solid tumors. FLICE-like inhibitory protein (FLIP) is an inhibitor of apoptosis mediated by death receptors. In this study, we identified a new link between the down-regulation of cellular FLIPL and ATO-induced autophagy. ATO induced the degradation of FLIPL in K562 and MGC803 cells, which was mediated by the ubiquitin–proteasome pathway. Moreover, the casitas B-lineage lymphoma-b (Cbl-b) was involved in this process, which interacted with FLIPL and promoted proteasomal degradation of FLIPL. Our findings lead to a better understanding of the mechanism of action of ATO, and suggest that a novel signaling pathway is required for ATO-induced autophagy in K562 and MGC803 cells. Structured summary of protein interactions FLIP-L physically interacts with CBL-B by anti bait coimmunoprecipitation ( View interaction )

Santacana M, Yeramian A, Velasco A, Bergada L, Gatius S, Garcia V, Azueta A, Palacios J, Dolcet X, Oliva E & Matias-Guiu X (2012) Histopathology 60, 460–471 Immunohistochemical features of post-radiation vaginal recurrences of endometrioid carcinomas of the endometrium: role for proteins involved in resistance to apoptosis and hypoxia Aims: Endometrioid carcinoma of the endometrium (EEC) is treated with surgery and radiotherapy. Post-radiation recurrences are associated with increased risk of metastases. Comparison of the expression of genes important in the development and progression of EEC, and others involved in resistance to apoptosis and hypoxia and adaptation to radiation, was performed between post-radiation vaginal recurrences (PVRs) and primary EECs. We tried to reproduce the results by exposing an EEC cell line to hypoxia and radiation. Methods and results: Immunohistochemistry and tissue microarrays were used to compare 24 PVRs with 82 primary EECs. PVRs exhibited increased expression of p53 (P

Exposure of live cells to shear flow induces major changes in cell shape, adhesion to the extracellular matrix, and migration. In the present study, we show that exposure of cultured multiple myeloma (MM) cells to shear flow of 4–36 dynes/cm2 triggers the extension of long tubular protrusions (denoted flow-induced protrusions, or FLIPs) in the direction of the flow. These FLIPs were found to be rich in actin, contain few or no microtubules and, apart from endoplasmic reticulum (ER)-like membranal structures, are devoid of organelles. Studying the dynamics of this process revealed that FLIPs elongate at their tips in a shear force-dependent manner, and retract at their bases. Examination of this force dependence revealed considerable heterogeneity in the mechanosensitivity of individual cells, most likely reflecting the diversity of the malignant B cell population. The mechanisms underlying FLIP formation following mechanical perturbation, and their relevance to the cellular trafficking of MM cells, are discussed. J. Cell. Physiol. 226: 3197–3207, 2011. © 2011 Wiley Periodicals, Inc.

Chemical shift-based water/fat separation, like iterative decomposition of water and fat with echo asymmetry and least-squares estimation, has been proposed for quantifying intermuscular adipose tissue. An important confounding factor in iterative decomposition of water and fat with echo asymmetry and least-squares estimation-based intermuscular adipose tissue quantification is the large difference in T(1) between muscle and fat, which can cause significant overestimation in the fat fraction. This T(1) bias effect is usually reduced by using small flip angles. T(1) -correction can be performed by using at least two different flip angles and fitting for T(1) of water and fat. In this work, a novel approach for the water/fat separation problem in a dual flip angle experiment is introduced and a new approach for the selection of the two flip angles, labeled as the unequal small flip angle approach, is developed, aiming to improve the noise efficiency of the T(1) -correction step relative to existing approaches. It is shown that the use of flip angles, selected such the muscle water signal is assumed to be T(1) -independent for the first flip angle and the fat signal is assumed to be T(1) -independent for the second flip angle, has superior noise performance to the use of equal small flip angles (no T(1) estimation required) and the use of large flip angles (T(1) estimation required).

