Hollow organosilica beads as reference particles for optical detection of extracellular vesicles.

Essentials Standardization of extracellular vesicle (EV) measurements by flow cytometry needs improvement. Hollow organosilica beads were prepared, characterized, and tested as reference particles. Light scattering properties of hollow beads resemble that of platelet-derived EVs. Hollow beads are ideal reference particles to standardize scatter flow cytometry research on EVs. SUMMARY: Background The concentration of extracellular vesicles (EVs) in body fluids is a promising biomarker for disease, and flow cytometry remains the clinically most applicable method to identify the cellular origin of single EVs in suspension. To compare concentration measurements of EVs between flow cytometers, solid polystyrene reference beads and EVs were distributed in the first ISTH-organized interlaboratory comparison studies. The beads were used to set size gates based on light scatter, and the concentration of EVs was measured within the size gates. However, polystyrene beads lead to false size determination of EVs, owing to the mismatch in refractive index between beads and EVs. Moreover, polystyrene beads gate different EV sizes on different flow cytometers. Objective To prepare, characterize and test hollow organosilica beads (HOBs) as reference beads to set EV size gates in flow cytometry investigations. Methods HOBs were prepared with a hard template sol-gel method, and extensively characterized for morphology, size, and colloidal stability. The applicability of HOBs as reference particles was investigated by flow cytometry with HOBs and platelet-derived EVs. Results HOBs proved to be monodisperse with a homogeneous shell thickness. Two-angle light-scattering measurements by flow cytometry confirmed that HOBs have light-scattering properties similar to those of platelet-derived EVs. Conclusions Because the structure and light-scattering properties HOBs resemble those of EVs, HOBs with a given size will gate EVs of the same size. Therefore, HOBs are ideal reference beads with which to standardize optical measurements of the EV concentration within a predefined size range.

[Iatrogenic meningitis after diagnosis lumbar puncture: 3 cases reports in the paediatric Children's Hospital of Tunis]. / Méningite bactérienne iatrogène après ponction lombaire de diagnostic: à propos de trois cas observés à l'hôpital d'enfants de Tunis.

We have collected cases of iatrogenic meningitis managed in the Children's Hospital of Tunis, between January 1998 and December 2006. Clinical information about each patient were collected, all bacterial samples were investigated in the microbiology laboratory of the hospital. Bacterial isolates were identified according to conventional criteria. In the interval under study, we recorded three cases of iatrogenic meningitis after lumbar puncture. Two cases occurred in newborn admitted for suspicion of neonatal infection and one in a 2-month-old infant admitted for exploration of hyperpyretic convulsion. In all patients, the initial cerebrospinal fluid was normal. All patients developed symptoms of acute meningitis within 72 hours after lumbar puncture; the second cerebrospinal fluid was, then, typical for purulent meningitis. The causal agents isolated in the three cases were Klebsiella pneumoniae, Enterobacter cloacae, and Serratia marcescens, all resistant to beta-lactams by extended spectrum beta-lactamase production. The use of quinolones was required in all cases. Different complications were recorded: hydrocephalus and brain abscess in one case, respiratory and hemodynamic failure managed in the intensive care unit in the second, and brain hygroma in the third case. This study shows high morbidity of iatrogenic meningitis. Simple aseptic precautions undertaken before the procedure of lumbar puncture can prevent such cases. The urgent need for increasing the awareness among medical personnel in hospitals of developing countries cannot be overemphasized.

Apoptosis-inducing factor mediates dopaminergic cell death in response to LPS-induced inflammatory stimulus: evidence in Parkinson's disease patients.

We show that intranigral lipopolysaccharide (LPS) injection, which provokes specific degeneration of DA neurons, induced caspase-3 activation in the rat ventral mesencephalon, which was mostly associated with glial cells. In contrast, nigral DA neurons exhibited AIF nuclear translocation in response to LPS. A significant decrease of the Bcl-2/Bax ratio in nigral tissue after LPS injection was observed. We next developed an in vitro co-culture system with the microglial BV2 and the DA neuronal MN9D murine cell lines. The silencing of caspase-3 or AIF by small interfering RNAs exclusively in the DA MN9D cells demonstrated the key role of AIF in the LPS-induced death of DA cells. In vivo chemical inhibition of caspases and poly(ADP-ribose)polymerase-1, an upstream regulator of AIF release and calpain, proved the central role of the AIF-dependent pathway in LPS-induced nigral DA cell death. We also observed nuclear translocation of AIF in the ventral mesencephalon of Parkinson's disease subjects.

