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Biomedical subjects

S Urbani

Publications and source records attributed to S Urbani.

At least 19 recordsLinked to original sources
PubMed2006

Use of CFDA-SE for evaluating the in vitro proliferation pattern of human mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSC) are multipotent progenitors retaining the capability to undergo multilineage differentiation, mostly towards all the mesodermal cellular lineages. MSC growing under standard conditions are composed of two main subpopulations with a characteristic distribution in the morphologic flow cytometric scatter: RS (recycling stem) cells (small, agranular) and m (mature) MSC (large, moderately granular cells). METHODS: MSC obtained from BM of healthy donors and expanded in culture were characterized by evaluating both the expression of conventional markers and differentiation potential. We used CFSE, a lipophilic dye that is taken up by cell membranes, to investigate separately the proliferative activity of RS cells and mMSC subsets. RESULTS: With flow cytometric analysis, RS cells and mMSC showed nearly the same immunophenotypic pattern, even if a significantly smaller percentage of RS cells expressed some of the classic mesenchymal Ag. The RS cell fraction was confirmed to have a higher proliferative potential and such a feature was particularly evident under certain culture conditions. DISCUSSION: CFSE has been shown as a reliable method for studying the proliferative activity of MSC subpopulations identified by flow cytometric analysis. The acquisition parameter strategy is crucial for the accuracy of the analysis.

Adipocytes↗
PubMed2005

Stress plays a role in the association between cognitive constructs and measures of eating disorders in male subjects.

OBJECTIVE: Several theorists have hypothesized that stressful situations may trigger abnormal eating and even eating disorders in predisposed people. The purpose of this study was to assess whether a stressful situation would reveal an association between perfectionism and low self-esteem, and measures of eating disorder symptoms in male high school students. METHOD: A sample of 61 male high school students completed the Eating Disorder Inventory, the Multidimensional Perfectionism Scale, and the Self Liking and Competence Scale three times: on an average school day, on the day of an exam and on the day the subjects received the results of that exam. Linear regression analysis was carried out to verify whether the dimensions of perfectionism were associated with the measures of eating disorders. RESULTS: Interoceptive awareness was associated with 'Bulimia' only during the stressful situation and with 'Drive for thinness' both in stress and non stress situations. Other results were contradictory and difficult to interpret. DISCUSSION: The results suggest that in nonclinical male individuals stress might bring out a previously absent association between some psychological predisposing factors for eating disorders and an actual desire or plan for ED related thoughts and behaviours. Such a finding suggests that stress may stimulate behaviours related to eating disorders in a predisposed personality. A central role may be played by interoceptive awareness in male subjects.

Adolescent↗
PubMed2005

[The treatment of diffuse cutaneous systemic sclerosis with autologous hemopoietic stem cells transplantation (HSCT): our experience on 2 cases].

OBJECTIVES: Autologous hematopoietic stem cell transplantation (HSCT) is a treatment option which may be considered for severe diffuse cutaneous systemic sclerosis (dcSSc) patients not responding to cyclophophamide (CY). We present two cases of dcSSc not responding to CY >10 g who were successfully treated with HSCT. PATIENTS AND METHODS: Two dcSSc patients were unresponsive to monthly i.v. pulse of CYC (0.75 g m²). Both patients had significant reduction of DLCO and mild-moderate pulmonary hypertension and HSCT was considered due to the rapid progression of the disease. Following informed consent and ethics committee approval, HSCT was performed. Mobilisation was performed with CY 4 g/m² and recombinant human granulocyte colony stimulating factor (rHu GCSF) followed by a successful apheresis (CD34+ cells, >7X106). Conditioning regimens were: CY 100mg/kg body weight plus thiotepa 10 mg/ kg in the first patient and CY 200 mg/kg in the second. Both graft products were CD34 selected. No arrhythmias occurred during the procedure and no other severe side effects were observed during hospitalisation. RESULTS: Follow up: Patients underwent a monthly follow up with physical examination, pulmonary function tests and echocardiography every 3 months. Chest CT has been performed 6 months post transplantation. The following was observed: skin score (from 40 to 10 for the first patient and from 38 to 12 for the second one), LVEF and pulmonary function remained stable, PAP decreased from 45 mmHg to 35 mmHg and from 40 to 32 mmHg. No late complications or cardiac toxicity was observed. CONCLUSION: These two dcSSc cases demonstrate that HSCT may be successfully performed without serious side effects in cases in whom despite a cumulative CY dose was ineffective. This suggests an "immunological threshold" effect which may be exploited in other severe, therapy refractory autoimmune cases.