Cardiac MR T(1) mapping is a promising quantitative imaging tool for the diagnosis and evaluation of cardiomyopathy. Here, we present a new preclinical cardiac MRI method enabling three-dimensional T(1) mapping of the mouse heart. The method is based on a variable flip angle analysis of steady-state MR imaging data. A retrospectively triggered three-dimensional FLASH (fast low-angle shot) sequence (3D IntraGate) enables a constant repetition time and maintains steady-state conditions. 3D T(1) mapping of the complete mouse heart could be achieved in 20 min. High-quality, bright-blood T(1) maps were obtained with homogeneous T(1) values (1764 ± 172 ms) throughout the myocardium. The repeatability coefficient of R(1) (1/T(1) ) in a specific region of the mouse heart was between 0.14 and 0.20 s(-1) , depending on the number of flip angles. The feasibility for detecting regional differences in ΔR(1) was shown with pre- and post-contrast T(1) mapping in mice with surgically induced myocardial infarction, for which ΔR(1) values up to 0.83 s(-1) were found in the infarct zone. The sequence was also investigated in black-blood mode, which, interestingly, showed a strong decrease in the apparent mean T(1) of healthy myocardium (905 ± 110 ms). This study shows that 3D T(1) mapping in the mouse heart is feasible and can be used to monitor regional changes in myocardial T(1), particularly in relation to pathology and in contrast-enhanced experiments to estimate local concentrations of (targeted) contrast agent.

Two strategies for fighting cancer by modulating FASL-induced apoptosis were modeled by 2D-cellular automata. Our models predict that cancer cells can be killed by maximizing the apoptosis via joint suppression of FLIP and IAP inhibitors by siRNA and SMAC proteins, respectively. It was also predicted that the presumed feedback loop CASP3-->CASP9-->|IAP in the intrinsic pathway accelerates the apoptosis, but does not change significantly the concentration of DFF40, the protein that decomposes DNA. The alternative strategy of preventing the killing of the immune system's T-cells, via minimizing their tumor-induced FAS-L apoptosis by overexpression of FLIP and IAP, was also shown to be promising with a predicted considerable synergy action of the two inhibitors. Dual suppression or overexpression of apoptosis inhibitors emerges thus as promising approach in the fight against cancer. Our modeling has also brought some light on the process of turning type-I cells into type-II ones, which emerges as compensatory mechanism in case of damaged or silenced FASL pathway by preserving about the same self-death level at only 10-12% lower performance rate.

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) class of ionotropic glutamate receptors comprises four different subunits: iGluR1/iGluR2 and iGluR3/iGluR4 forming two subgroups. Three-dimensional structures have been reported only of the ligand-binding core of iGluR2. Here, we present two X-ray structures of a soluble construct of the R/G unedited flip splice variant of the ligand-binding core of iGluR4 (iGluR4i(R)-S1S2) in complex with glutamate or AMPA. Subtle, but important differences are found in the ligand-binding cavity between the two AMPA receptor subgroups at position 724 (Tyr in iGluR1/iGluR2 and Phe in iGluR3/iGluR4), which in iGluR4 may lead to displacement of a water molecule and hence points to the possibility to make subgroup specific ligands.

It is increasingly accepted that cancer stem cells (CSCs) are rather resistant to apoptosis to various inducers, including the immunological apoptogen TRAIL. Here we show that cancer cells with high expression of CD133, a marker that is often associated with CSCs, are resistant to TRAIL-induced apoptosis, compared to their CD133-low counterparts. We show that this resistance can be ascribed to the high expression of FLIP, an inhibitor of the extrinsic apoptotic pathway, in CD133-high cells. Downregulation of FLIP by siRNA in CD133-high cells sensitised them to TRAIL killing. Thus, CD133-high cells may be resistant to TRAIL due to high expression of FLIP.

Aims: Disruption of apoptotic cell death has been implicated in tumour aggressiveness in colonic carcinogenesis. The Fas–Fas ligand (FasL) system is involved in the execution of apoptosis induced by the immune system. c-FLIP protein constitutes an inhibitor of Fas and other (TRAIL) death receptor-mediated apoptosis. The aim of this study was to investigate the simultaneous expression of Fas, FasL and c-FLIP in relation to standard clinicopathological parameters and patients' outcome in colorectal cancer. Methods and results: Levels of Fas, FasL and c-FLIP protein expression were quantified immunohistochemically in paraffin-embedded tissues from 90 patients. Immunopositivity was detected for Fas, FasL and c-FLIP in 71%, 35.5% and 68.8% of cases, respectively. Concurrent expression of Fas/FasL was seen in 28 samples (31%), of which 24 (85.7%) also displayed c-FLIP positivity (P = 0.04). c-FLIP overexpression (> 10%) tended to prevail marginally in higher stage tumours (P = 0.09). Additionally, FasL and c-FLIP adversely affected survival on both univariate (P = 0.001 and P = 0.0024, respectively) and multivariate analysis [hazard ratio (HR) 3.491, P = 0.005 and HR 2.960, P = 0.036, respectively]. Conclusions: The frequent expression and coexpression of Fas, FasL and c-FLIP in colorectal carcinoma implicates c-FLIP as an inhibitor of the Fas–FasL-induced death pathway in these tumours. Moreover, c-FLIP conveys independent prognostic information in the presence of classical prognosticators.

