In vitro exposure of human chondrocytes to pulsed electromagnetic fields.

The effect of pulsed electromagnetic fields (PEMFs) on the proliferation and survival of matrix-induced autologous chondrocyte implantation (MACI)-derived cells was studied to ascertain the healing potential of PEMFs. MACI-derived cells were taken from cartilage biopsies 6 months after surgery and cultured. No dedifferentiation towards the fibro- blastic phenotype occurred, indicating the success of the surgical implantation. The MACI-derived cultured chondrocytes were exposed to 12 h/day (short term) or 4 h/day (long term) PEMFs exposure (magnetic field intensity, 2 mT; frequency, 75 Hz) and proliferation rate determined by flow cytometric analysis. The PEMFs exposure elicited a significant increase of cell number in the SG2M cell cycle phase. Moreover, cells isolated from MACI scaffolds showed the presence of collagen type II, a typical marker of chondrocyte functionality. The results show that MACI membranes represent an optimal bioengineering device to support chondrocyte growth and proliferation in surgical implants. The surgical implant of MACI combined with physiotherapy is suggested as a promising approach for a faster and safer treatment of cartilage traumatic lesions.

TNF-related apoptosis-inducing ligand (TRAIL) and erythropoiesis: a role for PKC epsilon.

The regulation of the hematopoietic stem cell pool size and the processes of cell differentiation along the hematopoietic lineages involve apoptosis. Among the different factors with a recognized activity on blood progenitor cells, TRAIL - a member of the TNF family of cytokines - has an emerging role in the modulation of normal hematopoiesis.PKC(epsilon) levels are regulated by EPO in differentiating erythroid progenitors and control the protection against the apoptogenic effect of TRAIL. EPO-induced erythroid CD34 cells are insensitive to the apoptogenic effect of TRAIL between day 0 and day 3, due to the lack of specific surface receptors expression. Death receptors appear after day 3 of differentiation and consequently erythroid cells become sensitive to TRAIL up to day 9/10, when the EPO-driven up-regulation of PKC epsilon intracellular levels inhibits the TRAIL-mediated apoptosis, via Bcl-2. In the time interval between day 3 and 9, therefore, the number of erythroid progenitors can be limited by the presence of soluble or membrane-bound TRAIL present in the bone marrow microenvironment.

The response of human natural killer cells to interleukin-2.

Natural killer cells play a key role in the defence of organisms against virus infections and in the control of tumor onset. Interleukin-2 is a multifunctional inflammatory cytokine able to activate natural killer cells, essentially inducing cell proliferation, lymphokine-activated-killer cell generation and cytokine production. Here we discuss some signaling events generated by interleukin-2 in the cell nucleus of primary human natural killer cells, specifically focusing on the lipid signal transduction and the induction of the cyclic adenosine-5'-monophosphate response element binding protein transcription factor. The implications of these nuclear events in the response of natural killer cells to interleukin-2 are also discussed.

Hepatocyte growth factor-activated NF-kappaB regulates HIF-1 activity and ODC expression, implicated in survival, differently in different carcinoma cell lines.