Female↗
PubMed2001

Lamivudine treatment can overcome cytotoxic T-cell hyporesponsiveness in chronic hepatitis B: new perspectives for immune therapy.

The hepatitis B virus (HBV) cytotoxic T lymphocyte (CTL) response in patients with chronic HBV infection is generally weak or totally undetectable. This inability to mount protective CTL responses is believed to be a crucial determinant of viral persistence, and its correction represents an important objective of immune therapies for chronic hepatitis B. However, amplification of CTL responses in vivo may be ineffective if HBV-specific CD8 cells are either absent or nonresponsive to exogenous stimulation. In this study, we asked whether antiviral treatments able to inhibit viral replication and to reduce viral and antigen load can successfully reconstitute CTL responses creating the appropriate conditions for their therapeutic stimulation. For this purpose, the HBV-specific CTL response before and during lamivudine therapy was studied longitudinally in 6 HLA-A2-positive patients with HBeAg+ chronic hepatitis B. Both HBV-specific cytotoxic T cell activity measured by chromium release assay on peptide stimulation in vitro and CD8+ T cell frequency measured ex vivo by HLA-A2/peptide tetramer staining were significantly augmented by lamivudine therapy. This enhancement followed the reconstitution of CD4 reactivity and the decline of viral load induced by therapy. Our study shows that lamivudine treatment in chronic hepatitis B can restore CTL reactivity, making CTL susceptible to exogenous stimulation. This effect may enhance the probability that T cell-based immune therapies delivered after lamivudine treatment can successfully reconstitute a protective CTL response able to cure chronic HBV infection.

Adult↗
PubMed2001

Identification of immunodominant hepatitis C virus (HCV)-specific cytotoxic T-cell epitopes by stimulation with endogenously synthesized HCV antigens.

Hepatitis C virus (HCV)-specific CD8(+) cytotoxic T lymphocytes (CTL) are believed to play an important role in the pathogenesis of liver cell injury and viral clearance in HCV infection. Because HCV does not efficiently infect human cells in vitro and primary infected hepatocytes cannot be used as stimulator/target cells for CTL analysis, development of efficient systems to activate and expand CTL in vitro, reproducing antigen presentation to CTL occurring during natural infection, is mandatory to study CTL activity and to define the hierarchy of immunodominance of CTL epitopes. To achieve this goal, 5 different defective adenoviruses carrying structural and nonstructural HCV genes (core, core-E1-E2, E2, NS3-NS4A, NS3-NS5A) were used to induce the endogenous synthesis of HCV proteins in human adherent mononuclear cells in vitro and to allow their entry into the HLA class I cytosolic pathway of antigen processing. The cytolytic activity of peripheral blood lympho-mononuclear cells (PBMC) from HLA-A2(+) HCV-infected patients stimulated with recombinant adenovirus-infected cells was tested against target cells either pulsed with a panel of synthetic peptides containing the HLA-A2 binding motif or infected with recombinant vaccinia viruses carrying HCV genes. Our study defines a reproducible system to stimulate and expand HCV-specific CTL in vitro that mimics the conditions of antigen encounter in vivo. By this approach, we have identified several HLA-A2-restricted epitopes that should correspond to immunodominant HCV sequences recognized by CTL during natural infection. Therefore, these amino acid sequences represent ideal candidates for the design of therapeutic vaccines for chronic HCV infection.

Adenoviridae↗
PubMed2000

CD8+ T lymphocyte responses are induced during acute hepatitis C virus infection but are not sustained.

