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In previous studies we demonstrated that during hibernation cell nuclei contain structural constituents usually absent in euthermia. The rapid disappearance of such nuclear bodies upon arousal makes very difficult the in vivo investigation of the disassembly process, which could clarify their functions in nuclear metabolism in the hibernator. In the present study we subjected liver samples taken from hibernating edible dormice ( Glis glis) to different in vitro experimental conditions: at 4 degrees C, to preserve the hypothermic state of the hibernating organism; at 37 degrees C, to simulate the drastic increase in body temperature occurring during arousal; at 37 degrees C, in culture medium containing 10(-5) M delta opioid D-Ala2- D-Leu5 enkephalin, which mimics the activity of the hibernation induction trigger in hibernators. Electron microscopic analysis of hepatocyte nuclei at increasing incubation times revealed the subsequent steps of disassembly of coiled bodies, amorphous bodies and fibro-granular material, the unusual structural constituents accumulating during hibernation in these nuclei. We demonstrated that: (1) a temperature of 37 degrees C induces the disappearance of all nuclear bodies typical of hibernation in a few minutes; (2) both low temperature and hibernation-triggering opioid are able to slow down, although to different extents, the process of disassembly of nuclear bodies; (3) the fibro-granular material rapidly disappears during the early phases of incubation; while (4) coiled bodies and amorphous bodies progressively disassemble as fibrous material. Our results support previous hypotheses based on in vivo observations about a possible role for coiled bodies, amorphous bodies and fibro-granular material as storage/assembly sites of molecules needed for the rapid and massive resumption of transcriptional and post-transcriptional activities upon arousal and suggest a strict correlation between the dynamics and metabolic rate of nuclear bodies.

Background: Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes. Results and Conclusions We show that supravital propidium iodide (PI) assay stains adherent apoptotic cells, allowing flow cytometric quantification. Moreover, supravital exposure to PI without prior permeabilization identifies apoptotic cells as well as Annexin V and permits the simultaneous surface staining by FITC- and PE-conjugated monoclonal antibodies. As in the case of necrotic or permeabilized cells, fluorescence microscopy has revealed that PI staining of apoptotic cells is localized in the nucleus. This suggests that the binding of PI to the DNA/RNA structures is stable enough to withstand the trypsinization and/or washing procedures necessary to detach adherent cells., (Copyright 2001 Wiley-Liss, Inc.)

By means of immunofluorescence and immunoelectron microscopy we have studied the fate of different nucleolar components during the apoptotic process in camptothecin-treated HL60 cells. We have found that RNA polymerase I disappeared while UBF was associated with previously described fibrogranular threaded bodies. In contrast, fibrillarin, C23/nucleolin, and B23/nucleophosmin remained detectable in granular material present amid micronuclei of late apoptotic cells. Double immunolabeling experiments showed colocalization of both C23 and B23 with fibrillarin. Immunoblotting analysis showed that UBF was proteolytically degraded, whereas fibrillarin, C23/nucleolin, and B23/nucleophosmin were not. These results may help explain the presence of anti-nucleolar antibodies seen in various pathological disorders., (Copyright 2000 Wiley-Liss, Inc.)

2',3'-Dideoxycytidine is a powerful in vitro inhibitor of human immunodeficiency virus and is currently used in the treatment of acquired immunodeficiency syndrome. A long-term exposure of U937 monoblastoid cells to dideoxycytidine induces the selection of drug-resistant cells (U937-R). In previous studies, we investigated some important biochemical properties and functional activities, such as basal respiration, protein kinase C activity, superoxide anion release, and the level of reduced glutathione, which were found to be higher in the drug-resistant cell line, compared to the parental one. In the present study, we evaluated the response of the two cell lines to the induction of apoptosis by treatment with staurosporine and okadaic acid, which interfere with the protein kinase and phosphatase pathways, respectively. Moreover, knowing that GSH plays a crucial role in the regulation of nitric oxide-dependent apoptosis, U937-R and parental lines have been treated with SIN-1, which is known to generate significant amounts of O2 and nitric oxide. Resistant and parental cells have been analysed by light and electron microscopy and agarose gel electrophoresis of isolated DNA has been performed. The obtained results demonstrate a different susceptibility of U937-R cell line to apoptosis induced with the three triggers. U937-R cells show more advanced apoptotic features if compared with parental cells, after staurosporine treatment. Differently, the okadaic acid does not induce a different behaviour in the two models. On the contrary, the agent SIN-1 determines an increased number of apoptotic cells in the U937 line. The results suggest that a higher level of protein kinase C and glutathione could prevent programmed cell death in U937-R.

The aim of this study was to evaluate the ultrastructural features of human megakaryocytes cultured in vitro. For this purpose, pluripotent CD34(+) (cluster of differentiation 34) hematopoietic progenitor cells, obtained from the peripheral blood of healthy adult donors, were differentiated along the megakaryocytic lineage in liquid cultures by the addition of the megakaryocyte-specific growth factor thrombopoietin (TPO, 100 ng/ml). After only 6-8 days, virtually all of the CD34-derived cells expressed the early megakaryocytic CD61 antigen, while, after 15-16 days, most cells also expressed the late megakaryocytic CD42a antigen. Ultrastructural analysis of cells obtained after 7 days of culture showed aspects typical of developing megakaryocytes (MK), such as formation of platelet territories and cytoplasmic fragmentation. At later (15-16 day) culture times, two distinct cell populations were observed: fully developed megakaryocytes releasing platelets into the culture medium and senescent megakaryocytes, characterized by morphological features of apoptosis. Analysis of DNA fragmentation in these cells revealed that apoptosis in megakaryocytes occurred in the absence of the internucleosomic cleavage, which is characteristic of most, but not all, types of apoptosis in cells of hematopoietic origin. On the other hand, flow cytometry of the DNA content of senescent megakaryocytes showed a subdiploid peak that was likely due to a loss of micronuclei during processing., (Copyright 2000 Wiley-Liss, Inc.)

