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Issue/problem The Italian National Health System has implemented a National Control Program for Salmonella in poultry populations in order to reduce the prevalence of infection. Starting from 2019, the control program has introduced health measures against Salmonella breeding flocks of Gallus gallus, positive to Salmonella Infantis, an emerging serotype at European level, and widespread along the entire poultry primary production chain. Description of the problem Recent data from the European Union One Health 2019 Zoonoses Report published by EFSA, highlighted that most of the Salmonella isolates (70%) originated from broilers. Interestingly, Salmonella Infantis accounted for 29.7% of the total. Results In the framework of the Italian Salmonella National Control Program the isolation of Salmonella spp. is carried out according to ISO 6579-1: 2017 from faeces, dust and boot socks. During the 2015-2020 period, testing in the Italian Regions Abruzzo and Molise was performed on 2,791 samples and 5,039 sample units. The percentage of samples positive to Salmonella spp. in Abruzzo and Molise was 16,9% and 10,1%, respectively. Out of 395 Salmonella spp. strains isolated, Salmonella Infantis was the most prevalent, representing the 54,9% and 61,9% for Abruzzo and Molise, respectively, followed by Salmonella Livingstone (18,4%) for Abruzzo and (14,4%) for Molise. Lessons The record of Salmonella Infantis in the areas under study reflects the European trend highlighted by the EFSA 2019 Report and justifies the decision to increase the biosecurity measures in farms where this serotype is isolated and to proceed with destruction of positive animals according to the Regulation 1069/2009/EC. Key messages Salmonella Infantis represents a risk for human health due to the presence of antimicrobial resistance genes that confer resistance to relevant antibiotics used in the treatment of human infections. Biosafety guidelines and disinfection practices on poultry farms should be improved in order to reduce the spread of this serotype, also considering its capability to form biofilm.

Debilitating symptoms of schizophrenia often persist after sustained treatment with atypical antipsychotics. To date, clozapine has been the most effective of the atypical antipsychotics; however, negative symptoms may persist, indicating a critical need to develop augmenting treatment approaches.A retrospective chart review evaluated outcomes for 5 young adult inpatients with treatment-resistant schizophrenia who were prescribed off-label oxytocin (OT; 10 IU/sublingual, 1 time per day, to 20 IU/sublingual, 3 times per day) after their therapeutic response to clozapine plateaued (dose range: 200 to 600 mg). The augmented treatment was well tolerated and continued for at least 1 year after discharge from the hospital, with continued outpatient follow-up by the treating psychiatrist. Evaluation included the Positive and Negative Syndrome Scale and clinical review based on both self and parent/guardian reports.The augmentation of clozapine with sublingual OT in young adults with treatment-resistant schizophrenia appeared to reduce negative symptoms, maintain lowered positive symptoms, and increase occupational and social functioning (eg, return to work or school), as noted by family members.Future controlled, prospective studies should investigate the possibility that OT can significantly reduce negative symptoms of chronic psychotic illnesses that are inadequately responsive to clozapine or other antipsychotic medications alone.

BACKGROUND: The prevalence of schizophrenia in the United States ranges between 0.5% and 1%. This difficult-to-treat disorder is marked by the presentation of symptoms that are both positive (i.e. hallucinations) and negative (i.e. blunted affect), as well as disturbances in cognition and affect. Several second-generation antipsychotics (i.e. olanzapine, risperidone, etc.) have been utilized for their varying effects on the symptoms of schizophrenia, yet 20% to 60% of patients with schizophrenia are considered treatment-resistant. While clozapine has been shown to be the most effective antipsychotic, negative symptoms commonly persist in clozapine-treated patients. Research shows that oxytocin has neuromodulatory effects on social perception and also enhances empathy and attentional engagement in individuals with schizophrenia, suggesting it has therapeutic effects on negative symptoms. The present study presents a series of cases suggesting the efficacy of combining clozapine and oxytocin for the reduction of positive and negative symptoms. This work contributes to schizophrenia treatment research by reporting a novel, integrated pharmacological approach to treatment-resistant schizophrenia. METHODS: Retrospective chart reviews were conducted on the charts of 15 patients with treatment-resistant schizophrenia. The patients were all admitted to the persistent psychotic disorder unit at a private hospital between May 2014 and September 2017. The average treatment duration was 2.7 months with a range between 1 and 9 months. All patients were 18 years or older and met the DSM-5 criteria for schizophrenia. The Positive and Negative Syndrome Scale (PANSS) was used to assess the efficacy of the combination treatment. Clozapine was prescribed to all 15 patients after they had failed to improve in three different trials of other antipsychotic medications. Intranasal oxytocin (10 IU 2x per day; 20 IU 3x per day) was prescribed to 11 of the patients only after the improvement in positive symptoms on the PANSS with clozapine had plateaued. Due to the risk of intranasal substance use in all the patients, oxytocin was administered sublingually to ensure adequate and less variable absorption of the neuropeptide. RESULTS: Over the course of treatment, PANSS scores reflected significant changes (p