Hepatocyte growth factor (HGF)-stimulated Met signaling influences tumor survival, growth and progression, all processes involving the transcription factor NF-kappaB. NF-kappaB plays a complex role in the control of survival due to the influence of cellular factors acting downstream. We undertook a comparative investigation of two human breast carcinoma cells with different grades of malignancy and HepG2 hepatoma cells, which present a biphasic response to HGF (proliferation followed by apoptosis). We found evidence that HGF induced gene patterns characteristic of survival rather than apoptosis depending on the cell type. The ability of NF-kappaB to regulate expression of hypoxia-inducible factor-1alpha (HIF-1alpha), a survival/anti-apoptotic gene in cancer, seemed to be critical. In the HepG2 and MCF-7 (low invasive breast carcinoma) cell lines increased transcription and translation were responsible for HIF-1alpha induction after HGF. The regulation by NF-kappaB was mainly at the level of the 5'-UTR of the HIF-1alpha message. HIF-1 (alpha/beta heterodimer) was likely to transactivate Mcl-1, another anti-apoptotic gene. Opposite results were observed in MDA-MB-231 cells (highly invasive breast carcinoma), which have high NF-kappaB activity, further inducible by HGF, because HIF-1alpha mRNA expression and HIF-1 transactivating capacity were HGF-insensitive while the alpha subunit seemed to be degraded after HGF. However, ornithine decarboxylase (ODC) and heme oxygenase mRNA expression persistently increased. By transiently transfecting two ODC gene reporters we demonstrated that ODC is a target gene of NF-kappaB in HGF-treated tumor cells. By regulating HIF-1 activity and specific gene expression downstream, NF-kappaB may influence the survival threshold, with an impact on the fate of carcinoma cells after prolonged HGF treatment.

Distinct epitopes on formiminotransferase cyclodeaminase induce autoimmune liver cytosol antibody type 1.

Liver cytosol antibody type 1 (LC1) is regarded as a serologic marker of type 2 autoimmune hepatitis, in addition to liver kidney microsomal antibody type 1. Among 38 patients with type 2 autoimmune hepatitis, 23 were positive for LC1 antibodies. The antigen recognized by LC1 has been identified as a liver-specific 58-kd metabolic enzyme named formiminotransferase cyclodeaminase (FTCD). All 23 LC1-positive sera immunoprecipitated rat FTCD, and 22 gave an identity reaction with rat FTCD by immunodiffusion. No reaction was observed with sera from 10 patients with type 1 autoimmune hepatitis, 10 with primary biliary cirrhosis, 10 with chronic hepatitis C, and 10 healthy controls. By Western immunoblotting all 23 LC1-positive sera and all the controls tested negative, suggesting that all the antigenic epitopes were destroyed by denaturation. FTCD is a bifunctional protein composed of distinct globular FT and CD domains connected by a short linker. To identify epitopes that trigger the LC1 autoimmune response, we tested LC1 antibodies against FTCD constructs encoding the N-terminal FT domain (amino acids 1-339), or the C-terminal CD domain (amino acids 332-541). Of 20 sera positive against full-length FTCD, 8 (40%) recognized the FT domain and the CD domain, 7 (35%) recognized only the FT domain, and 5 (25%) did not recognize either construct. No sera reacted with only the CD domain. These data indicate that multiple regions of FTCD trigger the LC1 autoimmune response, and that LC1 reactivity is mainly directed to conformation-sensitive epitopes located in the FT region of FTCD.

Extracellular Tat activates c-fos promoter in low serum-starved CD4+ T cells.

The regulatory human immunodeficiency virus-1 (HIV-1) Tat protein shows pleiotropic effects on the survival and growth of both HIV-1-infected and uninfected CD4+ T lymphocytes. In this study, we have demonstrated that low concentrations (10 ng/ml) of extracellular Tat protein induce the expression of both c-fos mRNA and protein in serum-starved Jurkat CD4+ lymphoblastoid T cells. Using deletion mutants, we demonstrates that the SRE, CRE and, to a lesser extent, also the SIE domains (all placed in the first 356 bp of c-fos promoter) play a key role in mediating the response to extracellular Tat. Moreover, the ability of Tat to activate the transcriptional activity of c-fos promoter was consistently decreased by pretreatment with the ERK/MAPK kinase inhibitor PD98058. Activation of c-fos is functional as demonstrated by induction of the AP-1 transcription factor, which is involved in the regulation of critical genes for the activation of T lymphocytes, such as interleukin 2. The Tat-mediated induction of c-fos and AP-1 in uninfected lymphoid T cells may contribute to explain the immune hyperactivation that characterizes the progression to autoimmuno deficiency syndrome and constitutes the optimal environment for HIV-1 replication, occurring predominantly in activated/proliferating CD4+ T cells.

