Early nucleolar disorganization in Dictyostelium cell death.

Cell death occurs in all eukaryotes, but it is still not known whether some core steps of the cell death process are conserved. We investigated this using the protist Dictyostelium. The dissection of events in Dictyostelium vacuolar developmental cell death was facilitated by the sequential requirement for two distinct exogenous signals. An initial exogenous signal (starvation and cAMP) recruited some cells into clumps. Only within these clumps did subsequent cell death events take place. Contrary to our expectations, already this initial signal provoked nucleolar disorganization and irreversible inhibition of rRNA and DNA synthesis, reflecting marked cell dysfunction. The initial signal also primed clumped cells to respond to a second exogenous signal (differentiation-inducing factor-1 or c-di-GMP), which led to vacuolization and synthesis of cellulose encasings. Thus, the latter prominent hallmarks of developmental cell death were induced separately from initial cell dysfunction. We propose that (1) in Dictyostelium vacuolization and cellulose encasings are late, organism-specific, hallmarks, and (2) on the basis of our observations in this protist and of similar previous observations in some cases of mammalian cell death, early inhibition of rRNA synthesis and nucleolar disorganization may be conserved in some eukaryotes to usher in developmental cell death.

Effectiveness and tolerability of pegylated interferon alfa-2b in combination with ribavirin for treatment of chronic hepatitis C: the PegIntrust study.

BACKGROUND AND STUDY AIMS: Large international clinical trials conducted in the past 5 years rapidly improved the treatment of chronic hepatitis C; however, it is unclear whether the advances seen in clinical trials are being paralleled by similar improvements in routine clinical practice. PegIntrust is a Belgian community-based trial evaluating the sustained virological response. PATIENTS AND METHODS: Observational study of 219 patients receiving pegylated interferon alfa-2b (1.5 microg/kg/wk) and weight-based ribavirin (800-1200 mg/day) for 48 weeks. Primary study end point was sustained virological response (SVR), defined as undetectable HCV RNA 6 months after the completion of treatment. RESULTS: In total, 108 patients (49.3 %) had undetectable HCV RNA at the end of therapy, 91 (41.6%) attaining SVR. Of the 111 patients without an end-of-treatment response, 28 were non-responders, and 21 had virological breakthrough. In total, 134 patients attained early virological response (EVR); 88 (65.7%) of those patients attained SVR. In contrast, 82 (96.5 %) of the 85 patients who did not attain EVR also did not attain SVR. Age, fibrosis score and baseline viral load were identified as important predictors of treatment outcome. The most frequently reported serious adverse events resulting in treatment discontinuation were anemia (n = 10), fatigue/asthenia/malaise (n = 6) and fever (n = 3). CONCLUSION: Our data indicate that treatment of chronic hepatitis C with PEG-IFN alfa-2b plus weight-based ribavirin results in favourable treatment outcomes in a Belgian cohort of patients treated in community-based clinical practice.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes.

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009.

Different types of cell death are often defined by morphological criteria, without a clear reference to precise biochemical mechanisms. The Nomenclature Committee on Cell Death (NCCD) proposes unified criteria for the definition of cell death and of its different morphologies, while formulating several caveats against the misuse of words and concepts that slow down progress in the area of cell death research. Authors, reviewers and editors of scientific periodicals are invited to abandon expressions like 'percentage apoptosis' and to replace them with more accurate descriptions of the biochemical and cellular parameters that are actually measured. Moreover, at the present stage, it should be accepted that caspase-independent mechanisms can cooperate with (or substitute for) caspases in the execution of lethal signaling pathways and that 'autophagic cell death' is a type of cell death occurring together with (but not necessarily by) autophagic vacuolization. This study details the 2009 recommendations of the NCCD on the use of cell death-related terminology including 'entosis', 'mitotic catastrophe', 'necrosis', 'necroptosis' and 'pyroptosis'.

Autophagic or necrotic cell death triggered by distinct motifs of the differentiation factor DIF-1.

Autophagic or necrotic cell death (ACD and NCD, respectively), studied in the model organism Dictyostelium which offers unique advantages, require triggering by the same differentiation-inducing factor DIF-1. To initiate these two types of cell death, does DIF-1 act through only one or through two distinct recognition structures? Such distinct structures may recognize distinct motifs of DIF-1. To test this albeit indirectly, DIF-1 was modified at one or two of several positions, and the corresponding derivatives were tested for their abilities to induce ACD or NCD. The results strongly indicated that distinct biochemical motifs of DIF-1 were required to trigger ACD or NCD, and that these motifs were separately recognized at the onset of ACD or NCD. In addition, both ACD and NCD were induced more efficiently by DIF-1 than by either its precursors or its immediate catabolite. These results showed an unexpected relation between a differentiation factor, the cellular structures that recognize it, the cell death types it can trigger and the metabolic state of the cell. The latter seems to guide the choice of the signaling pathway to cell death, which in turn imposes the cell death type and the recognition pattern of the differentiation factor.