Cracking the death code: apoptosis-related histone modifications.

The degradation and compaction of chromatin are long-standing hallmark features of apoptosis. The histones, chief protein components of chromatin, are subjected to a wide range of post-translational modifications. An increasing body of evidence suggests that combinations of epigenetic histone modifications influence the overall chromatin structure and have clear functional consequences in cellular processes including apoptosis. This review describes the work to date on the post-translational modification of histones during apoptosis, their regulation by enzymatic complexes and discusses the existence of the apoptotic histone code.

Opposing effects of hMOF and SIRT1 on H4K16 acetylation and the sensitivity to the topoisomerase II inhibitor etoposide.

Various inhibitors of histone deacetylase (HDAC) activity can sensitize drug resistant cancer cells to chemotherapeutic agents. However, the mechanisms underlying such effects of distinct HDAC inhibitors (HDACi) remain poorly understood. Here we show that both the HDACi trichostatin A and valproic acid induced a sensitization of multidrug-resistant cancer cells to the topoisomerase II inhibitor etoposide/VP16. This effect was associated with increased acetylation of certain lysines on histones H3 and H4, including lysine 16 on histone H4 (H4K16). Overexpression of the histone acetyltransferase hMOF, known to target H4K16, was sufficient to mimic HDACi treatment on sensitization and H4K16 acetylation, and importantly, small-interfering RNA (siRNA)-mediated knockdown of hMOF abolished the HDACi-mediated sensitizing effects as well as the increase in H4K16 acetylation. Conversely, siRNA-mediated knockdown of the H4K16 deacetylase SIRT1 mimicked HDACi treatment whereas overexpression of SIRT1 abolished H4K16 acetylation and significantly reduced the sensitizing effects of HDACi. Interestingly, the effects of hMOF on H4K16 acetylation and sensitization to the topoisomerase II inhibitor could be directly counteracted by exogenous expression of increasing amounts of SIRT1 and vice versa. Our study results suggest that hMOF and SIRT1 activities are critical parameters in HDACi-mediated sensitization of multidrug-resistant cancer cells to topoisomerase II inhibitor and increased H4K16 acetylation.

LPS differentially affects vasoconstrictor responses: a potential role for RGS16?

The profound hypotension in septic shock patients is difficult to treat as it is accompanied by depressed constrictor responses to alpha1-adrenoceptor agonists. Bacterial lipopolysaccharide (LPS) is the main trigger for most of the cardiovascular alterations occurring in septic shock. In this study we investigated the effects of LPS exposure on vascular contractility in general and the role of Regulator of G protein Signalling (RGS) proteins in the LPS-induced vascular alterations. Exposure of rat aortic rings to various LPS concentrations (3, 10, 30 microg/ml) for 22 hours differentially affected agonist-induced contractile responses at four distinct G-protein coupled receptors (alpha1-adrenoceptors, angiotensin II, serotonin and endothelin-1 receptors). While the endothelin-1-induced contraction was unaffected by LPS pre-treatment, phenylephrine- and angiotensin II-induced contraction were significantly reduced whereas serotonin-induced contraction was significantly enhanced. Concomitantly, LPS treatment increased the RGS16 mRNA expression both in aortic rings and cultured vascular smooth muscle cells (VSMCs) but not that of RGS2, RGS3, RGS4 or RGS5. The significant increase in RGS16 mRNA expression in VSMCs by LPS was time- and concentration-dependent but independent of increased inducible NO synthase (iNOS) activity. The changes in RGS16 mRNA might contribute to the differential regulation of the contractile responses to vasoconstrictors upon LPS exposure.

Diversos efectos vasoconstrictores del LPS: un posible papel para RGS16? / LPS differentially affects vasoconstrictor responses: a potential role for RGS16?