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.

Apoptosis plays a fundamental role in shaping normal hematopoiesis. We have investigated the relationship existing between susceptibility to apoptosis and lineage commitment in hemopoietic cells. The presence and degree of apoptosis were investigated in myeloid (HL-60 and K562), T (Jurkat and MOLT-4), and B (CESS and Raji) lymphoid cell lines by using a variety of techniques-transmission electron and light microscopy, flow cytometry and DNA gel electrophoresis. The major achievement of this study is that hematopoietic cells respond to different chemical (staurosporin, tiazofurin, camptothecin) and physical (hyperthermia or hypothermia) stimuli by apoptosis in a lineage-related way. Moreover, with respect to the methods used to detect apoptosis, a strong correlation was observed between the presence of the hypodiploid peak determined by flow cytometry and the DNA laddering evaluated by gel electrophoresis, but both techniques failed to demonstrate the presence of apoptosis in some cases. We conclude that cells of different hematopoietic lineages mostly show a lineage-related behaviour in their apoptotic response to different stimuli, suggesting that the lineage commitment and the stage of differentiation can confer different sensitivities to specific apoptotic stimuli. Moreover, morphological techniques still represent the most reliable approach to detect apoptosis in hemopoietic cells.

Prostate-specific antigen (PSA) is considered a highly specific biochemical marker of the human prostate gland, and it currently is used for prostate cancer diagnosis and monitoring. Recently, PSA production and secretion were found in nondiseased and diseased cells, tissues, and fluids from women. In this study, we characterized the presence of PSA in two human neuroblastoma cell lines with biochemical, ultrastructural, and molecular approaches. Using reverse transcription-PCR, we identified PSA mRNA, and Western blotting revealed a substantial amount of complexed form of PSA protein, which is localized mainly in free ribosomes. Although the role of PSA in human neuroblastoma cell lines is still unknown, our study supports the hypothesis that this serine protease may be involved in controlling the growth of human brain tumor cells, adding more support to the notion that PSA is a widespread kallikrein-like protease with biological functions much more complex than recently thought.

The behaviour of plasma membrane was studied in UV-treated cells to investigate its involvement in apoptosis. It was studied in HL60 cells, in which DNA oligonucleosomic cleavage occurs, and in Molt-4 cells, which are characterised by a different fragmentation pattern. During the early stages of apoptosis, a membrane lipid rearrangement occurs, which involves phosphatidylserine translocation from the inner to the outer leaflet. This molecular alteration was investigated by annexin V-FITC binding, analysed by flow cytometry and confocal microscopy. It was correlated with transmission electron microscopy, subdiploid peak appearance and DNA fragmentation. Our data indicate that the plasma membrane represents an early apoptotic target, even if its alterations are not detectable by ultrastructural analysis, which indicates its good preservation until late apoptotic stages. In addition, the study of apoptotic cells with absent or inactivated endonuclease demonstrates the independence of this membrane mechanism from nuclear activity.

Background and Objective: The K562 cell line, derived from a chronic myeloid leukemia (CML) patient and expressing B3A2 bcr-abl hybrid gene, is known to be particularly resistant to apoptotic death. IFN-alpha treatment of CML patients impairs malignant cell clone, apparently protecting from progression to terminal blast crisis. The mechanisms underlying this kind of cell deletion are analyzed here by multiple technical approaches., Design and Methods: K562 cells, variably treated with IFN-alpha, were examined by agarose gel DNA electrophoresis, light and electron microscopy. The presence of bcr-abl rearrangement was revealed by RT-PCR., Results: At 4 day treatment both DNA ladder and apoptotic nuclear changes were identified, consistently in the presence of bcr-abl expression., Interpretation and Conclusions: Even cells expressing bcr-abl, such as K562, can be triggered to apoptosis. Therefore, this genetic condition, commonly preventing PCD, does not prevent IFN-alpha-mediated apoptosis. PCD seems thus to be the mechanism underlying IFN-alpha-treated K562 cell deletion and it could be the basis of malignant clone reduction in IFN-alpha treated CML patients.

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.

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.

Background: After marrow transplantation, the interaction of helper T lymphocytes from the donor with the patient alloantigens leads to cellular activation and release of IL-2 as initial events of the graft versus host reaction. A method for assessing the size of the pool containing allospecific helper T cells capable of producing IL-2 could be applied in the selection of better donors for marrow transplantation., Material and Methods: PBMC are added to replicate sets of wells each containing various amounts of EBV-LCL cells and PHA. After culture for some days the supernatant is removed from each well and added to IL-2 dependent CTLL-2 line. The proliferation of the CTLL-2 line is assessed by pulse labeling with 3H-thimidine. The precursor frequency of cell capable of producing IL-2 per ml/blood is estimated from the minimum X2 regression of the function of non-responding wells plotted as logarithmic function of the number of PBMC added per well., Results: Approximately 30-40% of PBMC are found to produce IL-2 under the following conditions in culture: the optimal PHA concentration is 1.25 micrograms/ml, the optimal number of stimulator EBV-LCL cells is 1 x 10(3) and 3 days of culture are required., Conclusion: Here we report a rapid and quantitative technique of limiting dilution analysis that can estimate the frequency of peripheral blood mononuclear cells capable of secreting interleukin-2 following interaction with specific alloantigen.

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.