Background The prevalence of schizophrenia in the United States ranges between 0.5% and 1%. This difficult-to-treat disorder is marked by the presentation of symptoms that are both positive (i.e. hallucinations) and negative (i.e. blunted affect), as well as disturbances in cognition and affect. Several second-generation antipsychotics (i.e. olanzapine, risperidone.) have been utilized for their varying effects on the symptoms of schizophrenia, yet 20% to 60% of patients with schizophrenia are considered treatment-resistant. While clozapine is shown to be the most effective antipsychotic, negative symptoms commonly persist in clozapine-treated patients. Research shows that oxytocin has neuromodulatory effects on social perception and enhances empathy and attentional engagement in individuals with schizophrenia, suggesting it may have therapeutic effects on negative symptoms. The present study presents a pilot prospective research study evaluating the efficacy of combining clozapine and sublingual oxytocin for the reduction of positive and negative symptoms. Methods Prospective research study evaluated 25 treatment resistant schizophrenic patients who were admitted to the persistent psychotic disorder unit at a private hospital, with an average treatment duration of 2.9 months with a range between 1 and 9 months. All have been followed as outpatient for up to 30 months after discharge. All patients were 18 years or older and met the DSM-5 criteria for schizophrenia. The Positive and Negative Syndrome Scale (PANSS) was used to assess the efficacy of the combination treatment. Clozapine was prescribed to all 25 patients after they had failed to improve in three different trials of other antipsychotic medications. Sublingual oxytocin (10 IU 2x per day; 20 IU 3x per day) was prescribed to 25 of the patients only after the improvement in positive symptoms on the PANSS with clozapine had plateaued. Due to a history of intranasal substance use in all the patients, oxytocin was administered sublingual to ensure adequate and less variable absorption of the neuropeptide. Results A time-series analysis demonstrated a significant decrease in PANSS scores across admission, stabilization of clozapine and stabilization of oxytocin (p Discussion The combined effect extends the current research of augmenting sublingual oxytocin and clozapine for individuals with previously treatment-resistant symptoms. Though each patient benefitted from clozapine alone, negative symptoms persisted. Patients, their families, and treatment program staff all observed a significant reduction in the patients’ anxiety and an improvement in the patients’ relatedness. While this case series cannot establish that oxytocin is responsible for the clinical improvements seen here, it does suggest that it may improve negative symptoms and social functioning in patients with treatment-resistant schizophrenia showing incomplete improvement with clozapine alone. The present study suggests the need for future research to explore the possibility that oxytocin can mitigate the negative symptoms of schizophrenia.