The profound hypotension in septic shock patients is difficult to treat as it isaccompanied by depressed constrictor responses to α1-adrenoceptor agonists. Bacteriallipopolysaccharide (LPS) is the main trigger for most of the cardiovascular alterationsoccurring in septic shock. In this study we investigated the effects of LPSexposure on vascular contractility in general and the role of Regulator of G proteinSignalling (RGS) proteins in the LPS-induced vascular alterations. Exposure of rataortic rings to various LPS concentrations (3, 10, 30 μg/ml) for 22 hours differentiallyaffected agonist-induced contractile responses at four distinct G-protein coupledreceptors (α1-adrenoceptors, angiotensin II, serotonin and endothelin-1 receptors).While the endothelin-1-induced contraction was unaffected by LPS pre-treatment,phenylephrine- and angiotensin II-induced contraction were significantly reducedwhereas serotonin-induced contraction was significantly enhanced. Concomitantly,LPS treatment increased the RGS16 mRNA expression both in aortic rings and culturedvascular smooth muscle cells (VSMCs) but not that of RGS2, RGS3, RGS4 orRGS5. The significant increase in RGS16 mRNA expression in VSMCs by LPS wastime- and concentration-dependent but independent of increased inducible NO synthase(iNOS) activity. The changes in RGS16 mRNA might contribute to the differentialregulation of the contractile responses to vasoconstrictors upon LPS exposure(AU)

El lipopolisacárido bacteriano LPS estáimplicado en la mayor parte de las alteracionescardiovasculares propias del shock séptico. Seinvestiga en este trabajo sobre los efectos de laexposición al LPS en la contractilidad vascularen general y sobre el papel de las proteínasreguladoras de la señalización de proteínas G(RGS) en las alteraciones vasculares inducidaspor el LPS. La exposición (22 h) de anillos aórticosde rata a diversas concentraciones de LPS(3, 10, 30 μg/ml) afecta de forma diferencial lasrespuestas contráctiles inducidas por la activaciónde 4 diferentes receptores acoplados (α1-adrenorreceptores, de angiotensinaII, de serotonina y ET-1 de endotelina).Asi, el pretratamiento con LPS no afecta lacontracción inducida por endotelina, mientrasque reduce la de fenilefrina y angiotensina II eincrementa la de serotonina. Además, el tratamientocon LPS aumenta la expresión deRNAm de RGS16 tanto en anillos aórticoscomo en células VSMC, pero no de RNAm deRGS2, RGS3, RGS4 y RGS5. Los cambios deRNAm de RGS16 podrían contribuir a laregulación diferencial de las respuestas contráctilesa los vasoconstrictores en presencia deLPS(AU)

[Antimicrobial susceptibility and serotype distribution of streptococcus pneumoniae isolates in children in Tunis]. / Sensibilité aux antibiotiques et distribution des sérotypes des souches de Streptococcus pneumoniae isolées chez l'enfant à Tunis.

Streptococcus pneumoniae is a major causative agent of severe infectious diseases. More than 90 pneumococcal serotypes are known, although most invasive and noninvasive diseases are associated with a much smaller number of serotypes. The aim of this study was to determine the antimicrobial susceptibility of S. pneumoniae isolates in children, the distribution of serogroups and serotypes, and the coverage by the serotypes included in the seven-valent pneumococcal conjugate vaccine toward pneumococcal disease. This study investigated 210 nonrepetitive isolates of S. pneumoniae isolated between 1998 and 2004. Antimicrobial susceptibility was tested using the disk diffusion method as determined by the CA-SFM guidelines. Penicillin susceptibility was determined using the oxacillin 5-microg disk screening test. The MICs of penicillin G, amoxicillin, and cefotaxime were determined using the E-test (ABBIODISK). Serotype was determined using rapid latex agglutination (Pneumotest Latex) and the capsular reaction test used antisera from the Staten Serum Institute. The evaluation of susceptibility to ss-lactamins showed that 52.8% of the strains were penicillin non susceptible strains (PNSs), 16.6% had decreased susceptibility to amoxicillin, and 8.5% to cefotaxime. Among noninvasive isolates, 55.2% were PNSs and 50.4% were invasive PNSs. The PNS strains were more frequently resistant to other antibiotics, with 68.4% resistance to erythromycin, 44.1% to trimethoprim-sulfamethoxazole, and 9.9% to chloramphenicol versus 32.3, 11.1, and 1%, respectively, in penicillin-susceptible strains. The predominant serogroups/serotypes of our study were 14 (22%), 23 (14.3%), 19 (11.9%), and 4 (8.5%). The study of the vaccine serotype distribution showed that the theoretical vaccinal coverage of the seven valent vaccines was 62.8% for all the isolates, 55.2% for the invasive isolates, and 67.9% for the PNSs.

