Campylobacter jejuni cell lysates differently target mitochondria and lysosomes on HeLa cells.

Campylobacter jejuni is the most common cause of bacterial gastroenteritis in humans. The synthesis of cytolethal distending toxin appears essential in the infection process. In this work we evaluated the sequence of lethal events in HeLa cells exposed to cell lysates of two distinct strains, C. jejuni ATCC 33291 and C. jejuni ISS3. C. jejuni cell lysates (CCLys) were added to HeLa cell monolayers which were analysed to detect DNA content, death features, bcl-2 and p53 status, mitochondria/lysosomes network and finally, CD54 and CD59 alterations, compared to cell lysates of C. jejuni 11168H cdtA mutant. We found mitochondria and lysosomes differently targeted by these bacterial lysates. Death, consistent with apoptosis for C. jejuni ATCC 33291 lysate, occurred in a slow way (>48 h); concomitantly HeLa cells increase their endolysosomal compartment, as a consequence of toxin internalization besides a simultaneous and partial lysosomal destabilization. C. jejuni CCLys induces death in HeLa cells mainly via a caspase-dependent mechanism although a p53 lysosomal pathway (also caspase-independent) seems to appear in addition. In C. jejuni ISS3-treated cells, the p53-mediated oxidative degradation of mitochondrial components seems to be lost, inducing the deepest lysosomal alterations. Furthermore, CD59 considerably decreases, suggesting both a degradation or internalisation pathway. CCLys-treated HeLa cells increase CD54 expression on their surface, because of the action of lysate as its double feature of toxin and bacterial peptide. In conclusion, we revealed that C. jejuni CCLys-treated HeLa cells displayed different features, depending on the particular strain.

When nitrosative stress hits the endoplasmic reticulum: Possible implications in oxLDL/oxysterols-induced endothelial dysfunction.

Oxidized LDL (oxLDL) and oxysterols are known to play a crucial role in endothelial dysfunction (ED) by inducing endoplasmic reticulum stress (ERS), inflammation, and apoptosis. However, the precise molecular mechanisms underlying these pathophysiological processes remain incompletely understood. Emerging evidence strongly implicates excessive nitric oxide (NO) production in the progression of various pathological conditions. The accumulation of reactive nitrogen species (RNS) leading to nitrosative stress (NSS) and aberrant protein S-nitrosylation contribute to NO toxicity. Studies have highlighted the involvement of NSS and S-nitrosylation in perturbing ER signaling through the modification of ER sensors and resident isomerases in neurons. This review focuses on the existing evidence that strongly associates NO with ERS and the possible implications in the context of ED induced by oxLDL and oxysterols. The potential effects of perturbed NO synthesis on signaling effectors linking NSS with ERS in endothelial cells are discussed to provide a conceptual framework for further investigations and the development of novel therapeutic strategies targeting ED.

Endothelial cells, endoplasmic reticulum stress and oxysterols.

Oxysterols are bioactive lipids that act as regulators of lipid metabolism, inflammation, cell viability and are involved in several diseases, including atherosclerosis. Mounting evidence linked the atherosclerosis to endothelium dysfunction; in fact, the endothelium regulates the vascular system with roles in processes such as hemostasis, cell cholesterol, hormone trafficking, signal transduction and inflammation. Several papers shed light the ability of oxysterols to induce apoptosis in different cell lines including endothelial cells. Apoptotic endothelial cell and endothelial denudation may constitute a critical step in the transition to plaque erosion and vessel thrombosis, so preventing the endothelial damaged has garnered considerable attention as a novel means of treating atherosclerosis. Endoplasmic reticulum (ER) is the site where the proteins are synthetized and folded and is necessary for most cellular activity; perturbations of ER homeostasis leads to a condition known as endoplasmic reticulum stress. This condition evokes the unfolded protein response (UPR) an adaptive pathway that aims to restore ER homeostasis. Mounting evidence suggests that chronic activation of UPR leads to cell dysfunction and death and recently has been implicated in pathogenesis of endothelial dysfunction. Autophagy is an essential catabolic mechanism that delivers misfolded proteins and damaged organelles to the lysosome for degradation, maintaining basal levels of autophagic activity it is critical for cell survival. Several evidence suggests that persistent ER stress often results in stimulation of autophagic activities, likely as a compensatory mechanism to relieve ER stress and consequently cell death. In this review, we summarize evidence for the effect of oxysterols on endothelial cells, especially focusing on oxysterols-mediated induction of endoplasmic reticulum stress.