A necrotic cell death model in a protist.

While necrotic cell death is attracting considerable interest, its molecular bases are still poorly understood. Investigations in simple biological models, taken for instance outside the animal kingdom, may benefit from less interference from other cell death mechanisms and from better experimental accessibility, while providing phylogenetic information. Can necrotic cell death occur outside the animal kingdom? In the protist Dictyostelium, developmental stimuli induced in an autophagy mutant a stereotyped sequence of events characteristic of necrotic cell death. This sequence included swift mitochondrial uncoupling with mitochondrial 2',7'-dichlorofluorescein diacetate fluorescence, ATP depletion and increased oxygen consumption. This was followed by perinuclear clustering of dilated mitochondria. Rapid plasma membrane rupture then occurred, which was evidenced by time-lapse videos and quantified by FACS. Of additional interest, developmental stimuli and classical mitochondrial uncouplers triggered a similar sequence of events, and exogenous glucose delayed plasma membrane rupture in a nonglycolytic manner. The occurrence of necrotic cell death in the protist Dictyostelium (1) provides a very favorable model for further study of this type of cell death, and (2) strongly suggests that the mechanism underlying necrotic cell death was present in an ancestor common to the Amoebozoa protists and to animals and has been conserved in evolution.

Redundant cell death mechanisms as relics and backups.

Here we review recent observations indicating the existence of redundant cell death mechanisms. We speculate that this redundancy reflects a particular evolutionary history for cellular demise. Autophagic or apoptotic elements might have been added to a primordial death mechanism, initially improving cell dismantling and later acquiring the ability to act themselves as death effectors. The resulting redundancy of cell death mechanisms has pathophysiological implications.

Reversible myopathy during successful treatment with pegylated interferon and ribavirin for acute hepatitis C.

There is no standard approved treatment for acute hepatitis C and the combination of pegylated interferon-alpha and ribavirin, currently recognized as the standard of care for chronic hepatitis C, has not been evaluated for acute hepatitis C. Adverse events induced by interferon therapy are numerous but myopathy is rare and has not been described with the use of pegylated interferon-alpha. We report the case of a 33-year-old Caucasian man who was successfully treated for acute hepatitis C with the combination of pegylated interferon-alpha2b and ribavirin, and who during treatment developed myopathy which proved reversible.

Hypotonic cell swelling stimulates permeability to cAMP in a rat colonic cell line.

This study characterized the membrane permeability to cAMP in a cell line derived from the rat colon (CC531(mdr+)) by comparison of fluxes of 3H-cAMP, 3H-8-bromo-cAMP, 3H-taurine, 3H-adenosine and 3H-5'AMP under various experimental conditions including cell membrane depolarization and hypotonic cell swelling. Cell volume was modified by changing the osmolality and composition of the extracellular medium. Incubation in iso- and hypotonic KCl media induced graded increases in cell volume and stable activation of volume-sensitive channels that was reflected in an increased efflux of 3H-taurine. Incubation in hypotonic KCl solution also enhanced the efflux of 3H-8-Br-cAMP (a non-hydrolysable analogue of cAMP). Both the efflux of 3H-taurine and of 3H-8-Br-cAMP were inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB, 100 microM) suggesting the involvement of volume-sensitive anion channels. To gain further insight into the route mediating cAMP permeability, the uptakes of 3H-cAMP, 3H-8-Br-cAMP and 3H-taurine were determined over short (5-min) periods. Uptakes of these substrates demonstrated close similarities: comparable increases were observed that correlated with the increases in cell volume in iso- and hypoosmotic KCl media; they were inhibited strongly by NPPB (100 microM) and metabolic inhibitors (deoxyglucose, 20 mM together with the mitochondrial uncoupler carbonylcyanide p-(trifluoromethoxy)phenylhydrazone, FCCP, 10 microM) while barely reduced by dipyridamole (100 microM) and they were not affected by adenosine (1 mM). In contrast, the uptakes of 3H-adenosine and 3H-5'AMP had strikingly different properties; they were insensitive to cell swelling; barely inhibited by NPPB (100 microM) and metabolic inhibitors (deoxyglucose and FCCP) while strongly reduced by dipyridamole (100 micro M). Unlike the uptakes of 3H-cAMP, 3H-8-Br-cAMP and 3H-taurine, the uptakes of 3H-adenosine and 3H-5'AMP were reduced in Na(+)-free media, suggesting the presence in this cell line of two different adenosine carriers, one sodium-dependent and one sodium-independent. Taken together the present data show that in this rat colonic cell line, cAMP permeability is increased by cell swelling in hypotonic KCl medium and inhibited by NPPB and metabolic inhibitors. The similarity of these characteristics to those of taurine permeability suggests the involvement of a volume-sensitive anion pathway.