S Kawakita1, F Marotta2, Y Naito3, U Gumaste4, S Jain5, J Tsuchiya1, E Minelli21Biokenkyusho Research Laboratory, Shizuoka, Japan; 2WHO-cntr for Biotechnology and Natural Medicine, University of Milan, Milan, Italy; 3Immunology Research Institute and Clinic, Nagoya, Japan; 4Agharkar Research Institute, Pune, Maharashtra, India; 5Department of Food Science and Human Nutrition, University of Illinois, Urbana-Champaign, IL, USA Abstract: The aim of this study was to test the combined effect of a quality-controlled red clover extract (RCE) standardized to contain 40% isoflavones by weight (genistein, daidzein, biochanin A, and formononetin present as hydrolyzed aglycones) together with a modified alkaline supplementation on bone metabolic and biomechanical parameters in an experimental model of surgically-induced menopause. Sprague–Dawley female rats were maintained under controlled standard conditions of light and fed with conventional food of standard calcium content and no alfalfa or soybean components. Rats were randomized into four groups: Group A represented normal rats (sham operated) while three other groups were ovariectomized (OVX) and fed for three months as follows: standard food (group B), 6 mg/kg/day food mixed with RCE (Group C), or given 6 mg/kg/day of RCE plus a modified alkaline supplementation (BP) through a nasogastric tube at a dose of 16 mg (group D). The animals were killed 90 days after surgery. As compared to group B, RCE or RCE + BP treatments brought about significantly higher level of estradiol and mitigated the weight loss of the uterus and improved maximum load of the femoral neck. Osteocalcin level showed an over 65% increase in group B but both RCE and RCE + BP treatments prevented such abnormality with a significantly better result in RCE + BP group which virtually normalized such parameter as well as urinary excretion of DPD. Group C and D reduced the over 20% loss of bone mineral density and bone mineral content/body weight ratio observed in untreated post-ovariectomy group. Untreated ovariectomy caused about 48% decrease of cancellous bone mass in the femoral neck while this abnormality was prevented at similar extent by both RCE and RCE + BP treatments. Ovariectomy determined an over 80% increase of bone alkaline phosphatase (BALP) level but both RCE and RCE + BP treatments significantly mitigated such variable. The BALP decrease yielded by the combined RCE + BP treatment was statistically lower than RCE alone. Taken together these data show that red clover preparation in dosages amenable to clinical practice do improve OVX-induced osteoporosis while a mild metabolic alkalosis might further synergize some therapeutic aspects. Keywords: red clover, alkaline supplementation, osteoporosis, ovariectomy

Thirty-three terminal cancer patients suffering from extremely severe drug-resistant pain were treated with Scrambler Therapy, a new bioengineering-based method for treating oncological/ neuropathic pain. Preliminary tests of the method’s effectiveness were performed by measuring pain intensity (visual analogue scale (VAS)) before and after each treatment session, and the duration of absence of pain in the hours following each single application, as well as by recording the variation in painkiller consumption. Each patient subsequently continued to receive treatment until the end point was reached, verifying whether the method retained the effectiveness displayed during the reference period. The VAS statistics before and after each treatment session, and of the baseline VAS prior to treatment and at the end of the reference cycle were subjected to a paired t-test (statistical significance). Both VAS references indicated a significant decrease in pain intensity, with p

Les auteurs rapportent deux observations de patients japonais souffrant de pancreatite chronique hereditaire : une femme de 46 ans atteinte de pancreatite chronique et son fils aine, qui fut symptomatique des l’âge de 18 ans et dont le diagnostic de pancreatite chronique calcifiante a ete pose a l’âge de 22 ans. Ces observations conduisent a une discussion de la prevalence de la pancreatite familiale dans la population japonaise.

To provide an epidemiologic interpretation of a suspected outbreak of food poisoning caused by Salmonella enterica subspecies enterica serovar Berta strains isolated from humans and from the leftovers of the implicated foods (cream, dairy-based desserts and eggs).We have correlated the similarity between the strains through genotyping with Pulsed Field Gel Electrophoresis (PFGE), studying antimicrobial sensitivity patterns and epidemiological investigation. The clonal origin of the outbreak was confirmed by all laboratory tests. PFGE analysis of the restriction profiles obtained with XbaI and SpeI revealed a certainly correlation from the strains isolated from the various sources, while the antimicrobial sensitivity pattern was the same in all cases, with all strains sensitive to all antibiotics tested.Poor hygiene conditions in the facility concerned, lack of hygiene in food handling, high summer temperatures and positive cultures from asymptomatic staff could all be implicated in the infection, with food being the means through which it spread.This study describes the first outbreak of food poisoning caused by Salmonella enterica subspecies enterica serovar Berta (Salmonella Berta) reported in Italy. It confirms the importance of correlating epidemiological investigations with genotyping and phenotyping to understand the dynamics of infection.