Cellular immune responses are likely to play a key role in determining the clinical outcome in acute infection with hepatitis C virus (HCV), but the dynamics of such responses and their relationship to viral clearance are poorly understood. In a previous study we have shown highly activated, multispecific cytotoxic T lymphocyte responses arising early and persisting in an individual who subsequently cleared the virus. In this study the HCV-specific CD8+ lymphocytes response has been similarly analyzed, using peptide-HLA class I tetramers, in a further nine individuals with documented acute HCV infection, six of whom failed to clear the virus. Significant populations of virus-specific CD8+ lymphocytes were detected at the peak of acute hepatic illness (maximally 3.5% of CD8+ lymphocytes). Frequencies were commonly lower than those seen previously and were generally not sustained. Early HCV-specific CD8+ lymphocytes showed an activated phenotype in all patients (CD38+ and HLA class II+), but this activation was short-lived. Failure to sustain sufficient numbers of activated virus-specific CD8+ lymphocytes may contribute to persistence of HCV.

Acute Disease↗
PubMed1999

Conserved hepatitis C virus sequences are highly immunogenic for CD4(+) T cells: implications for vaccine development.

The HLA class II-restricted T-cell response to hepatitis C virus (HCV) antigens is believed to influence the final outcome of hepatitis C, because it is vigorous in patients who recover from acute hepatitis C, but it is weak in those who develop a chronic infection. For this reason, exogenous stimulation of T-cell responses in chronic HCV infection may represent a strategy to cure patients with chronic hepatitis C by approximating the vigor of their T-cell reactivity to that of patients who succeed in recovering from hepatitis. It may also be a preventive approach to avoid spread of the virus by facilitating the development of a vigorous protective response at the very early stages of infection. T-cell-based vaccines composed of immunodominant, promiscuous, and conserved T-cell epitopes may represent a powerful tool to achieve optimal stimulation of the T-cell reactivity. To identify HLA class II-restricted T-cell epitopes useful for this purpose, 22 subjects with acute HCV infection were studied and followed for an average time of 29 months. Eight of them recovered from hepatitis, and 14 developed a chronic infection. Overlapping 20-mer peptides covering the entire core and NS4 antigens and a panel of peptides representing highly conserved regions of core, NS3, NS4, and NS5 were used. By direct peripheral blood T-cell stimulation and by fine-specificity analysis of HCV-specific T-cell lines and clones, highly immunogenic T-cell epitopes were identified within core, NS3, and NS4. All these epitopes are immunodominant and highly conserved among the known HCV isolates. Moreover, they are promiscuous, because they can be presented to T cells by different HLA class II molecules. Immunodominance, sequence conservation, and promiscuity make these epitopes ideal components of preventive or therapeutic T-cell-based vaccines against HCV.

Acute Disease↗
PubMed1998

Lamivudine treatment can restore T cell responsiveness in chronic hepatitis B.

High viral and/or antigen load may be an important cause of the T cell hyporesponsiveness to hepatitis B virus (HBV) antigens that is often observed in patients with chronic HBV infection. Reduction of viral and antigen load by lamivudine treatment represents an ideal model for investigating this hypothesis. HLA class II restricted T cell responses and serum levels of HBV-DNA, HBsAg, and HBeAg were studied before and during lamivudine treatment in 12 patients with hepatitis B e antigen positive chronic active hepatitis B to assess possible correlations between viral and/or antigen load and vigor of the T cell response. Cell proliferation to HBV nucleocapsid antigens and peptides and frequency of circulating HBV nucleocapsid-specific T cells were assessed to characterize CD4-mediated responses. A highly significant enhancement of the CD4-mediated response to HBV nucleocapsid antigens was already detectable in most patients 7-14 d after the start of lamivudine treatment. This effect was dramatic and persistent in 10 patients but undetectable in 2. It occurred concomitant with a rapid and marked reduction of viremia. Interestingly, lamivudine also enhanced the responses to mitogens and recall antigens, showing that its effect was not limited to HBV-specific T cells. In conclusion, an efficient antiviral T cell response can be restored by lamivudine treatment in patients with chronic hepatitis B concurrently with reduction of viremia, indicating the importance of viral load in the pathogenesis of T cell hyporesponsiveness in these patients. Since lamivudine treatment can overcome T cell hyporeactivity, combining lamivudine with treatments directed to stimulate the T cell response may represent an effective strategy to induce eradication of chronic HBV infection.

Adult↗
PubMed1998

Antiviral cell-mediated immune responses during hepatitis B and hepatitis C virus infections.