FLICE-like inhibitory protein (FLIP), a naturally occurring caspase-inhibitory protein that lacks the critical cysteine domain necessary for catalytic activity, is a negative regulator of Fas-induced apoptosis. Decreased FLIP levels sensitize tumor cells to Fas- and TRAIL-mediated apoptosis; however, the cellular mechanisms regulating FLIP expression have not been defined. Here, we examined the roles of the PKC and NF-kappaB pathway in the regulation of FLIP in human colon cancers. FLIP mRNA levels were increased in Caco-2 cells by treatment with PMA; actinomycin D completely inhibited the induction of FLIP by PMA, indicating transcriptional regulation. PKC inhibitors Gö6983 and Ro-31-8220 blocked PMA-stimulated FLIP expression. Pretreatment with the PKCdelta-selective inhibitor rottlerin or transfection with PKCdelta siRNA inhibited PMA-induced FLIP expression, which identifies a role for PKCdelta in FLIP induction. Treatment with the proteasome inhibitor, MG132, or the NF-kappaB inhibitor (e.g., PDTC and gliotoxin), or overexpression of the superrepressor of IkappaB-alpha inhibited PMA-induced upregulation of FLIP. Moreover, PMA-induced NF-kappaB transactivation was blocked by GF109203x. In conclusion, our results demonstrate a critical role for PKCdelta/NF-kappaB in the regulation of FLIP in human colon cancer cells.

NF-kappaB activation plays a crucial role in anti-apoptotic responses in response to the apoptotic signaling during tumor necrosis factor (TNF)-alpha stimulation. TNF-alpha induces apoptosis sensitive to the hepatitis B virus (HBV) infected cells, despite sustained NF-kappaB activation. Our results indicate that the HBV infection induces sustained NF-kappaB activation, in a manner similar to the TNF-alpha stimulation. However, these effects are not merely combined. Computational simulations show that the level of form of the IKK complex activated by phosphorylation (IKK-p) affects the dynamic pattern of NF-kappaB activation during TNF-alpha stimulation in the following ways: (i) the initial level of IKK-p determines the incremental change in IKK-p at the same level of TNF-alpha stimulation, (ii) the incremental change in IKK-p determines the amplitudes of active NF-kappaB oscillation, and (iii) the steady state level of IKK-p after the incremental change determines the period of active NF-kappaB oscillation. Based on experiments, we observed that the initial level of IKK-p was upregulated and the active NF-kappaB oscillation showed smaller amplitudes for a shorter period in HepG2.2.15 cells (HBV-producing cells) during TNF-alpha stimulation, as was indicated by the computational simulations. Furthermore, we found that during TNF-alpha stimulation, NF-kappaB-regulated anti-apoptotic genes were upregulated in HepG2 cells but were downregulated in HepG2.2.15 cells. Based on the previously mentioned results, we can conclude that the IKK-p-level changes induced by HBV infection modulate the dynamic pattern of active NF-kappaB and thereby could affect NF-kappaB-regulated anti-apoptotic gene expressions. Finally, we postulate that the sensitive apoptotic response of HBV-infected cells to TNF-alpha stimulation is governed by the dynamic patterns of active NF-kappaB based on IKK-p level changes.

The properties of the glutamate receptor subunits 1–4 (GluR1–4) are influenced by the alternative splicing of two homologous and mutually exclusive exons flip and flop. The flip form is most abundant during early development, while the flop form is dominant in adults. From transfections with a GluR2 mini-gene we show that flip is the preferred splice form in all tested cell lines, but coexpression of the SR-proteins ASF/SF2 and SC35 increases the flop to flip splice ratio. The increased flop incorporation depends on ASF/SF2- and SC35-dependent enhancer elements located in the flop exon, which stimulate the splicing between the flop exon and the preceding exon 13.

