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R Scarpioni, M Ricardi, V Albertazzi, S De Amicis, F Rastelli, L Zerbini Department of Nephrology and Dialysis, Azienda Unità Sanitaria Local (AUSL) Hospital “Guglielmo da Saliceto”, Piacenza, Italy Abstract: Amyloidosis refers to the extracellular tissue deposition of fibrils composed of low-molecular-weight subunits of a variety of proteins. These deposits may result in a wide range of clinical manifestations depending upon their type, location, and the amount of deposition. Dialysis-related amyloidosis is a serious complication of long-term dialysis therapy and is characterized by the deposition of amyloid fibrils, principally composed of β2 microglobulins (β2M), in the osteoarticular structures and viscera. Most of the β2M is eliminated through glomerular filtration and subsequent reabsorption and catabolism by the proximal tubules. As a consequence, the serum levels of β2M are inversely related to the glomerular filtration rate; therefore, in end-stage renal disease patients, β2M levels increase up to 60-fold. Serum levels of β2M are also elevated in several pathological conditions such as chronic inflammation, liver disease, and above all, in renal dysfunction. Retention of amyloidogenic protein has been attributed to several factors including type of dialysis membrane, prolonged uremic state and/or decreased diuresis, advanced glycation end products, elevated levels of cytokines and dialysate. Dialysis treatment per se has been considered to be an inflammatory stimulus, inducing cytokine production (such as interleukin-1, tumor necrosis factor-α, interleukin-6) and complement activation. The released cytokines are thought to stimulate the synthesis and release of β2M by the macrophages and/or augment the expression of human leukocyte antigens (class I), increasing β2M expression. Residual renal function is probably the best determinant of β2M levels. Therefore, it has to be maintained as long as possible. In this article, we will focus our attention on the etiology of dialysis-related amyloidosis, its prevention, therapy, and future solutions. Keywords: chronic inflammation, long-term dialysis, β2 microglobulin, high-flux dialysis membrane, mineral bone disease

Abstract Background We performed a time-course microarray experiment to define the transcriptional response to carboplatin in vitro, and to correlate this with clinical outcome in epithelial ovarian cancer (EOC). RNA was isolated from carboplatin and control-treated 36M2 ovarian cancer cells at several time points, followed by oligonucleotide microarray hybridization. Carboplatin induced changes in gene expression were assessed at the single gene as well as at the pathway level. Clinical validation was performed in publicly available microarray datasets using disease free and overall survival endpoints. Results Time-course and pathway analyses identified 317 genes and 40 pathways (designated time-course and pathway signatures) deregulated following carboplatin exposure. Both types of signatures were validated in two separate platinum-treated ovarian and NSCLC cell lines using published microarray data. Expression of time-course and pathway signature genes distinguished between patients with unfavorable and favorable survival in two independent ovarian cancer datasets. Among the pathways most highly induced by carboplatin in vitro, the NRF2, NF-kB, and cytokine and inflammatory response pathways were also found to be upregulated prior to chemotherapy exposure in poor prognosis tumors. Conclusion Dynamic assessment of gene expression following carboplatin exposure in vitro can identify both genes and pathways that are correlated with clinical outcome. The functional relevance of this observation for better understanding the mechanisms of drug resistance in EOC will require further evaluation.