Overcoming chemoresistance of non-small cell lung carcinoma through restoration of an AIF-dependent apoptotic pathway.

Non-small cell lung carcinomas (NSCLCs) are typically resistant against apoptosis induced by standard chemotherapy. We evaluated the effects of the two potential antitumor agents of the lamellarin class on a highly apoptosis-resistant NSCLC cell line. Both the marine alkaloid lamellarin-D and its synthetic amino derivative PM031379 induced the activation of Bax, the mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF), as well as the activation of caspase-3. However, only PM031379 triggered cell death and sign of nuclear apoptosis coupled to the nuclear translocation of AIF. Depletion of AIF with small interfering RNA or microinjection of a neutralizing anti-AIF antibody largely prevented PM031379-induced cytotoxicity, underscoring the essential contribution of AIF to NSCLC killing. Using NSCLC cells lacking mitochondrial DNA, we showed that the generation of mitochondrial reactive oxygen species (ROS) was crucial for the PM031379-induced translocation of AIF to the nucleus and subsequently cell death. Pretreatment of NSCLC cells with menadione, a mitochondrial ROS generator, was able to restore the deficient chemotherapy-induced apoptosis of NSCLC cells. Altogether, these data suggest that mitochondrial ROS generation is crucial for overriding the chemoresistance of NSCLC cells. Moreover, this study delineates the unique mechanism of action of lamellarins as potential anticancer agents.

Combinatorial action of the HDAC inhibitor trichostatin A and etoposide induces caspase-mediated AIF-dependent apoptotic cell death in non-small cell lung carcinoma cells.

Commonly used regimens in cancer therapy rely on the induction of apoptotic cell death, and drug resistance can be attributed, at least in part, to a disabled apoptotic program. Non-small cell lung carcinomas (NSCLC), exhibit an intrinsic resistance to chemotherapy. Here, we show that co-treatment with etoposide (VP16) and the pan-histone deacetylase (HDAC) inhibitor trichostatin A (TSA), but not valproic acid (VPA), induced apoptotic cell death in drug-resistant NSCLC cells. Co-treatment, but not single treatment, with VP16 and TSA induced apoptosis in a caspase-dependent manner accompanied by a crucial decrease in Bcl-xL expression allowing Bax activation and subsequent initiation of the apoptosis inducing factor (AIF)-dependent death pathway. Importantly, AIF proved to be required for the effects of TSA/VP16 as RNA knockdown of AIF resulted in a complete abolishment of TSA/VP16-induced apoptotic cell death in drug-resistant NSCLC cells. Our results thus provide evidence for the requirement of both caspase-dependent and caspase-independent apoptotic pathways in TSA/VP16-mediated death of drug-resistant NSCLC cells, and extend previous suggestions that HDAC inhibitors in combination with conventional chemotherapeutic drugs could be valuable in the treatment of NSCLC cancer and other malignancies in which Bcl-xL is overexpressed.

The cell cycle inhibitor p57(Kip2) promotes cell death via the mitochondrial apoptotic pathway.

The p57(Kip2) gene belongs to the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors and has been suggested to be a tumor suppressor gene, being inactivated in various types of human cancers. However, little is known concerning p57(Kip2) possible interplay with the apoptotic cell death machinery and its possible implication for cancer. Here, we report that selective p57(Kip2) expression sensitizes cancer cells to apoptotic agents such as cisplatin, etoposide and staurosporine (STS) via a mechanism, which does not require p57(Kip2)-mediated inhibition of CDK. Translocation of p57(Kip2) to mitochondria occurs within 20 min after STS application. In fact, p57(Kip2) primarily promotes the intrinsic apoptotic pathways, favoring Bax activation and loss of mitochondrial transmembrane potential, consequent release of cytochrome-c into cytosol, caspase-9 and caspase-3 activation. In accordance, Bcl2 overexpression or voltage-dependent anion channel (VDAC) inhibition is able to inhibit p57(Kip2) cell death promoting effect. Thus, in addition to its established function in control of proliferation, these results reveal a mechanism whereby p57(Kip2) influences the mitochondrial apoptotic cell death pathway in cancer cells.

Hepatitis B genotypes, precore and core promoter mutants circulating in Tunisia.