The cellular homologues of the viral anti-apoptotic v-FLIP proteins exist as a long (c-FLIP(L)) and a short (c-FLIP(S)) splice variant. While c-FLIP(S) and v-FLIP are composed solely of two death effector domains, c-FLIP(L) contains an (inactive) caspase-like domain in addition to these two death effector domains, thereby structurally resembling pro-Caspase-8. Both c-FLIP(L) and c-FLIP(S) suppress apoptosis by inhibiting Caspase-8 activation, although at different levels of pro-Caspase-8 processing. To analyze the consequences of deregulated c-FLIP(S) expression in vivo, we established lck FLIP(S)-transgenic mice overexpressing the transgene in thymocytes and in mature T cells. As expected, CD95L-induced apoptosis was impaired in lck FLIP(S)-transgenic T cells, indicating the functionality of the FLIP(S) transgene. Remarkably, activation-induced cell death of transgenic T cells was unaffected, despite the observed inhibition of CD95-induced T cell death. Thymic and splenic cell numbers as well as CD4/CD8 cellularity were normal in lck FLIP(S)-transgenic animals, which in contrast to CD95-deficient mice do not accumulate Thy1(+) B220(+) CD4(-) CD8(-) peripheral T cells. c-FLIP(S) overexpression leads to a significant decrease in activation-induced T cell proliferation in vitro. Despite the capacity of FLIP(S) to inhibit CD95-induced apoptosis, T cell lymphomagenesis is not observed in lck FLIP(S)-transgenic mice. Interestingly, the Vbeta8(+) memory T cell pool is enlarged upon staphylococcal enterotoxin B injections, suggesting a specific in vivo function for FLIP(S) in the maintenance of restimulated T cells.

AMPA-type glutamate receptor (GluR) channels are the most abundant excitatory transmitter receptors of the central nervous system. Four subunits with different posttranscriptional modifications and flip/flop splice variants are known. In vivo they occur as tetrameric heteromeric receptors. In the present study we analyzed the time course of desensitization (τD) and resensitization (τrec) kinetics of different homomeric (coassembly of splice or editing variants of one subunit) and heteromeric (coassembly of different subunits) GluR channels. We found that τD had intermediate values depending on the amount of cDNA of the respective subunit at all heteromeric and homomeric GluR channels tested. The same holds true for τrec except GluR2 flip channels were coexpressed with GluR1 channels. In this case, τrec had values close to that of fast resensitizing GluR2 flip channels, even in the case of an abundance of GluR1 cDNA. Synapse 55:176–182, 2005. © 2005 Wiley-Liss, Inc.