Prostate-specific antigen synthesis and secretion by human placenta: a physiological kallikrein source during pregnancy.

Prostate-specific antigen (PSA), a kallikrein-like serine protease until recently thought to be prostate specific, has been demonstrated in various nonprostatic tissues and body fluids. PSA has been also found in human endometrium and amniotic fluids, even if the significance of this novel expression is unclear. In this study, we have demonstrated by multiple techniques that human placental tissue, obtained at delivery from normal full-term pregnancies, synthesizes and secretes PSA. RT-PCR showed the presence of PSA messenger ribonucleic acid; biochemical, chromatographic, and immunological studies revealed the expression of both free and complexed PSA forms; immunoelectron microscopy indicated the syncytiotrophoblast as the site of PSA synthesis and secretion. Moreover, in vitro experiments demonstrated that PSA production and secretion are up-regulated by 17beta-estradiol, a pregnancy-related steroid hormone. These results suggest that human placenta is a source of the PSA present in amniotic fluid and maternal serum during pregnancy.

Interaction between the mu-agonist dermorphin and the delta-agonist [D-Ala2, Glu4]deltorphin in supraspinal antinociception and delta-opioid receptor binding.

1. In rats, the interaction between the mu-opioid agonist dermorphin and the delta-opioid agonist [D-Ala2, Glu4]deltorphin was studied in binding experiments to delta-opioid receptors and in the antinociceptive test to radiant heat. 2. When injected i.c.v., doses of [D-Ala2, Glu4]deltorphin higher than 20 nmol produced antinociception in the rat tail-flick test to radiant heat. Lower doses were inactive. None of the doses tested elicited the maximum achievable response. This partial antinociception was accomplished with an in vivo occupancy of more than 97% of brain delta-opioid receptors and of 17% of mu-opioid receptors. Naloxone (0.1 mg kg-1, s.c.), and naloxonazine (10 mg kg-1, i.v., 24 h before), but not the selective delta-opioid antagonist naltrindole, antagonized the antinociception. 3. In vitro competitive inhibition studies in rat brain membranes showed that [D-Ala2, Glu4]deltorphin displaced [3H]-naltrindole from two delta-binding sites of high and low affinity. The addition of 100 microM Gpp[NH]p produced a three fold increase in the [D-Ala2, Glu4]deltorphin Ki value for both binding sites. The addition of 10 nM dermorphin increased the Ki value of the delta-agonist for the high affinity site five times. When Gpp[NH]p was added to the incubation medium together with 10 nM dermorphin, the high affinity Ki of the delta-agonist increased 15 times. 4. Co-administration into the rat brain ventricles of subanalgesic doses of dermorphin and [D-Ala2, Glu4]deltorphin resulted in synergistic antinociceptive responses. 5. Pretreatment with naloxone or with the non-equilibrium mu-antagonists naloxonazine and beta-funaltrexamine completely abolished the antinociceptive response of the mu-delta agonist combinations. 6. Pretreatment with the delta-opioid antagonists naltrindole and DALCE reduced the antinociceptive response of the dermorphin-[D-Ala2, Glu4]deltorphin combinations to a value near that observed after the mu-agonist alone. At the dosage used, naltrindole occupied more than 98% of brain delta-opioid receptors without affecting mu-opioid-receptors. 7. These data suggest that in the rat tail-flick test to radiant heat, mu- and delta-opioid agonists co-operate positively in evoking an antinociceptive response. Although interactions between different opioid pathways cannot be excluded, in vitro binding results indicate that this co-operative antinociception is probably mediated by co-activation of the delta-opioid receptors at the cellular level by the mu- and delta-agonist.

Hyperthermia triggers apoptosis and affects cell adhesiveness in human neuroblastoma cells.

Hyperthermia is a known apoptotic inducer and has been recently utilized in combination with chemo-and/or radiotherapy in cancer treatment. In this study we have described its effect on SK-N-MC human neuroblastoma tumor cells, a line which grows as a double adherent and floating population. Considering this particular culture behavior, we also investigated the relationship between hyperthermia and cell adhesiveness by evaluating integrin expression, namely CD11a, which is, as known, closely correlated to cell adhesion properties. By a multiple, ultrastructural and flow cytometrical approach, we have demonstrated that hyperthermia, while triggering apoptosis, also determines a CD11a surface expression decrease in apoptotic and living cells. We thus suggest a further role for this treatment, which, affecting adhesion mechanisms, could down-regulate metastatic diffusion.