Evaluation of stool antigen detection for diagnosis of Helicobacter pylori infection in adults.

OBJECTIVE: The aims of this study were to evaluate the performance of Helicobacter pylori stool antigen test in the diagnosis of H. pylori infection. METHOD: The study included 63 out patients attending the ULB-Lothier Clinic between January 1 and July 31, 2002. They underwent an upper endoscopy, as well as biopsies for histological examination and for culture of H. pylori. Stool samples of these patients were collected either the day of the endoscopy or within 24 hours and tested for H. pylori antigen (HpSA Test) RESULTS: The mean age of study patients included 29 men and 34 women was 51(+/- :16) years. H. pylori infection was detected in 29 cases (46 %) by culture and histology, and in 31 cases (49.2 %) by detection of the antigen in the feces. In 27 patients, all methods were positive whereas 5 in they provided discrepant results. Compared to the reference methods (culture and histology), the HpSA test had a sensitivity of 96.5% and a specificity of 91.2%. PPV of 90.3% and NPV of 96.8%. CONCLUSION: The good correlation found between the results of the HpSA test and the methods based on endoscopy supports its use as an alternative to invasive methods of diagnosis of H. pylori infection and therapeutic follow-up.

Endoscopic histoacryl obliteration vs. propranolol in the prevention of esophagogastric variceal rebleeding: a randomized trial.

BACKGROUND AND STUDY AIMS: The obliteration of esophageal and/or gastric varices using Histoacryl is highly effective in controlling active bleeding. However, it is not known whether repeated injections are useful for the long-term eradication of esophagogastric varices. The aim of the study was to compare endoscopic Histoacryl obliteration with propranolol in the secondary prevention of esophagogastric variceal bleeding. PATIENTS AND METHODS: Between August 1995 and February 1999, 41 patients with a first bleeding from esophageal (n = 31) or gastric (n = 10) varices were included in the study. After primary hemostasis with obliteration using Histoacryl, patients were randomly allocated either to undergo complete Histoacryl obliteration of the remaining varices (group A, n = 21) or to long-term propranolol administration (group B, n = 20), for the prevention of rebleeding. RESULTS: The two groups were well matched for age, sex, etiology of cirrhosis, Child-Pugh score, renal function, and infection at the time of admission. The median follow-up was 31.9 months (4.8 - 74.7) for group A and 23.2 months (3.0 - 70.0) for group B. Initial hemostasis was achieved in 40/41 patients (97 %). No significant difference was observed between groups A and B with regard to the incidence of early rebleeding (during the first 6 weeks; 5/21 and 3/20), bleeding-related deaths by 6 weeks (3/21 and 6/20), long-term rebleeding (11/21 and 5/20), or overall number of deaths (9/21 and 9/20). The incidence of complications was higher in group A (10/21) than group B (2/20) (P < 0.03). CONCLUSIONS: Repeated injections of Histoacryl with the aim of eradicating esophagogastric varices are associated with more complications compared with beta-blocker administration, with similar results in terms of rebleeding rate and survival in the long term.

Individual variations in the correlation between erythemal threshold, UV-induced DNA damage and sun-burn cell formation.