Objective Hyperplasia of fibroblast-like synoviocytes (FLS) contributes to chronic inflammation and joint destruction in rheumatoid arthritis (RA). FLICE-inhibitory protein (FLIP) is an antiapoptotic protein that might prevent apoptotic elimination of FLS in response to death ligands such as tumor necrosis factor α (TNFα) or Fas ligand, which are present in RA synovium. Previous studies on FLIP expression by osteoarthritis (OA) and RA FLS have shown variable results, and the specific role of FLIP as an apoptosis inhibitor in these cells remains unclear. We undertook this study to investigate the expression and antiapoptotic function of FLIP in FLS. Methods We studied the expression of FLIP by immunohistochemistry and immunoblotting in synovial tissues or cultured FLS from RA and OA patients. FLS apoptosis was induced by an agonistic anti-Fas monoclonal antibody and FLS were then quantified. We studied the effects of cycloheximide (CHX), TNFα, and FLIP antisense oligonucleotide on FLIP expression and FLS apoptotic susceptibility. Results FLIPL was the isoform mainly expressed in lining synoviocytes and cultured FLS. Synovial tissues and cultured FLS from OA and RA tissues displayed similar patterns and levels of expression of FLIP. Fas-induced apoptosis was variable in different FLS lines, but differences between OA and RA groups were not detected. TNFα induced increases in FLIPL and FLIPS expression and protected RA FLS from apoptosis, while CHX induced the opposite effects. Down-regulation of FLIP by antisense oligonucleotide strongly sensitized RA FLS to Fas-mediated apoptosis. Conclusion Apoptosis susceptibility and FLIP expression are similar in OA and RA FLS. Down-regulation of FLIP sensitizes RA FLS to Fas-mediated apoptosis and may be a valuable tool for targeting RA FLS hyperplasia.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in some, but not all cancer cells. To assess the regulation of TRAIL-resistance in the human gastric cancer cells, we examined TRAIL sensitivity, TRAIL receptor expression, and intracellular signaling events induced by TRAIL. All the gastric cancer cell lines tested were susceptible to TRAIL to some extent, except for SNU-216 cell line, which was completely resistant. TRAIL receptor expression was not related to the TRAIL-sensitivity. Of the cell lines tested, SNU-216 showed the highest level of constitutively active Akt and the short form of FLICE inhibitory protein (FLIP(S)). Treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or with the protein synthesis inhibitor cycloheximide induced a suppression of constitutive Akt activation in SNU-216 cells and a concomitant decrease in the expression of FLIP(S). The reduction of Akt activity by LY294002 affected the transcriptional level of FLIP(S), but not the mRNA stability. As a result, LY294002 or cycloheximide significantly enhanced TRAIL-induced apoptosis. Moreover, the overexpression of constitutively active Akt in the TRAIL-sensitive cell line, SNU-668, rendered the cell line resistant to TRAIL. In addition, infection of the same cell line with retrovirus expressing FLIP(S) completely inhibited TRAIL-induced apoptosis by blocking the activation of caspase-8. Therefore, our results suggest that Akt activity promotes human gastric cancer cell survival against TRAIL-induced apoptosis via upregulation of FLIP(S), and that the cytotoxic effect of TRAIL can be enhanced by modulating the Akt/FLIP(S) pathway in human gastric cancers.

Summary. KG1a cells (CD34+/38–) express FAS and TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand) receptors but are resistant to FAS-ligand and TRAIL/APO2-L (apoptosis antigen-2 ligand)-induced apoptosis. KG1a cells are sensitized to FAS-induced apoptosis by chelerythrin, an inhibitor of protein kinase C (PKC). As cytoplasmatic adaptor molecules of FAS, e.g. FLIP {Fas-associated death domain protein (FADD)-like interleukin 1 beta-converting enzyme [FLICE (caspase-8)-inhibitory protein]}, also modulate TRAIL signals, we determined whether chelerythrin affected TRAIL-mediated apoptosis. Chelerythrin by itself induced apoptosis in KG1a cells, and apoptosis was associated with activation of caspase-8. While TRAIL alone failed to activate caspase-8 or induce apoptosis, the addition of TRAIL to chelerythrin-treated cells significantly enhanced cleavage of caspase-8 and apoptosis. Chelerythrin-pretreated KG1a cells showed decreased phosphorylation of protein kinase C (PKC)-ζ and downregulation of both FLIP long and FLIP short proteins. Downregulation of FLIP and induction of apoptosis were partially abrogated by pretreatment with the specific caspase-8 inhibitor, Z-IETD-FMK. The decrease in FLIP protein expression induced by chelerythrin was accompanied by a progressive increase in mRNA levels of both FLIP long and FLIP short. CD34+ precursors from normal human marrow were also sensitive to chelerythrin but, in contrast to KG1a cells, were not sensitized to TRAIL-mediated apoptosis. Thus, resistance to TRAIL-induced apoptosis in leukaemic KG1a cells but not in normal CD34+ precursors was overcome in the presence of chelerythrin. The mechanism appeared to involve inhibition of PKC. Central targets were FLIP long and FLIP short, and their interactions with caspase-8. Whether such a pathway can be exploited to selectively target leukaemic progenitor cells remains to be determined.

In this review, we consider the role caspases play in cell death downstream of death receptors and cell intrinsic death mechanisms. In particular, we focus on these mechanisms in antigen-induced cell death, a mechanism which regulates the number of surviving T cells at the end of an immune response. The relative role of the apoptosome as an amplifier rather than an initiator of apoptosis is considered. Several factors that regulate the susceptibility to activation-induced cell death are considered. These factors emanate from the stimulation of the T-cell receptors and include multiple pathways. Recent work has shown that death receptor signaling can play an interesting role in cell proliferation in both humans and animals. These recent findings are discussed in the light of models of death receptor signaling.