HIV-1 Tat protects CD4+ Jurkat T lymphoblastoid cells from apoptosis mediated by TNF-related apoptosis-inducing ligand.

We have here investigated the effect of TNF-related apoptosis-inducing ligand (TRAIL), a new member of the TNF cytokine superfamily, on the survival of Jurkat lymphoblastoid cell lines stably transfected with plasmids expressing the wild-type or mutated (Cys22) human immunodeficiency virus type 1 (HIV-1) tat gene. Jurkat cells transfected with wild-type tat were resistant to TRAIL-mediated apoptosis, while Jurkat cells mock-transfected with the control plasmid or with a mutated nonfunctional tat cDNA were highly susceptible to TRAIL-mediated apoptosis. Also, pretreatment with low concentrations (10-100 ng/ml) of extracellular synthetic Tat protein partially protected Jurkat cells from TRAIL-mediated apoptosis. Taken together, these results demonstrated that endogenously expressed tat and, to a lesser extent, extracellular Tat block TRAIL-mediated apoptosis. Since it has been shown that primary lymphoid T cells purified from HIV-1-infected individuals are more susceptible than those purified from normal individuals to TRAIL-mediated apoptosis, our findings underscore a potentially important role of Tat in protecting HIV-1-infected cells from TRAIL-mediated apoptosis.

Stroma-derived factor 1alpha induces a selective inhibition of human erythroid development via the functional upregulation of Fas/CD95 ligand.

CXC chemokine receptor 4 (CXCR4), the high-affinity receptor for stroma-derived factor 1alpha (SDF-1alpha), shows distinct patterns of expression in human CD34+ haematopoietic progenitor cells induced to differentiate in vitro along the granulocytic and erythroid lineages. In serum-free liquid cultures supplemented with stem cell factor (SCF), interleukin 3 (IL-3) and granulocyte colony-stimulating factor, the expression of surface CXCR4 progressively increased in cells differentiating along the granulocytic lineage. The addition in culture of 200 ng/ml of SDF-1alpha, a concentration which maximally activated intracellular Ca2+ flux, only modestly affected the expression levels of CD15 and CD11b granulocytic antigens, as well as the total number of viable cells. On the other hand, in liquid cultures supplemented with SCF, IL-3 and erythropoietin, SDF-1alpha induced the downregulation of glycophorin A erythroid antigen, accompanied by a progressive decline in the number of viable erythroblasts. Moreover, in semisolid assays, SDF-1alpha significantly reduced the number of plurifocal erythroid colonies (erythroid blast-forming units; BFU-E), whereas it did not affect granulocyte-macrophage colony-forming units (CFU-GM). We also demonstrated that the inhibitory effect of SDF-1alpha on glycophorin A+ erythroid cell development was mediated by the functional upregulation of CD95L in erythroid cultures. These data indicate that SDF-1alpha plays a role as a negative regulator of erythropoiesis.

Aerobic thermophilic treatment of sewage sludge at pilot plant scale. 1. Operating conditions.

The aerobic thermophilic treatment process of sewage sludge was studied at different bioreactor scales in a pilot plant installation. Since, for a satisfactory sludge disinfection, the Swiss legislation requires minimal incubation times of all volume elements, the bioreactors were operated in repetitive batch mode (draw and fill). Different retention times and frequencies of the volume changes were applied in order to prove the capability of the particular operation modes in assuring high degradative potential. The main enzymatic activity involved during the aerobic treatment was proteolysis: the RQ values ranged between 0.8 and 0.9 depending on the applied operating conditions. Although not in a linear manner, the efficiency of the microflora decreased as the bioreactor scale increased, when this increase corresponded with a reduction of the specific power input. The sludge oxidation rates can be tuned by some process operating conditions such as the volume change frequency, the changed volume quantities and the retention times. It was possible to improve the microbial degradative efficiency by an increased frequency of the changes, while the mean retention time influenced in particular the ultimate product quality, described as residual organic matter content of the sludge. The microflora present was also satisfactorily active at mean hydraulic retention times of less than 10 h. The organic matter concentration of the inlet sewage sludge plays an important role: it influences the aerobic degradation process positively.