Early tracheostomy for primary airway management in the surgical critical care setting.

During a 12-month period, 264 patients with multiple injuries who required mechanical ventilation were admitted to the surgical intensive care unit. One hundred twenty patients (46%) were disengaged from the ventilator, and 38 patients (14%) died. Of the remaining 106 patients (40%) 51 patients (group I) were to receive tracheostomy within 1 to 7 days, and 55 patients (group II) underwent late (8 or more days after admission) tracheostomy. Multiple variables in four categories (admission, operative, ventilatory, and outcome) were analyzed prospectively to define the impact that early tracheostomy had on duration of mechanical ventilation, intensive care stay, and hospital stay. Morbidity and mortality rates of the procedures were assessed. Early tracheostomy, in a homogeneous group of critically ill patients, is associated with a significant decrease in duration of mechanical ventilation, as well as shorter intensive care unit and hospital stays, compared with translaryngeal endotracheal intubation. There were no deaths attributable to tracheostomy, and overall morbidity of the procedures was 4%. We conclude that early tracheostomy has an overall risk equivalent to that of endotracheal intubation. Furthermore, early tracheostomy shortens days on the ventilator and intensive care unit and hospital days and should be considered for patients in the intensive care unit at risk for more than 7 days of intubation.

Disappearance rate of serum calcitonin after total thyroidectomy for medullary thyroid carcinoma.

We studied the half-life of serum calcitonin (CT) in patients subjected to total thyroidectomy for medullary thyroid carcinoma (MTC). One patient showed a rapid serum CT component with a half-life of 3 hours and a slow component with a half-life of 30 hours; in another case only the 30-hour component was found. By chromatography of tumor extracts, we found that all the immunoreactive CT had a molecular weight of 3,600. After surgery, normalization of serum CT was achieved within 15 days in 4 patients, at 3 months and at 6 months in 2 other patients, while 1 patient never normalized. Normalization of serum CT after surgery is not an index of definitive cure in MTC, as demonstrated by one patient who relapsed 3 months after normalization of serum CT. However, as a general rule, patients who reach undetectable serum CT levels soon after surgery, are those having the best prognosis.

The onset of CML in blastic crisis: molecular features.

The lack of distinguishing characteristics between lymphoid blastic crisis (BC) of Philadelphia (Ph)+ chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) remains an exciting dilemma. Indeed, the genetic defect of approximately half of Ph+ ALL patients is identical to that identified in CML. Here we report the case of one patient admitted with immunological and molecular patterns indicative of Ph+ ALL. The patient was brought into complete remission by chemotherapy and was transplanted with an HLA identical sibling donor, but relapsed a few months later with immunological and molecular evidence of BC of CML, displaying myeloid markers and lacking lymphoid antigens and immunoglobulin heavy chain (IgH) rearrangement. This suggest a CML case with an initial BC without a previous chronic phase or supervening on a subclinical CML.

Intracellular detection of Bcl-2 and p53 proteins by flow cytometry: comparison of monoclonal antibodies and sample preparation protocols.

Several techniques have been proposed for flow cytometric evaluation of intracellular antigens. This approach is particularly important for detection at the single cell level of proteins which correlate to tumour progression. Bcl-2 and p53 are two of the most relevant proteins. In the present study we have compared five different cell fixation-permeabilisation protocols and nine fluorochrome-conjugated (FITC or PE) monoclonal antibodies (mAb): four mAb directed against Bcl-2 and five against p53. For detection of Bcl-2 we have analysed three Bcl-2 positive cell lines (K562, Daudi and MCF-7), and peripheral blood samples obtained from nine healthy subjects. To distinguish internal positive (lymphocytes) and negative control cells (granulocytes), it was necessary to perform simultaneous detection of surface and intracellular antigens. For detection of p53 three cell lines, two p53 positive (Raji and CEM) and one p53 negative (HL-60), were analysed. Using these cells we have performed a combined analysis of the efficiency of monoclonal antibodies and sample preparation techniques. In conclusion, clones 124-FITC and Bcl-2/100-PE (Bcl-2), and clones BP53,12-FITC and G59-12-PE (p53) provided the highest specific fluorescence intensity of the respective markers independent of cell preparation protocols. Importantly, our results show that mAb background may depend on the specific fixation/permeabilisation kit and that mAb titration using negative and positive control cells is essential to determine the specificity and the sensitivity of the mAb used.