AMPA-type glutamate receptors (AMPAR) display a high variability in functional properties, which determine the time course of excitatory postsynaptic potentials. They are assembled as tetramers of GluR subunits 1-4 of different splice variants and nuclear edited isoforms. Presently, the kinetics of activation, desensitization and recovery from desensitization of human AMPARs (GluR1, 3 and 4 flip and flop, and GluR2 flip and flop in R and G edited forms, respectively) transiently expressed in HEK293 cells were studied with patch-clamp techniques and ultra fast agonist application. Activation time constants were identical for all receptors (0.13 ms). The GluR2 flip G variant showed the slowest desensitization (10.8 ms), GluR4 flip the fastest (1.6 ms). Recovery from desensitization varied between 3.1 ms (GluR4 flip) and 178 ms (GluR1 flip). To determine functional interactions between subunits in heteromeric receptors the GluR1 flip and the GluR2 flip R were coexpressed. The time constant of desensitization increased linearly from 2.5 ms (GluR1 flip homomers) to 6.8 ms (GluR2 flip R homomers) with the amount of GluR2 flip R cDNA transfected. Recovery followed a monoexponential time course and had a time constant of 178 ms in GluR1 flip homomeric expression. In all GluR1 flip/GluR2 flip heteromers and in GluR2 flip R homomers desensitization recovered with a time constant of approximately 50 ms. Thus, subunit interaction seems likely during recovery but not desensitization. Both parameters might influence the ability of AMPA receptors to mediate glutamate induced chronic excitotoxicity in neurodegenerative diseases.

Contract grant sponsor: the Fondo de Investigacion Sanitaria (F.I.S.); Contract grant number: 94/0864; Contract grant sponsor: CICYT; Contract grant numbers: SAF 96-0336, SAF 97-0117; Contract grant sponsors: the CIRIT (Generalitat de Catalunya) (to the Department of Neurochemistry (IIBB/CSIC) as Grup de Recerca de Qualitat)

Polar lipids must flip rapidly, and often bidirectionally, across membranes to support cellular life. For example, phospholipids are synthesized on the cytoplasmic face of the endoplasmic reticulum...

Peripheral blood-derived monocytes spontaneously undergo apoptosis mediated by Fas-Fas ligand (FasL) interactions. Activation of monocytes by LPS or TNF-alpha prevents spontaneous monocyte apoptosis through an unknown mechanism. Here, we demonstrate that LPS and TNF-alpha up-regulate Flip and suppress spontaneous Fas-FasL mediated monocyte apoptosis and caspase 8 and 3 activation. Flip was responsible for this protection, since inhibition of Flip by antisense oligonucleotides in the presence of LPS or TNF-alpha restored monocyte sensitivity to spontaneous apoptosis. We also investigated whether the PI3K pathway contributes to the suppression of spontaneous monocyte apoptosis mediated by LPS and TNF-alpha. Monocytes treated with a reversible PI3K inhibitor (LY294002) displayed enhanced apoptosis, while LPS and TNF-alpha partially protected against apoptosis mediated by LY294002. However, direct suppression of Fas-FasL interactions by addition of neutralizing anti-FasL antibody did not further suppress LY294002-induced apoptosis in the presence of LPS or TNF-alpha. Collectively, these data demonstrate that LPS or TNF-alpha protect monocytes from death receptor-mediated apoptosis through the up-regulation of Flip, but not apoptosis initiated by inhibition of the PI3K pathway.

Derangement of either apoptosis or cell division is known to play an important role in tumorigenesis. Fas-mediated apoptosis on normal and leukaemic T cells is finely tuned by inhibitory proteins, such as FAP-1, FLIP and survivin, and defective caspase isoform which can attenuate the function of its intact caspase as a decoy molecule. However, complex involvement of such inhibitors in tumour biology relating to apoptotic pathology remains unclear in the neoplasms. We report the aberrant expression of FAP-1, FLIP and survivin mRNAs on leukaemic T cells from adult T-cell leukaemia (ATL) patients. Among these inhibitors, only survivin was aberrantly expressed in all ATL cases, but not in any normal peripheral blood mononuclear cells (PBMCs). Furthermore, survivin mRNA expression level was characteristic in each subtype of ATL and represented an important determinant for ATL prognosis. However, the apoptotic effector of casp-8, which is essential in Fas-mediated signal transduction, was dominant in defective casp-8 rather than intact casp-8 in ATL cells, suggesting a favourable biological situation for escape from apoptosis. Taken together, ATL cells probably possess many different regulatory mechanisms in order to attenuate Fas-mediated signalling and subsequently expand their populations under escape from apoptosis. Among these inhibitors, survivin is a useful bio-marker to assess tumour biology and may be a potential new target for apoptosis-based selective therapy in neoplasms as the expression is a general feature of neoplasia, but not normal tissues.