Cell-mediated immune responses to hepatitis B (HBV) and hepatitis C virus (HCV) antigens are vigorous and multispecific in acute, self-limited infections. Moreover, the prevalent cytokine pattern of circulating virus-specific T cells from patients who recover spontaneously from acute hepatitis is Th1-like. Longitudinal analysis of the T cell response to HCV antigens from the early stages of HCV infection in patients who recover from hepatitis and those who do not indicates that weaker responses and a prevalent Th2 pattern of cytokine production is associated with viral persistence and chronic evolution of disease. Although similar sequential studies are missing in hepatitis B, the observation that HBV-specific T cell responses are very weak or totally undetectable in the peripheral blood of patients with long-lasting chronic hepatitis B suggests that strength and quality of virus-specific T cell responses at the early stages of infection may influence the final outcome of both hepatitis B and C. While T cell hyporesponsiveness seems to be an important determinant for HBV persistence once chronic hepatitis has developed, this mechanism appears to be less critical in chronic HCV infection, because the vigor and quality of HCV-specific T cell responses seem to improve as a function of the duration of infection. This is shown by the finding that HCV-specific CD4- and CD8-mediated responses are easily detectable in the peripheral blood of patients with long-lasting chronic hepatitis C and that production of Th1 cytokines predominates within their livers. HCV therefore seems to be able to persist even in the face of an active T cell response and to acquire the capacity to survive within a host environment apparently unfavorable to its persistence. The high variability of HCV may explain its efficiency in escaping immune surveillance.

Hepatitis B↗
PubMed1996

An effective solution for prolonged preservation of cultured human pulmonary artery endothelial cells.

Pulmonary endothelium is considered the compartment most susceptible to preservation damage. This investigation was designed to analyze the efficacy of an original, University of Parma low-potassium-albumin solution (SPAL UP) on cultured human pulmonary artery endothelial cells (HPAEC) and to compare its effects with those of University of Wisconsin solution (UW) and Euro-Collins solution (EC). Cryopreserved HPAEC tertiary cultures were inoculated at the density of 5000 cells/cm2 in 9-cm2 well-plates; subcultures were then incubated at 10 degrees C for 6 hr and 16 hr in 2 ml/well of SPAL UP, UW, and EC. The HPAEC viability after incubation was assessed by evaluating the total protein content and the expression of cytotoxicity, and by analyzing the rate of protein synthesis and expression of cellular functionality after stress. Results after 6 hr of preservation showed that SPAL UP had a less significant cytotoxic effect than EC, exerted a less depressing effect on cellular metabolism, and enhanced functional recovery of endothelial cells compared with UW. At the second time interval (16 hr), SPAL UP provided a less cytotoxic effect than UW; besides, SPAL UP-induced cytotoxicity was similar to that of warm control. In conclusion, in vitro preliminary data regarding the use of SPAL UP in HPAEC preservation suggest its suitability as solution for prolonged lung protection.

Cells, Cultured↗
PubMed1996

Activation of heat-shock transcription factor 1 by hypertonic shock in 3T3 cells.

The exposure of 3T3 cells to a medium made hypertonic by the addition of NaCl induced activation of a heat-shock transcription factor (HSF). This activation, as monitored by gel-mobility-shift assays, occurred within 10 min of hypertonic shock and was dose-dependent in relation to the osmotic strength of the medium up to 0.7 osM. Competition analysis indicated that the effect of hypertonic shock on HSF binding activity was specific. The magnitude of the heat-shock element (HSE)-HSF binding induced by incubating the cells in a 0.7 osM medium was comparable in intensity and time course with that induced by a 44 degrees C heat shock. Following removal of the stressors, the decrease in HSF-HSE binding was more rapid in hypertonicity-shocked than in heat-shocked cells. Treatment of the cells with cycloheximide did not inhibit HSF-HSE binding, indicating that the activation was independent of new protein synthesis. By using a specifically directed polyclonal serum, HSF1 was identified as the transcription factor involved in the hypertonicity-induced activation. HSF was also activated when a membrane-impermeable osmolyte such as sucrose was used to increase the osmolarity of the medium. However, no HSF-HSE binding was observed after addition of glycerol (a freely membrane-permeable osmolyte) in excess. There was a temporal relationship between the hypertonicity-induced volume decrease, the increase in the intracellular K+ concentration and the induction of HSF-HSE binding. In contrast, an increase in the intracellular Na+ concentration was not required to induce HSF-HSE binding. However, unlike the heat-shock response, the activation of HSF by hypertonic shock did not lead to elongation of the RNA transcript of heat-shock protein 70.