5-(2-Ethyl-phenyl)-3-(3-methoxy-phenyl)-1H-[1,2,4]triazole (DL-111-IT) and related compounds induce apoptotic patterns in cultures of human tumor cell lines.

5-(2-Ethyl-phenyl)-3-(3-methoxy-phenyl)-1H-[1,2,4]triazole (DL-111-IT) and related compounds were extensively studied as anti-gestational agents and some of these molecules were also described as inhibitors of ornithine decarboxylase. Polyamine depletion has been frequently related to the induction of apoptosis and consequently we investigated DL-111-IT and analogs for this effect in myeloid (HL60), neuroblastic (SK-N-MC) and epithelial (BeWo) human tumor cell lines, by means of electron microscopy and DNA electrophoresis. HL60 and SK-N-MC appeared notably sensitive to apoptosis, whereas BeWo responsiveness was variable and frequently associated with necrosis. Our results indicate that the contragestational effect of DL-111-IT and analogs is associated with apoptotic deletion of chorionic tissue and that these molecules, due to their effect on human tumor cell lines, can be considered as antiblastic lead compounds.

C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization.

In this study, the differentiation of C2C12 cells, a primary line of murine myoblasts, was investigated by a multiple technical approach. Undifferentiated cells, and those at intermediate and final differentiation times, were studied at the reverted microscope, by conventional and confocal immunofluorescence, and by transmission and scanning electron microscopy. The general monolayer architecture changed during differentiation from fusiform or star-shaped cells to elongated confluent cells, finally originating long, multinucleated myotubes. Sarcomeric actin and myosin are present also in undifferentiated myoblasts, but progressively acquire a structured pattern up to the appearance of sarcomeres and myofibrils at about 5 days after differentiation induction. Myotubes show a particular positivity for actin and myosin, and M-cadherin, an adhesion molecule characteristic, as known, of satellite cells, also seems to be involved in their assembling. Rare apoptotic patterns, as evidenced by the TUNEL technique, appear during myoblast maturation.

Autologous bone marrow purging with LAK cells.

In this study we will demonstrate that LAK cells, in vitro, can lyse hematologic neoplastic cells with a minor toxicity of the staminal autologous marrow cells. In fact, after bone marrow and LAK co-culture at a ratio of 1/1 for 8 hours, the inhibition on the GEMM colonies resulted to be 20% less compared to the untreated marrow. These data made LAK an inviting agent for marrow purging in autologous bone marrow transplantation.

Apoptotic pathways depend on the target enzymatic activity and not on the triggering agent.

Molt-4 human leukemia cells were triggered to apoptosis by various agents with different mechanisms of action. Staurosporine, a protein kinase C (PKC) inhibitor; camptothecin, a topoisomerase I blocking drug; and tiazofurin, an inhibitor of inosine 5'-phosphate dehydrogenase (IMPDH), were used. Ultrastructural analysis showed morphologic changes characteristic of apoptosis that were very similar for all three agents. Nevertheless, DNA oligonucleosomic fragmentation was not detectable by agarose gel electrophoresis. However, a genomic DNA cleavage appeared after pulse-field gel electrophoresis (PFGE) in cells treated with these agents for 24 h. Furthermore, in situ nick translation (NT) showed a finely spotted nuclear labelling in staurosporine-treated cells and a compact fluorescence after camptothecin incubation. In tiazofurin-treated cells an intermediate pattern was found. Therefore, apoptotic agents with different mechanisms of action induced the formation of large genomic DNA fragments and very similar ultrastructural changes. Therefore, both phenomena and the closely related apoptosis progression depend on target cell machinery and not on the triggering agent.

A multiple technical approach to the study of apoptotic cell micronuclei.

Apoptotic micronuclei have been studied, in different cell types, from a morphologic and functional point of view. Conventional electron microscopy, in various staining conditions, selective cytochemistry for DNA, and freeze fracture for the analysis of chromatin fiber organization and size were performed. In situ TdT and Pol I immunofluorescent techniques were carried out to detect double- and single-strand DNA breaking points by confocal laser scanning microscopy. Apoptotic cell ultrathin cryosections were also performed and were analysed by field emission in lens scanning electron microscopy. Double/single strand massively cleaved DNA was detected in micronuclei, with a highly supercoiled, uniformly packed, very dense arrangement.