Death receptor-mediated apoptosis is involved in the regulation of immune responses and in the maintenance of immunological tolerance. FLICE-inhibitory proteins (FLIPs) are important modulators of death receptor-mediated apoptosis. To date, the FLIP family encompasses multiple members, of which some are reported to be antiapoptotic and others pro-apoptotic. This led us to investigate the activity of several FLIP proteins in vitro. Concomitant with the cloning of various FLIP isoforms, a new and unexpected member of the FLIP family, denoted FLIPR, was isolated from the human Burkitt lymphoma B-cell line Raji. During the characterization of FLIPR, the genomic sequence of human FLIP was found in the NCBI GenBank. This enabled us to present the complete exon-intron constellation of the human FLIP gene and the generation of all known human FLIP isoforms by alternative splicing. We show that the human FLIP gene with a size of approximately 48 kb, consists of at least 14 exons and can give rise to 11 distinct isoforms by alternative splicing. When studying the activity of some of these isoforms, including FLIPR, they all efficiently inhibited Fas-mediated apoptosis in A20 B lymphoma cells by impeding caspase-8, -3 and -7 activity as well as poly(ADP-ribose) polymerase (PARP) cleavage.

Electrophysiological recordings have been used to characterize responses mediated by AMPA receptors expressed by cultured rat cortical and spinal cord neurones. The EC(50) values for AMPA were 17 and 11 microM, respectively. Responses of cortical neurones to AMPA were inhibited competitively by NBQX (pK(i)=6.6). Lower concentrations of NBQX (< or =1 microM) also potentiated the plateau responses of spinal cord neurones to AMPA, which could be attributed to a depression of desensitization to AMPA. GYKI 52466 inhibited responses of spinal cord neurones to AMPA to about twice the extent of responses of cortical neurones. Blockade of AMPA receptor desensitization by cyclothiazide (CTZ) potentiated responses of spinal cord neurones (6.8 fold) significantly more than responses of cortical neurones (4.8 fold). Responses of cortical neurones to KA were potentiated 3.5 fold by CTZ, while responses of spinal cord neurones were unaffected. Ultra-fast applications of AMPA to outside-out patches showed responses of spinal cord neurones desensitized by 97.5% and exhibit marked inward rectification, whereas cortical neurones desensitized by 91% and exhibited slight outward rectification. The time constants of deactivation and desensitization were about twice as fast in spinal cord than cortical neurones. In cortical neurones, single-cell RT - PCR showed GluR2 and GluR1 accounted for 91% of all subunits and were expressed together in 67% of neurones, predominantly as the flip variants (78%). GluR2 was detected alone in 24% of neurones. GluR3 and GluR4 were present in only 14 and 29% of neurones, respectively. For spinal cord neurones, GluR4(o) was detected in 81% of neurones, whereas predominantly flop versions of GluR1, 2 and 3 were detected in 38, 13 and 13% of neurones, respectively. These expression patterns are related to the respective pharmacological and mechanistic properties.