Breast cancer cells MCF-7 and MDA-MB-231 were treated with Tamoxifen (5 μM) or Paclitaxel (1 μM) or with a combination of the two drugs. Herein, we have employed gas chromatography coupled with mass spectroscopy to identify metabolic changes occurring as response to different drug treatments. We report the identification of sixty-one metabolites and overall the two studied cell lines showed a distinct metabolomic profile from each other. Further data analysis indicates that a total of 30 metabolites were significantly differentially abundant in MCF-7 drug-treated cells, most of the metabolic changes occurred when cells were treated with either Tamoxifen (15) or Paclitaxel (25). On the other side, a total of 31 metabolites were significantly differentially abundant in MDA-MB-31 cells with drug treatment. Similarly, to MCF-7 most of the metabolic changes occurred when cells were treated with either Tamoxifen (19) or Paclitaxel (20). In conclusion, this report demonstrates that Tamoxifen and/or Paclitaxel treatment have a pronounced effect on the main metabolic pathways in both breast cancer (BC) cell lines (MCF-7 and MDA-MB231), which could be used as a foundation for future investigations to understand the possible effect of these drugs on different metabolic pathways. SIGNIFICANCE: Metabolic profiling of cancer cells is a promising tool in tumor diagnosis, biomarker discovery and drug treatment protocols, since cancer cells exhibit altered metabolism when compared to normal cells. Although numerous studies have reported the use of various OMICs applications to investigate breast cancer cells, very few of these have performed thorough screening of metabolites in such cells. Our investigation highlights the first study to characterize MCF7 and MDA-MB-231 cancer cells treated with Tamoxifen and/or Paclitaxel and to identify the affected metabolic pathways. Such findings might play an important role in revealing the molecular bases of the underlying mechanism of action of these two frontline anti-breast cancer drugs., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The developmentally important T-box transcription factor TBX3, is overexpressed in several cancers and contributes to tumorigenesis as either a tumour promoter or tumour suppressor. For example, TBX3 promotes cell proliferation, migration and invasion of chondrosarcoma cells but inhibits these processes in fibrosarcoma cells. This suggests that the cellular context influences TBX3 oncogenic functions, but the mechanism(s) involved has not been elucidated. COL1A2 encodes type I collagen and, like TBX3, plays important roles during embryogenesis and can act as either oncogene or tumour suppressor. Here we explore the possibility that COL1A2 may be a TBX3 target gene responsible for mediating its opposing oncogenic roles in chondrosarcoma and fibrosarcoma cells. Results from qRT-PCR, western blotting, luciferase reporter and chromatin immunoprecipitation assays show that TBX3 binds and activates the COL1A2 promoter. Furthermore, we show that TBX3 levels are regulated by AKT1 and that pseudo-phosphorylation of TBX3 at an AKT consensus serine site, enhances its ability to activate COL1A2. Importantly, we demonstrate that COL1A2 mediates the pro- and anti-migratory effects of TBX3 in chondrosarcoma and fibrosarcoma cells respectively. Our data reveal that the AKT1/TBX3/COL1A2 axis plays an important role in sarcomagenesis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Promoting Responsible Research and Innovation (RRI) is a major strategy of the "Science with and for Society" work program of the European Union's Horizon 2020 Framework Programme for Research and Innovation. RRI aims to achieve a better alignment of research and innovation with the values, needs, and expectations of society. The RRI strategy includes the "keys" of public engagement, open access, gender, ethics, and science education. The Structural Transformation to Attain Responsible BIOSciences (STARBIOS2) project promotes RRI in 6 European research institutions and universities from Bulgaria, Germany, Italy, Slovenia, Poland, and the United Kingdom, in partnership with a further 6 institutions from Brazil, Denmark, Italy, South Africa, Sweden, and the United States., Objective: The project aims to attain RRI structural change in 6 European institutions by implementing action plans (APs) and developing APs for 3 non-European institutions active in the field of biosciences; use the implementation of APs as a learning process with a view to developing a set of guidelines on the implementation of RRI; and develop a sustainable model for RRI in biosciences., Methods: The project comprises interrelated research and implementation designed to achieve the aforementioned specific objectives. The project is organized into 6 core work packages and 5 supporting work packages. The core work packages deal with the implementation of institutional APs in 6 European institutions based on the structural change activation model. The supporting work packages include technical assistance, learning process on RRI-oriented structural change, monitoring and assessment, communication and dissemination, and project management., Results: The project is funded by Horizon 2020 and will run for 4 years (May 2016-April 2020). As of June 2018, the initial phase has been completed. The participating institutions have developed and approved APs and commenced their implementation. An observation tool has been launched by the Technical Assistance Team to collect information from the implementation of APs; the Evaluation & Assessment team has started monitoring the advancement of the project. As part of the communication and dissemination strategy, a project website, a Facebook page, and a Twitter account have been launched and are updated periodically. The International Scientific Advisory Committee has been formed to advise on the reporting and dissemination of the project's results., Conclusions: In the short term, we anticipate that the project will have a considerable impact on the organizational processes and structures, improving the RRI uptake in the participating institutions. In the medium term, we expect to make RRI-oriented organizational change scalable across Europe by developing guidelines on RRI implementation and an RRI model in biosciences. In the long term, we expect that the project would help increase the ability of research institutions to make discoveries and innovations in better alignment with societal needs and values., International Registered Report Identifier (irrid): DERR1-10.2196/11745., (©Vittorio Colizzi, Daniele Mezzana, Pavel V Ovseiko, Giovanni Caiati, Claudia Colonnello, Andrea Declich, Alastair M Buchan, Laurel Edmunds, Elena Buzan, Luiz Zerbini, Dimitar Djilianov, Evanthia Kalpazidou Schmidt, Krzysztof P Bielawski, Doris Elster, Maria Salvato, Luiz C Jr Alcantara, Antonella Minutolo, Marina Potestà, Elena Bachiddu, Maria J Milano, Lorna R Henderson, Vasiliki Kiparoglou, Phoebe Friesen, Mark Sheehan, Daniela Moyankova, Krasimir Rusanov, Martha Wium, Izabela Raszczyk, Igor Konieczny, Jerzy P Gwizdala, Karol Śledzik, Tanja Barendziak, Julia Birkholz, Nicklas Müller, Jürgen Warrelmann, Ute Meyer, Juliane Filser, Fernanda Khouri Barreto, Carla Montesano. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 07.03.2019.)