Aerobic thermophilic treatment of sewage sludge at pilot plant scale. 2. Technical solutions and process design.

The performance of the ATS process depends essentially on the oxygen transfer efficiency. Improvement of the mass transfer capacity of a bioreactor allowed to reduce the incubation time necessary to attain sludge stabilization. It is important to use equipment with a high aeration efficiency such as an injector aeration system. The ratio between the total oxygen consumption and the organic matter degradation (delta COD) ranged between 0.4 and 0.8 in the pilot plant, whereas 1.23 was found in completely mixed bioreactors (Bomio, 1990). No significant improvement of the bacterial degradation efficiency was attained with a specific power input exceeding 6-8 kW m-3. A mean residence time of less than 1 d allowed organic matter removals up to 40% with specific power consumption of 10 kWh kg-1 COD oxidized. The sludge hygienization is one of the objectives and benefits of the thermophilic treatment: not only temperature but also the total solids content were important factors affecting inactivation of pathogens. The inactivation rate was promoted by the increase of temperature, while the residual colony forming units decreased with reducing the total solids content of sewage sludge. It is concluded that continuous operation mode would not affect the quality of the hygienization but could display the high degradation potential of the aerobic system.

Managing and displaying different time granularities of clinical information.

We approach the need of representing by powerful graphical user-interface the granularity of the temporal clinical information. In this work we present some contributions, to suitably display such temporal clinical information. The graphical representation relies on a temporal clinical data model able to manage data having different granularities.

Factors affecting homologous overexpression of the Saccharomyces cerevisiae lanosterol 14 alpha-demethylase gene.

The Saccharomyces cerevisiae Lanosterol 14 alpha-demethylase (14DM) gene was overexpressed in S. cerevisiae using promoter sequences of the highly expressed S. cerevisiae glyceraldehyde-3-phosphate dehydrogenase (TDH3) gene. To investigate factors affecting 14DM overproduction, the levels of 14DM-specific RNAs, apoprotein, and heme protein, respectively, were determined and the 14DM-specific RNA levels compared with the RNA levels originating from the endogenous TDH gene(s). The quantitative measurements revealed that the 14DM steady-state RNA levels reached were some three- to five-fold below the theoretically expected values. With a view towards further improving expression of the 14DM gene, the spacing between the TDH3 promoter and the AUG was adjusted precisely and to rule out possible toxic effects exerted by the 14DM protein, the TDH3 promoter was placed under galactose regulation by introducing an UASG segment. Furthermore, the effects of the gene copy number on 14DM overproduction were investigated. From the analysis of the improved expression constructs five conclusions could be reached: (1) expression from the native 14DM gene is comparable to the expression driven by the TDH3 promoter-14DM fusion construct on single copy plasmid vectors; (2) expression from the TDH3 promoter-14DM construct on single-copy vectors is nearly as efficient as expression from the corresponding endogenous TDH3 gene; (3) the gene copy number has an effect on the relative expression levels of the TDH3 promoter-14DM constructs; (4) the steady-state amounts of protein produced are very nearly proportional to gene dosage; and (5) protein toxicity does not have a major impact on 14DM production. The maximum yield of 14DM was in the order of 7% of the total yeast protein and the maximum production of functional 14DM heme protein appears to be limited by the availability of heme.

[Comparison of amiodarone and quinidine in the conversion to sinusal rhythm of atrial fibrillation of recent onset]. / Confronto tra amiodarone e chinidina nella conversione a ritmo sinusale della fibrillazione atriale di recente insorgenza.