Objective The chronic inflammation and progressive joint destruction observed in rheumatoid arthritis (RA) are mediated in part by macrophages. A paucity of apoptosis has been observed in RA synovial tissues, yet the mechanism remains unknown. The present study sought to characterize the expression of Fas, Fas ligand (FasL), and Fas-associated death domain–like interleukin-1β–converting enzyme–inhibitory protein (FLIP), and to quantify the apoptosis induced by agonistic anti-Fas antibody, using mononuclear cells (MNC) isolated from the peripheral blood (PB) and synovial fluid (SF) of RA patients. Methods The expression of Fas, FasL, and FLIP and apoptosis induced by agonistic anti-Fas antibody in MNC from the PB and SF of RA patients were determined by flow cytometry. Immunohistochemistry employing a monospecific anti-FLIP antibody was performed on RA and osteoarthritis (OA) synovial tissue. Results CD14-positive monocyte/macrophages from normal and RA PB and from RA SF expressed equivalent levels of Fas and FasL. Furthermore, unlike the CD14-positive PB monocytes, RA SF monocyte/macrophages were resistant to the addition of agonistic anti-Fas antibody. In contrast, both CD14-positive PB and SF monocyte/macrophages were sensitive to apoptosis mediated by a phosphatidylinositol 3-kinase inhibitor. Intracellular staining of the caspase 8 inhibitor, FLIP, in CD14-positive SF monocyte/macrophages revealed a significant up-regulation of FLIP compared with normal and RA PB monocytes. Immunohistochemical analysis of synovial tissue from RA and OA patients revealed increased FLIP expression in the RA synovial lining compared with the OA synovial lining. Furthermore, FLIP expression was observed in the CD68-positive population in the RA synovial lining. Forced reduction of FLIP by a chemical inhibitor resulted in RA SF macrophage apoptosis that was enhanced by agonistic anti-Fas antibody, indicating that FLIP is necessary for SF macrophage survival. Conclusion These data suggest that up-regulation of FLIP in RA macrophages may account for their persistence in the disease. Thus, the targeted suppression of FLIP may be a potential therapeutic strategy for the amelioration of RA.

Objective. To examine the relationship between apoptosis and the expression of antiapoptotic proteins in the pathogenesis of experimental inflammatory arthritis. Methods. Clinical and histologic assessment of adjuvant-induced arthritis (AIA) was performed over a 42-day period. The induction of apoptosis was measured by TUNEL analysis, and the antiapoptotic proteins, Bcl-2 and FLIP, were examined by immunohistochemistry with the use of monospecific antibodies. The percentage of Bcl-2- and FLIP-positive cells was correlated with histologic markers of AIA. Results. Arthritis developed by day 14 following adjuvant injection. Few TUNEL-positive cells were observed between days 0 and 21, indicating that apoptosis did not occur at these time points. An increase in the number of TUNEL-positive cells was observed at day 28, particularly outside sites of cartilage or bone erosion, which dramatically declined by day 35. Immunohistochemical analyses of Bcl-2 and FLIP revealed that the synovium was positive for Bcl-2 and FLIP on day 0. On day 14, Bcl-2 was present at the sites of early erosions and correlated with the erosion and inflammation scores. FLIP was also highly expressed at sites of erosion and was localized to the pannus starting on day 21. Although TUNEL positivity peaked at day 28, a time point in which Bcl-2 and FLIP were present, the areas that displayed intense positivity for expression of Bcl-2 and FLIP were TUNEL negative. In addition, the number of neutrophils in the synovial lining and pannus significantly decreased from day 28 to day 35, suggesting that the cells undergoing apoptosis were neutrophils. Furthermore, at day 42 when TUNEL-positive cells were absent, Bcl-2 expression was diminished, while FLIP remained highly expressed in the pannus. Conclusion. The overall percentage of TUNEL-positive cells in the ankle was

During inflammatory reactions in the central nervous system (CNS), resident macrophages, the microglia, are exposed to Th1 cell-derived cytokines and pro-apoptotic Fas ligand (FasL). Despite the presence of TNF-alpha and IFN-gamma, both being capable of sensitizing microglia to FasL, apoptosis of microglia is not a hallmark of inflammatory diseases of the CNS. In the present study, TGF-beta is found to counteract the effect of TNF-alpha and IFN-gamma to sensitize microglia to FasL-mediated apoptosis. Resistance to Fas-mediated apoptosis by TGF-beta does not correlate with a down-regulation of Fas expression. As a key inhibitor of Fas-mediated apoptosis, we found expression of the cellular FLICE-inhibitory protein (c-FLIP) to be induced by TGF-beta in resting as well as in activated microglia. Induction of FLIP was found to depend on a mitogen-activated protein kinase kinase (MKK)-dependent pathway as shown by the use of the specific MKK-inhibitor PD98059. The presence of FLIP strongly interfered with FasL-induced activation of caspase-8 and caspase-3 preventing subsequent cell death. The presented data provide the first evidence for a TGF-beta-mediated FLIP in macrophage-like cells and suggest a mode of action for the anti-apoptotic role of TGF-beta in the CNS.