Background: Interest around Nordic Walking (NW) has increased in recent years. However, direct comparisons of NW with normal walking (W), particularly in ecologically valid environments is lacking. The aim of our study was to compare NW and W, over long distances in a natural mountain environment. Methods: Twenty one subjects (13 male/8 female, aged 41 ± 12 years, body mass index BMI 24.1 ± 3.7), walked three distinct uphill paths (length 2.2/3.4/7 km) with (NW) or without (W) walking poles over two separate days. Heart rate (HR), energy expenditure (EE), step length (SL), walking speed (WS), total steps number (SN) and rating of perceived exertion (RPE) were monitored. Results: HR (+18%) and EE (+20%) were higher in NW than in W whilst RPE was similar. SN (-12%) was lower and SL (+15%) longer in NW. WS was higher (1.64 vs. 1.53 m s -1 ) in NW. Conclusions: Our data confirm that, similarly to previous laboratory studies, differences in a range of walking variables are present between NW and W when performed in a natural environment. NW appears to increase EE compared to W, despite a similar RPE. Thus, NW could be a useful as aerobic training modality for weight control and cardiorespiratory fitness., Competing Interests: The authors declare no conflict of interest.

The T-box transcription factor, TBX3, plays an important role in embryonic development, and haploinsufficiency of TBX3 causes ulnar-mammary syndrome. Overexpression of TBX3, on the other hand, is associated with several cancers, and preliminary evidence suggests that increased levels of TBX3 may inhibit cell proliferation but promote tumor migration and invasion. Although this suggests that deregulated levels of TBX3 are deleterious in development and promotes disease, very little is known about the signaling pathways that regulate TBX3 expression. Here we show that overexpressing TBX3 inhibits proliferative ability while promoting the migration of breast epithelial cells. We demonstrate that the transforming growth factor β1 (TGF-β1) pathway up-regulates TBX3 protein and mRNA levels and show a detailed transcriptional mechanism by which this occurs. Using in vitro and in vivo assays, we show that Smad3/4 and JunB bind and cooperatively regulate TBX3 promoter activity through a Smad-binding element at -67 base pairs. Further, we show that TBX3 plays a pivotal role in mediating the antiproliferative and promigratory role of TGF-β1 in breast epithelial and skin keratinocytes. This study identifies the TGF-β1 signaling pathway as a potentially important player in the regulation of TBX3 in development and cancer.