The effectiveness of amiodarone and quinidine in converting atrial fibrillation of recent onset (less than three weeks) to sinus rhythm was compared in a randomized, open-label study. Patients with signs of heart failure determining a NYHA class 3 or 4, acute myocardial infarction, unstable angina pectoris, sick sinus syndrome, Wolff-Parkinson-White syndrome, conduction disturbances, dysthyroidism, or undergoing concomitant therapy with antiarrhythmic drugs, were excluded from the study. Sixty-eight consecutive patients were randomized to receive amiodarone (group A) or quinidine (group B). Group A was treated with amiodarone intravenously as a bolus of 5 mg/Kg over a 20 min period followed by a 15 mg/Kg infusion during the first 24 hours and then orally at a dose of 0.4 g every 6 hours. Group B was treated with quinidine sulphate orally at a dose of 0.2 g every 6 hours during the first day; 0.4 g every 6 hours the second day and 0.6 g every 6 hours during the third day of therapy. Quinidine was preceded by rapid intravenous digitalization depending on the patient's clinical status so as to obtain a ventricular rate of about 100 beats/min, with subsequent oral digitalis administration in maintenance doses. Both treatments were continued until conversion or for a maximum of three days. If the sinus rhythm was not restored, patients underwent electrical cardioversion. Drug efficacy was assessed on the basis of conversion to sinus rhythm. Six patients converted to sinus rhythm with intravenous digitalization alone and were excluded from the comparison between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic effects of transdermal nitroglycerin in stable angina pectoris: a within-patient, placebo-controlled study.

The efficacy of transdermal nitroglycerin patches, releasing 20 mg of active substance over a period of 24 h (TDN 20), was investigated in 10 patients with stable exercise-induced angina pectoris. The study was divided into 3 periods: the first part was an acute, within-patient, crossover, double-blind, placebo-controlled study, in which patients performed a cycloergometric exercise test 4 and 24 h after the application of the patches (TDN 20 or placebo). During the 2nd period, patients were given TDN 20, in single blind conditions, for 4 weeks and another exercise test was performed, on the last day, 4 and 24 h after patch application. Finally, after a one-day placebo wash-out, a second acute study similar to the first was performed. Four h after dosing, exercise duration to 1 mm ST segment depression was 441 s and 314 s (p less than 0.01) for TDN 20 and placebo, respectively (first acute study), 394 s for TDN 20 after chronic treatment (p less than 0.001 vs acute placebo) and 472 and 354 s (p less than 0.001) for TDN 20 and placebo, respectively (second acute study). No difference in exercise duration to 1 mm ST segment depression was found between TDN 20 and placebo, 24 h after administration, in any of the periods. Blood pressure significantly decreased and heart rate significantly increased 4 h after TDN dosing (in comparison with placebo) in both the acute studies, but no difference was observed after chronic TDN treatment. In conclusion, TDN 20 increases exercise tolerance 4 h after the application of both acute and chronic treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

[Prognostic meaning of the echo-dipyridamole test in recent non-Q myocardial infarct]. / Significato prognostico del test eco-dipiridamolo nell'infarto miocardico non Q recente.

The aim of this study was to evaluate the usefulness of the dipyridamole-echocardiography test soon after non Q wave myocardial infarction. Forty-two consecutive patients admitted to the Coronary Care Unit for a first episode of a non Q wave myocardial infarction were enrolled. Dipyridamole-echocardiography test and exercise stress test were performed on 29/42 patients without clinical or electrocardiographic evidence of residual ischaemia, before hospital discharge (from 7 to 15 days after admission). They were followed-up for 1 to 15 months (mean 11.9) or until one of the following clinical end points occurred: recurrence of myocardial infarction, angina or cardiac death. Over a period of 4 minutes, 0.56 mg/kg of dipyridamole was infused intravenously. The test was considered positive when a new transient wall motion abnormality was confirmed by two independent observers. According to these criteria a positive test was observed in 7/29 patients (24%) and a negative one in 22/29 (76%). The exercise stress test was positive in 12/27 patients (44%). Subsequent coronary events occurred in all the patients (100%) with positive dipyridamole-echocardiography test and in 7/22 patients (32%) with negative dipyridamole-echocardiography test (p less than 0.001). Five out of the 7 patients with positive test who underwent coronary angiography showed multivessel coronary artery disease. The sensitivity, specificity and positive predictive value of dipyridamole-echocardiography test for the identification of patients at risk for subsequent coronary events were respectively 50%, 100%, 100%, while for exercise stress test these values were 83%, 86% and 75%.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of a transdermal patch system containing nitroglycerin on exercise tolerance in patients with angina pectoris. Double-blind, placebo controlled, comparison with slow-release nifedipine and verapamil.