3T3 Cells↗
PubMed1996

Cell susceptibility to apoptosis by glutamine deprivation and rescue: survival and apoptotic death in cultured lymphoma-leukemia cell lines.

Human leukemia/lymphoma cells maintained in culture medium without provision of fresh nutrients lose viability and die by a process resembling apoptosis within a few days. Upon incubation in an FCS-supplemented RPMI 1640 medium containing 2 mM L-glutamine CEM, Namalwa, HL-60 and U937 cells, seeded at initial densities of 0.2 to 1 x 10(6) cells/ml, ceased growing within 3-5 days and progressively entered an apoptotic pathway, as assessed by nucleosomal DNA fragmentation and morphology. Both the major energy-source nutrients in the medium, glucose and glutamine, became rapidly exhausted during the incubation. Further studies were performed using CEM cells. Incubation in glutamine-free or glucose-free medium renewed every 24 h showed that glutamine deprivation is associated with cell death by apoptosis independent of energetic failure, whereas glucose deprivation is followed by rapid loss of mitochondrial function with sharp drop of intracellular ATP and cell death by necrosis. A 12-24 h incubation in glutamine-depleted medium was required to direct the cells toward the apoptotic pathway. Growth arrest followed by apoptotic death was detected in CEM cells when medium glutamine concentration remained below 0.3-0.4 mM for at least 24 h, but a reinstatement of medium glutamine to 2 mM within this period rescued the cells from growth arrest and death.

Apoptosis↗
PubMed1996

An original application of plasma expanders: heart-lung preservation.

The lack of an ideal heart-lung preservation solution is one of the principal factor that limits the wide spread of transplantation. The aim of this work was to investigate the efficacy of Haemaccel (HM) on isolated human pulmonary artery endothelial cells comparing its effects with those of University of Wisconsin (UWS). Subcultures of HPAEC were inoculated at the density of 5,000 cells per cm2 in 9 cm2 well-plates. Cells were incubated with HM and UWS for 6 hrs at 10 degrees C. Cellular viability was analysed by the total protein content (cytotoxicity index) and by the rate of protein synthesis (metabolic index). The results showed that HM and UWS did no show a significant differences in the toxicity when compared with the control; on the contrary, HM seems to determine a less inhibitory effect on cellular metabolism permitting a more rapid cellular metabolic recovery than UWS. Thus, HM appears to be more suitable for the preservation of isolated HPAEC than UWS.

Adenosine↗
PubMed1995

Methodology for the assessment of lung protection. Human pulmonary artery endothelial cell preservation using haemaccel.

This investigation was designed to show an original methodology for the assessment of lung preservation and to analyze the efficacy of a low potassium polygelin solution (haemaccel [HM]) on isolated human pulmonary artery endothelial cells. The effects of HM were compared with those of low potassium dextran (LPD), Belzer (University of Wisconsin [UWS]), and Euro-Collins solutions. The viability of the endothelial cultures was assessed by means of both total protein content and recovery of metabolic cellular function expressed as the protein synthesis rate after 6 hr and 16 hr of incubation at 10 degrees C. Our results failed to show any significant difference in the total protein content for HM, LPD, and UWS, both after 6 and 16 hr of incubation; however, the Euro-Collins-preserved sample revealed a significant drop in this parameter as early as 6 hr after the start. This finding was regarded as a clear indication of cellular cytotoxicity. In contrast, the metabolism recovery capacity of the cells varied significantly between HM and UWS at 6 hr and among HM, LPD, and UWS at 16 hr; at 6 hr, however, no significant difference was observed between HM and LPD. In conclusion, HM appears to exert a more significant effect on human pulmonary artery endothelial cell metabolism recovery than do the other fluids, thus suggesting its suitability as a long-term pulmonary perfusate.

Cell Division↗
PubMed1995

A new extracellular type solution for lung preservation: "in vitro" comparison with Beltzer, low potassium Dextran and Euro-Collins solutions by means of human lung fibroblasts.