Hepatitis B virus (HBV) is characterized by genetic heterogeneity, including genotypes and mutations. Eight genotypes (A-H) have been identified throughout the world with a characteristic geographical distribution. Previous studies also suggest that the viral genotypes may correlate with differences in clinical features of the infection. Two types of mutations were particularly described, precore and basal promoter mutations; they may play an important role in the clinical outcome of HBV infection. The aim of this study was to investigate the prevalence of HBV genotypes and HBV variants in Tunisia, and their eventual association with severity of liver disease. Using a molecular method, HBV genotypes, precore and basal core promoter mutations were determined in 56 asymptomatic carriers and in 82 patients with histologically verified chronic liver disease and hepatocellular carcinoma (HCC). Three genotypes (D, A, and E) were detected; the prevalence was 80%, 8%, and 9%, respectively. No significant difference was observed for genotype D with clinical status. HBV mutants were detected in 93% of cases, precore mutants were the most prevalent. Basal core promoter mutants were observed in 61% of cases, they were frequently characterized by a double mutation in 1762 and 1764. Co-infection by these two types of mutants was detected in 50% of cases. Genotype D was the most prevalent HBV genotype in Tunisia. High circulation of precore and basal core promoter mutants are common in chronic hepatitis B infection in Tunisia.

Induction of genotoxic and cytotoxic damage by aclarubicin, a dual topoisomerase inhibitor.

The anthracycline aclarubicin (ACLA) is an intercalative antibiotic and antineoplastic agent that efficiently binds to DNA, leading to a secondary inhibition of the catalytic activity of topoisomerase II (topo II) on DNA. Besides this activity, ACLA has been reported to exert a concomitant poisoning effect on topo I, in a fashion similar to that of the antitumor drug camptothecin and its derivatives. As a consequence of this dual (topo II catalytic inhibiting/topo I poisoning) activity of ACLA, the picture is somewhat confusing with regards to DNA damage and cytotoxicity. We studied the capacity of ACLA to induce catalytic inhibition of topo II as well as cytotoxic effects and DNA damage in cultured Chinese hamster V79 cells and their radiosensitive counterparts irs-2. The ultimate purpose was to find out whether differences could be observed between the two cell lines in their response to ACLA, as has been widely reported for radiosensitive cells treated with topo poisons. Our results seem to agree with the view that the radiosensitive irs-2 cells appear as hypersensitive ACLA as compared with radiation repair-proficient V79 cells. The recovery after ACLA treatment was also followed-up, and the irs-2 mutant was found to be less proficient than V79 to repair DNA strand breaks induced by ACLA.

DNA strand breaks induced by the anti-topoisomerase II bis-dioxopiperazine ICRF-193.

The bis-dioxopiperazine ICRF-193 has long time been considered as a pure topoisomerase II catalytic inhibitor able to exert its inhibitory effect on the enzyme without stabilization of the so-called cleavable complex formed by DNA covalently bound to topoisomerase II. In recent years, however, this concept has been challenged, as a number of reports have shown that ICRF-193 really "poisons" the enzyme, most likely through a different mechanism from that shown by the classical topoisomerase II poisons used in cancer chemotherapy. In the present investigation, we have carried out a study of the capacity of ICRF-193 to induce DNA strand breaks, as classical poisons do, in cultured V79 and irs-2 Chinese hamster lung fibroblasts using the comet assay and pulsed-field gel electrophoresis (PFGE). Our results clearly show that ICRF-193 readily induces breakage in DNA through a mechanism as yet poorly understood.

Physicians' perceptions of menopause and prescribing practices in Morocco.

OBJECTIVE: To investigate the prescribing practices of Moroccan physicians around menopause. METHODS: A survey was carried out on a representative sample of physicians in the capital city Rabat. The sample included general practitioners, gynecologists, cardiologists and rheumatologists, practicing in both public and private facilities. The instrument consisted of close- and open-ended questions about the socio-demographic characteristics of physicians, their patient population, their prescribing practices, and their perceptions of menopause and the different medical approaches to managing the symptoms and risks associated with it. RESULTS: Most of the physicians interviewed are positively inclined towards the notion of prevention and in favor of hormonal treatment, and approximately half report that they have prescribed hormone therapy. Gynecologists and male physicians prescribe hormones more frequently, as well as physicians who are at private facilities. These findings are discussed in relation to the physicians' perceptions of the menopause transition. CONCLUSION: There are considerable variations in prescribing practices and perceptions of menopause among Moroccan physicians.