Insulin and IGF binding protein (IGFBP)-1 are linked by negative association. Somatostatin (SS) reduces insulin secretion by acting on pancreatic beta-cell and also by decreasing GH secretion. SS analogues in acromegaly reduce total IGF-I levels inhibiting GH hypersecretion, but they also reduce free IGF-I bioactivity increasing IGFBP-1 levels by inducing insulin decrease. In 13 acromegalic patients we studied GH, IGF system, insulin, and glucagon levels at baseline and at 7 days, 1 and 6 months under treatment with slow release (SR)-lanreotide (LAN) (60 mg im monthly). The hormonal and metabolic response to arginine (ARG) (0.5 g/kg iv in 30 min) was also studied at each time point. LAN decreased GH, total IGF-I, and IGFBP-3 levels at each time point. Insulin and glucagon levels were reduced, while IGFBP-1 and free IGF-I levels were increased by LAN at day 7 and after 1 month only. LAN did not modify the GH, insulin, glucagon, glucose, and IGFBP-1 responses to ARG. At each time point ARG-induced insulin increase was coupled to increase in glucagon and IGFBP-1 levels. This study shows that acromegalic patients under chronic treatment with LAN display: a) inhibition of GH and total IGF-I levels, not coupled to persistent decrease in free IGF-I levels; b) persistent decrease in IGFBP-3 but transient decrease and increase in insulin and IGFBP-1, respectively; c) unchanged hormonal and metabolic response to ARG. Our findings also show that ARG stimulates IGFBP-1 despite marked increase in insulin secretion; this escape from the negative relationship linking insulin and IGFBP-1 would likely reflect the ARG-induced glucagon increase. (J. Endocrinol. Invest. 35: 372-377, 2012) (c) 2012, Editrice Kurtis

The destruction by phagocytic cells of ingested microorganisms is best ensured by an intact respiratory burst with production of superoxide anion and other metabolites. The aim of the present study was to investigate the status of the reticulo-endothelial system, as assessed by superoxide anion generation of blood monocytes, in 18 patients with chronic pancreatitis (CP) as compared to 15 with chronic renal failure (CRF), 14 with diabetes mellitus (DM) and to 20 healthy volunteers. Macrophage suspensions (1 × 10 6 ) were prepared from blood samples withrawn after an overnight fast were tested for conventional phagocytosis function (carbon partical ingestion: percent and intensity) and for superoxide anion generation (chemiluminescent activation: cpm of light integrated intensity). Conventional phagocytosis function tests did not show any differemce among the groups examined. However, the peak value of generated susperoxide anion (× 10 4 cpm) was significantly lower in CP patients as compared to either healthy controls (14.9 ± 4.3 vs. 28.3 ± 4.7, P P P P

Huit cas de tumeurs insulaires (cinq insulinomes, deux gastrinomes et un glucagonome) ont ete diagnostiques au cours des onze dernieres annees au Centre National du Cancer a Tokyo. Un pancreatogramme a ete obtenu par CPRE dans quatre cas. Deux d’entre eux presentaient une interruption du canal pancreatique principal au niveau corporeal, un des patients avait une interruption et un aspect mouchete de la branche correspondant au processus incine. Un autre presentait, au niveau caudal, une dilatation irreguliere isolee du canal pancreatique. Les auteurs commentent les aspects caracteristiques des images pancreatographiques de ces tumeurs. Jusqu’a present, le diagnostic de ces tumeurs pancreatiques etait pose principalement sur base de donnees endocrinologiques et radiologiques. Neanmoins, a partir des donnees rapportees, il semble que dans la localisation des tumeurs insulaires, la CPRE presente une valeur diagnostique equivalente a celle de l’angiographie et de la tomodensitometrie computee.

Three hepatitis C patients out of 15 who received recombinant interferon-alpha 2a (rIFN-alpha 2a) therapy developed high levels of neutralizing antibody coincident with clinical relapse. On analysis, antibodies in their sera were found to also produce some neutralization of lymphoblastoid IFN. Purified lymphoblastoid IFN was separated chromatographically into 10 fractions, each containing one or two IFN-alpha subtypes, and these were used individually to examine the subtype specificity of the neutralizing antibodies in the patients' sera. All three sera neutralized all the subtypes present in the 10 lymphoblastoid IFN fractions. These findings may explain why some patients who develop antibody-mediated resistance to treatment with rIFN-alpha 2 preparations are unable to respond again when treated instead with a human cell derived preparation containing many IFN-alpha subtypes.