UNLABELLED: The attempt to synthesise efficacious solution to prolong lung preservation is, at present one of the most interesting challenges in transplant research. Recently, several issues emphasise the central role of ionic composition of lung-flush solutions, underlining, however, that colloid-free solutions are clearly detrimental. We have been studying a complex extracellular type solution (SPAL UP) synthesised to minimise the pathological events that occur during both preservation and reperfusion period. We report the results of toxicity of SPAL UP on normal human fibroblasts obtained from foetal lung (WI-38). WI-38 cells were seeded at 1.4 x 10(4)/cm2 in disposable plastic 12-well plates. After 3 days, cells were incubated in SPAL UP, Beltzer (UWS), Low Potassium Dextran (LPD) and Eurocollins (ECS) solutions for 6 hours at 10 degrees C. Cellular viability was evaluated by the rate of protein synthesis exploiting the incorporation of 35S-Methionine (2 microCi/ml) in growth medium with 10 mM unlabelled Methionine during 30 minutes incubation at 37 degrees C. The results were expressed as nmol. 35S-Methionine/mg of proteins/minute, and presented as means +/- SD of data of three (n = 3) well for each solution studied. RESULTS: the viability at time 0 before incubation (considered as control) was 1.65 +/- 0.1; after hypothermic preservation the data were respectively as follow: SPAL UP 0.51 +/- 0.09; UW 0.24 +/- 0.02; ECS 0.19 +/- 0.01; LDP 0.19 +/- 0.05. CONCLUSIONS: in this "in vitro" model SPAL UP solution provides a significantly (p < 0.05) better cell preservation than regular UW, ECS and LPD solutions.

Adenosine↗
PubMed1994

Haemmaccel, a key for lung preservation?

Several and different solutions have been used for lung preservation but, at present, fluids and solutions are quite alike. In the last years extracellular type solutions have been progressively tested in experimental researches and have shown a better protection vs intracellular one. In this research we have studied the effect of a low-potassium solution normally used as plasma expander (Haemmaccel, HM) on isolated foetal human fibroblasts (WI-38). HM has been compared with Belzer solution (UWS) after 16 hrs incubation at 10 degrees C and low-potassium solutions with dextran 2% and 5% after 6 hrs and 16 hrs incubation at 10 degrees C. Wi-38 cells have been seeded at the density of 9x10(4)/cm2 onto plastic well plates. Cellular viability was measuring using the rate of protein synthesis through the incorporation of 3H leucine in growth medium during a 1 hr incubation at 37 degrees C. The results were expressed as nmol 3 H leu/mg of proteins/minute and presented as means +/- SD; the comparison has been performed by the one way variance analysis test. After 16 hrs incubation at 10 degrees C HM preserves WI-38 cells significantly better than UWS. Comparing HM with LPD 2% and 5% a significant difference both at 6 hrs and at 16 hrs was observed. Our preliminary in vitro results confirm that low-potassium solutions are less toxic on isolated lung cells than intracellular one. Polygelin contained in HM could be considered a suitable colloidal substance that determine a better preservation if compared with Low-Potassium Dextran solutions.

Culture Techniques↗
PubMed1994

Verapamil used in lung transplantation: could it have a cytoprotective effect during the ischemic phase of pulmonary preservation? Study on alveolar type II cells.

It has been shown that Verapamil (V) improves lung preservation, but it is not clear whether the effect is due to vasodilation, with a more homogeneous cooling effect during hypothermic preservation, or a direct cytoprotection. Attempting to verify this, we studied the cellular viability of rats' pneumocytes type II primary cultures separately incubated in the first experiment for 6 hours in Ringer Lactate (RL) +/- V (10 mg/l) at 25 degrees C and at 4 degrees C, and in the second experiment for the same period in RL, EuroCollins (EC) and Collins (CL) solution +/- V (10 mg/l) at 4 degrees C. We analyzed the influence of the drug using two methods: the total protein content in each culture and the metabolic function of the cells using the rate of protein synthesis by means of 35 S methionine uptake assay. All data showed that there were no significant statistical difference (Student t-test, p > 0.05) in the use of verapamil during alveolar type II cells either in normothermic or hypothermic preservation. Thus we conclude that the protection of this calcium channel blocker during lung preservation is probably due to the effect on pulmonary vessels rather than to a direct cytoprotective effect on the lung cells.

Animals↗