A linear correlation between erythema intensity and DNA damage upon exposure to UV has not been firmly established. Many of the deleterious effects of UV exposure do occur after exposure to suberythemal doses. After DNA damage, cells undergo DNA repair. It is commonly accepted that when the burden of damage is beyond the repair capacities, the cell undergoes programmed cell death or apoptosis. The aim of this study is to quantify the amount of UV-induced DNA damage (estimated via the measurement of DNA repair or unscheduled DNA synthesis or UDS) and cellular damage (estimated via the determination of the density of sunburn cells or SBC). If DNA damage and erythema are correlated, similar intensity of UDS and similar density of SBC should be found in volunteers irradiated with a UV dose equal to two minimal erythema doses (MED). Our results show that in 15 different individuals the same relative dose (2 MEDs) provokes UDS values, which vary within a factor of 4. An even larger variability affects SBC counts after the same relative dose. When DNA damage or SBC are plotted versus the absolute dose (i.e. the dose expressed in J/m(2)), there is a rough correlation (with several exceptions) between dose and extent of UDS and SBC counts. It seems possible to divide the volunteers into two subpopulations with different susceptibilities to UV damage. It is well known that UDS and SBC measurements are often affected by large experimental indeterminacy, yet, the analysis of our results makes it plausible to suggest that for the triggering of erythema, a common threshold value for DNA damage or for SBC count are not to be found. In conclusion, the erythema response seems to be loosely correlated with DNA damage. This suggests that the protection offered by the sunscreens against DNA damage, the molecular basis of UV-induced mutagenesis, might not be related to the sun protection factor (SPF) indicated on the label of sunscreens, which is evaluated using the erythema as an endpoint.

Inhibition of basolateral cAMP permeability in the toad urinary bladder.

1. The effect of sulphonylurea drugs on hydrosmotic flow across toad urinary bladder epithelium was re-evaluated in the present study. Glibenclamide, added to the basolateral medium, significantly enhanced the osmotic flow induced by low doses of antidiuretic hormone (ADH) or forskolin (FK), while it inhibited the effect of exogenous cyclic adenosine monophosphate (cAMP) or its non-hydrolysable bromo derivative, 8-Br-cAMP, added to the basolateral medium. These opposite effects of glibenclamide on the transepithelial osmotic flow can be explained by a reduction of cAMP permeability across the basolateral membrane of the epithelium. The decrease in cAMP permeability leads, according to the direction of the cAMP gradient, to firstly an enhanced osmotic flow when cAMP is generated intracellularly by addition of ADH and FK, glibenclamide reducing cAMP exit from the cell, and secondly a decreased osmotic flow in response to cAMP (and 8-Br-cAMP) added to the basolateral medium, glibenclamide inhibiting, in this case, their entry into the cell. 2. The demonstration that glibenclamide actually inhibits the basolateral cAMP permeability rests on the fact that firstly it decreases the release of cAMP into the basolateral medium by about 40 %, at each concentration of ADH or forskolin tested, secondly it increases the cAMP content of paired hemibladders incubated in the presence of ADH or FK, when intracellular degradation was prevented by phosphodiesterase inhibition, and thirdly it decreases also the uptake of basolateral 8-Br-[3H]cAMP into paired toad hemibladders. 3. Taken together, the present data demonstrate that glibenclamide inhibits the toad urinary bladder basolateral membrane permeability to cAMP, most probably by a direct interaction with a membrane protein not yet indentified but distinct from the sulphonylurea receptor.

Signal transduction. FasL binds preassembled Fas.

The binding of a ligand to its receptor has always been viewed as the trigger for signal transduction to ensue. However, as Golstein explains in his Perspective, new findings (Chan et al. and Siegel et al.) suggest that the Fas receptor preassembles into trimers without the help of its ligand, and that this preassembly conditions ligand binding, and thus subsequent signal transduction of a death signal.

Interdigital cell death can occur through a necrotic and caspase-independent pathway.

Programmed cell death in animals is usually associated with apoptotic morphology and requires caspase activation. Necrosis and caspase-independent cell death have been reported, but mostly in experimental conditions that lead some to question their existence it in vivo. Loss of interdigital cells in the mouse embryo, a paradigm of cell death during development [1], is known to include an apoptotic [2] and caspase-dependent [3] [4] mechanism. Here, we report that, when caspase activity was inhibited using drugs or when apoptosis was prevented genetically (using Hammertoe mutant mice, or mice homozygous for a mutation in the gene encoding APAF-1, a caspase-activating adaptor protein), interdigital cell death still occurred. This cell death was negative for the terminal-deoxynucleotidyl-mediated dUTP nick end-labelling (TUNEL) assay and there was no overall cell condensation. At the electron microscopy level, peculiar 'mottled' chromatin alterations and marked mitochondrial and membrane lesions, suggestive of classical necrotic cell death, were observed with no detectable phagocytosis and no local inflammatory response. Thus, in this developmental context, although caspase activity confers cell death with an apoptotic morphotype, in the absence of caspase activity an underlying mechanism independent of known caspases can also confer cell death, but with a necrotic morphotype. This cell death can go undetected when using apoptosis-specific methodology, and cannot be blocked by agents that act on caspases.