Discuss exhaustively the clinical aspects of hepatorenal syndrome (HRS), laboratory data, the progress made in understanding the pathogenesis, and treatment.The work is a result of the research conducted in the framework of the most important studies on HRS.Functional renal failure (FRF) is the most severe manifestation of hepatorenal syndrome, and becomes manifest with a decreased renal perfusion along with oliguresis, reduced excretion of sodium, high urinary osmolarity, azotemia and creatinemia. Liver transplantation restores normal renal function in patients affected by HRS; this suggests the functional origin of the disease. Portal hypertension determines systemic arterial vasodilatation (peripheral vasodilatation theory), with non absolute but effective hypovolemia in terms of its ability to release high circulating levels of vasoactive substances (PG vasodilators, endotoxins, nitric oxide, calcitonin-GPP, glucagon and PAF). Following vasodilatation, a number of systems are triggered to restore peripheral resistance, such as the renin-angiotensin-aldosterone system, the sympathetic systems and the antidiuretic hormone. In some subjects, the activation of these hormones is sufficient to restore resistance and fill the dilated vascular bed, with the subsequent inactivation of the systems themselves. Conversely, in a subgroup of cirrhosis patients with severe portal hypertension, the activation of water retention and vasoconstrictive systems is not sufficient to restore an effective volemia. These subjects present hypotension, effective hypovolemia, high renin levels, high norepinephrine levels and high antidiuretic hormone levels. These substances cause in a sequence, at the renal level, water and sodium retention, progressive vasoconstriction, reduction of plasma flow and GF, initially countered by intrarenal defense mechanisms such as PG, prostacyclins and callicreins. The FRF also sets in because the kidney, in its context, produces determinant endogenous ischemia-inducing substances like tromboxanes, endothelines and leukotrienes. Kidney transplantation is the only treatment.

Natural immune responses, both cellular and humoral, are not capable of terminating HCV infection in most patients. The aim of this study was to evaluate: a) the importance of the immune system in the pathogenesis of chronic HCV infection; b) analysis of successful immunoresponses in persons infected with C virus; c) immuno mechanisms in the progression of hepatic damage; d) different cytokine profiles from patients with persistent and self-limited hepatitis C virus infection; e) development of new antiviral strategies when virus is resistant to interferon treatment. The inadequate T helper1 (Th1) immunity as well as the weak HCV-specific T-cell response at the site of inflammation is associated with failure to clear the virus and a chronic course of disease. The production of interleukin 12 (IL-12) is critical for induction of Th1 immunity, directed towards elimination of intracellular pathogenes and viruses. The core protein of HCV seems to have a suppressive action on IL-12 production at the transcriptional level. The specific Th1 cell defect is correlated with insufficient Th and CTL responses, and lower production of type 1 cytokine (IL-2, IFN-gamma, lymphokine-activated killer cells). Taken together, these results are probably responsible for non-eradication of HCV infection. Particularly the effects of interferon-gamma may include inhibition of HCV virion production by an effect on viral RNA and protein synthesis, enhancement of immune lysis of HCV infected cells, inhibition of hepatic fibrosis by an effect on TGF-beta, and an effect on HCV induced carcinogenesis. These data suggest an HCV-related cellular immune defect in patients with hepatitis C that can be restored in most patients by IL-12. New approaches using a combination of nucleoside analogs or other strategies, such as immune intervention (DNA vaccine, stimulation of the Th1 response) or gene therapy (antisense oligonucleotides dominant negative mutants) should therefore be evaluated in animal models to optimize the current antiviral protocols.