The antianginal efficacy of nitroglycerin (NTG), given in a new transdermal therapeutic system (TTS), was compared with that of nifedipine and verapamil, both in slow-release (SR) formulation, in a randomized, double-blind, placebo-controlled study, carried out in 8 patients with stable exercise-induced angina pectoris. TTS NTG 40 cm2 (releasing 20 mg of NTG over 24 hours), nifedipine 20 mg SR, verapamil 120 mg SR and placebo were given once on 4 consecutive days according to a 4 X 4 latin-square design, twice replicated. A cycloergometric symptom-limited exercise test was performed 4 and 8 hours after the administration of each drug. Four hours post-dosing, mean exercise duration was 407 sec. after placebo and 523 (+28%) and 485 (+ 19%) sec. after TTS NTG and nifedipine SR respectively, while at the 8th hour it was 375 sec. after placebo, and 515 (+ 37%) and 457 (+ 21%) sec. after TTS NTG and nifedipine SR. Exercise duration after verapamil was similar to that after placebo. In comparison with placebo maximal workload and total work performed were significantly higher on TTS NTG and on nifedipine at both times of observation, but no significant differences were seen after verapamil. Peak exercise systolic blood pressure was nearly identical after all the treatments tested. Peak exercise heart rate and pressure rate product were both significantly higher on TTS NTG, as well as on nifedipine, in comparison with placebo, while values after verapamil did not differ from those after placebo.(ABSTRACT TRUNCATED AT 250 WORDS)

[Drug therapy combinations in angina pectoris]. / Associations médicamenteuses dans l'angine de poitrine.

The critical level of myocardial oxygen consumption at which anginal pain will occur can be assessed in patients with coronary artery disease. We studied the results of exercise ECG in patients with effort angina, administered placebo, nitrate derivatives, betablockers, and calcium inhibitors, alone and in different associations. Nitrate derivatives and calcium inhibitors appeared to be more effective than betablockers. The study demonstrated the utility of the association of betablockers with other drugs with a more effective anti-anginal action but without the hypotensive or antiarrhythmic effects of betablockers which are often very desirable in the treatment of coronary artery disease.

[Verapamil in effort angina: a multi-centre study]. / Il verapamil nell'angina da sforzo: valutazione policentrica.

We have performed a multi-centre study with 47 outpatients in order to evaluate the efficacy of Verapamil (V) in the treatment of stable effort angina, and to compare the effect of two different doses of the drug (240 and 360 mg/die). The protocol consisted of a first period of Placebo, followed by the double-blind randomized cross-over administration of Placebo (P) and Verapamil (V) in doses of 240 and 360 mg/die. The symptomatology, the consumption of TNG, the ECG pattern at rest and during exercise, the maximum exercise tolerance during exercise and the rate of recovery were evaluated at the end of each 1 month period. V. provided a significant reduction of the number of angina attacks and of the consumption of TNG pills with improvement of symptomatology. The maximum exercise performance improved without changes in maximum rate pressure double product. A decrease of double product was observed at rest and during the recovery period. The higher dose of V. (360 mg/die) provides a better improvement in the number of angina attacks, in the symptomatology, in the double product at rest, and in the rate of recovery than the lower dose (240 mg/die). Thus these data indicate that V. provides anti-anginal efficacy by reducing myocardial oxygen demand, and increases exercise tolerance in effort angina patients.