To discuss exhanstively: complex molecular and cellular mechanism in Hepatocellular Carcinoma (HCC); effect of chronic inflammation and cirrhosis, accompained by regenerative process, on the development of HCC; genetic instability of liver cells of regenerating nodules; the relative role of hepatitis C virus (HCV) and hepatitis B virus (HBV) in hepatocarcinogenesis; tumorigenicity of aflatoxin B1 (AFB1); gene expression profiles in HCC; liver tumors and host defense; future perspectives of HCC treatment.We reviewed the most important studies on HCV.HCC is an aggressive malignancy with poor prognosis and is one of the most common tumor in the world. In the majority of cases, HCC is found in conjunction with cirrhosis of the liver. Chronic inflammation and cirrhosis, accompagnied by regenerative process, function as a tumor promoter, providing a common pathway from chronic HBV or HCV infection to HCC. The direct etiologic role of HBV and HCV for HCC is obscure. Tumor progression may be brought about in HCC by mutation of the p53 tumor suppressor gene. The prevalences of p53 mutations is similar in HBV-associated and HCV-associated HCCs. Another mechanisms of host defense are the production of transforming growth factor beta1 (TGFbeta1), and the induction of cytotoxic T lymphocytes; the failure of there mechanisms permits the process of hepatocarcinogenesis. Treatment with alpha interferon of chronic hepatitis is necessary to delary or prevent the progression to liver cirrhosis and development of HCC. Various therapies, such radical operation, intra-arterial chemoembolization, percutaneous intratumoral ethanol injection, radio-frequency ablation, have been employed, but there is still non satisfactory treatment. Recent advances in recombinant and gene delivery thechnologies suggest that gene therapy may be a promising alternative to explore. Furthemore, immunotherapy may become a modality for patients with HCC. Clinical application of vaccine immunotherapy with NY-ESO-1 derived peptides in HLA-A2 positive HCC patients will be possible.

Infection by hepatitis C virus (HCV) generally determines an asymptomatic acute hepatitis which becomes chronic in about 90% of cases. In order to contribute data on the prevalence and the transmission of HCV infection and its associated conditions, anti-HCV seropositivity records in a large sample of a population living in a rural area in Southern Italy were collected and examined.Data were obtained from the registers of local general practitioners operating in 4 neighbouring countries which make up the region analysed. Information on established or potential risk factors for HCV transmission was obtained by means of a questionnaire.More than half of the entire population of the examined region (19,800 subjects, 60%) had a record for an anti-HCV blood testing. Out of these 19,800 subjects, 2,213 were found to be seropositive, with a resulting overall anti-HCV prevalence higher than that reported for the whole country (11.1% vs 3%). Genotype 1b was the most commonly detected (86%). Anti-HCV prevalence was significantly higher in the 50-59 and 60-69 year age groups than in other age groups. The results of multiple regression analysis showed that blood transfusion, use of glass syringes, surgical interventions, promiscuous use of tooth-brush, promiscuous use of sharp-edged instruments and lowest number of years of schooling were all independent predictors of anti-HCV positive. No association was found with family history of liver disease and alcohol consumption. A total 46.6% of the subjects had chronic hepatitis, 24.4% had cirrhosis, 1.8% had hepatocellular carcinoma and cirrhosis and 27.2% were "asymptomatic" (with normal serum ALT levels and no histological features of chronic hepatitis despite HCV viremia).The most striking result of the study was that the high levels of HCV endemicity was not frequently associated with apparent evidence of parenteral exposure, suggesting that HCV spread in the community can even occur mostly through inapparent parenteral routes.

The oil extracted from olive paste is a turbid and opalescent must and contains impurities that can compromise its quality since they facilitate hydrolysis, fermentation and rancidity. Although filtration of this oil removes these otherwise damaging substances, it can also cause small changes in the oil. In this paper membrane cross-flow filtration is proposed as a different and innovative filtration process for extra virgin olive oil. The preliminary results reported here show that the removal of damaging substances through membrane filtration can be achieved in a single step, without the addition of filter aids (therefore reducing oil loss). Membrane filtration also does not alter the chemical composition of the oil.

We advocate the need to exploit formal methods in the development of critical applications on top of RT-CORBA, a recently defined real-time extension of CORBA. We illustrate our approach using the TRIO formal notation. First, we provide a model of the core features of RT CORBA and of the real-time event service. Then we formalize the requirements of a simple application for supervision and control, and we outline the object architecture of its implementation based on the RT-CORBA platform. Finally we show how the above model (RT-CORBA and service plus application objects) can be employed in the proof that the application requirements are actually fulfilled.

AC133+ cells may represent an alternative source of transplantable haemopoietic progenitor cells to CD34+ cells. Here, we have addressed the characterization of umbilical cord blood (UCB) AC133+ cells and compared their immunophenotypic and functional features with those of UCB CD34+ cells. UCB AC133+ and CD34+ cell fractions were purified by magnetic cell sorting, analysed by flow cytometry, tested for their content in blast cell colony-forming units (CFU-Bl), erythroid and granulocyte-macrophage colony-forming units before and after expansion in the presence of various haemopoietic growth factor combinations. Median AC133+ cell yield was 62.3%, and median AC133+ population purity was 97.9%. AC133+ cells were found to contain significantly more CFU-Bl than CD34+ cells; furthermore, the replating efficiency, i.e. the number of CFU-Bl capable of generating secondary colonies, was higher in the former than in the latter cells. Both AC133+ and CD34+ cells displayed an increased ability to give rise to committed progenitors after 7-day expansion in liquid cultures. These data suggest that the AC133+ cell subset is a heterogeneous pool of immature and more differentiated cells that can be maintained and expanded in well-defined culture conditions. In comparison with CD34+ cells, UCB AC133+ cells appear to contain a higher number of early haemopoietic progenitors.

An Experimental Design technique applied for the training of Artificial Neural Networks is presented. The methodology is oriented to maximize the information achievable during experimental tests by guiding them in a structured way and to avoid the overtraining and the overfitting of the Network. The adopted technique is known as Active Learning and is based on the selection of the most significant training examples from an existing set of experimental data. Each experiment, feeding the Neural Network, is chosen as the one which brings the highest content of information through the maximization of an information functional based on the Shannon's Entropy concept. The results for the estimation of the Mechanical Efficiency and the Load Torque for a Spark Ignition Engine exhibit high precision levels with a lower number of experiments with respect to the use of conventional strategies for selecting the training data set.

The feasibility of gene marking or gene therapy protocols making use of purified CD34+ cells greatly depends on the efficiency of their stable transduction. The great potential of umbilical cord blood as a source of CD34+ cells combined with the availability of advanced cell purification procedures prompted us to evaluate whether incubation with growth factors might influence the type of cells effectively transduced by retroviral vectors.Isolated, at least 95% pure, CD34+ cells were infected with the LXSN murine retrovirus carrying the neomycin-resistance gene. Different schedules of CD34+ cell infection were performed with or without incubation for different times in the presence of Interleukin-3 (IL-3), Interleukin-6 (IL-6) and stem cell factor (SCF). Efficiency of transduction was evaluated by clonogenic assays, semiquantitative PCR and RT-PCR analyses performed either immediately or after 7 day expansion of CD34+ cells in liquid culture in the presence of erythropoietin (EPO), IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF).The results obtained indicated that the amount of transduced cells increased with the lenght of incubation with growth factors, either before or during infections. However, different types of cells were transduced depending on the duration of stimulation and infection. Thus, following one week culture of CD34+ cells in the presence of EPO, IL-3 and GM-CSF the clonogenic potential was affected dyshomogeneously. Precisely, with a single 3-hour infection performed after 12 hours of stimulation with growth factors, the clonogenic potential of the transduced cells greatly increased after one week in culture. In contrast, with a 48 hour infection, the transduced cells completely lost their clonogenic potential after one week in culture.These results demonstrate that a reasonably high transduction efficiency of purified CD34+ cells can be achieved with short schedules of incubation/infection in the absence of stroma or extracellular matrix.

In vitro induced megakaryocytic differentiation/maturation of megakayocyte (meg) progenitors represents an important tool for investigating cytokine-induced in vitro thrombocytopoiesis. We have developed an assay which allows the in situ study of human meg progenitor-derived colonies, cultured on a plasma clot in the presence of cytokines. Plates were immunostained by using an anti-alpha IIb beta 3 monoclonal antibody and an alkaline phosphatase-labeled secondary antibody. alpha IIb beta 3-bearing cells were stained an intense red and were clearly differentiated from the negative cells. Processed plates were stable for some weeks at 4 degrees C. The described procedure is easy to perform and allowed us to enumerate the meg colonies and assess colony morphology and cell ploidy.