The adjustment of pulmonary oxygen uptake (VO2p), heart rate (HR), limb blood flow (LBF), and muscle deoxygenation [HHb] was examined during the transition to moderate-intensity, knee-extension exercise in six older adults (70 ± 4 years) under two conditions: normoxia (FIO₂ = 20.9 %) and hypoxia (FIO₂ = 15 %). The subjects performed repeated step transitions from an active baseline (3 W) to an absolute work rate (21 W) in both conditions. Phase 2 VO₂p, HR, LBF, and [HHb] data were fit with an exponential model. Under hypoxic conditions, no change was observed in HR kinetics, on the other hand, LBF kinetics was faster (normoxia 34 ± 3 s; hypoxia 28 ± 2), whereas the overall [HHb] adjustment (τ' = TD + τ) was slower (normoxia 28 ± 2; hypoxia 33 ± 4 s). Phase 2 VO₂p kinetics were unchanged (p < 0.05). The faster LBF kinetics and slower [HHb] kinetics reflect an improved matching between O₂ delivery and O₂ utilization at the microvascular level, preventing the phase 2 VO₂p kinetics from become slower in hypoxia. Moreover, the absolute blood flow values were higher in hypoxia (1.17 ± 0.2 L min(-1)) compared to normoxia (0.96 ± 0.2 L min(-1)) during the steady-state exercise at 21 W. These findings support the idea that, for older adults exercising at a low work rate, an increase of limb blood flow offsets the drop in arterial oxygen content (CaO₂) caused by breathing an hypoxic mixture.

This study tested the hypothesis that the DMAX (for maximal distance) method could be applied to ratings of perceived exertion (RPE), to propose a novel method for individual detection of the lactate threshold (LT) using RPE alone during an incremental test to exhaustion. Twenty-one participants performed an incremental test on a cycle ergometer. At the end of each stage, lactate concentration was measured and the participants estimated RPE using the Borg CR100 scale. The intensity corresponding to the fixed lactate values of 2 or 4 mmol · L-1 (2mM and 4mM), the ventilatory threshold (VT), the respiratory-compensation point (RCP), and the instant of equality of pulmonary gas exchange (RER=1.00) were determined. Lactate (DMAX La) and RPE (DMAX RPE) thresholds were determined using the DMAX method. Oxygen uptake (VO2), heart rate, and power output measured at DMAX RPE and at DMAX La were not statistically different. Bland-Altman plots showed small bias and good agreements when DMAX RPE was compared with the DMAX La and RER=1.00 methods (bias = -0.05% and -2% of VO2max, respectively). Conversely, VO2 from the DMAX RPE method was lower than VO2 at 4 mM and at RCP and was higher than VO2 at 2 mM and at VT. VO2 at DMAX RPE was strongly correlated with VO2 at DMAX La (r = .97), at RER=1.00 (r = .97), at 2 mM (r = .85), at 4 mM (r = .93), at VT (r = .95), and at RCP (r = .95). The combination of the DMAX method with the RPE responses permitted precise and individualized estimates of LT using the DMAX method.

The present study was designed to investigate whether absolute work rate (WR) affects Phase I pulmonary oxygen uptake (Vo(2)(p)) duration during moderate-intensity (Mod) exercise and to compare two methods for estimating Phase I Vo(2)(p) duration (P(I-Dur)). Fourteen males (24 ± 5 yr) each completed 4-8 repetitions of Mod transitions from 20 W to 50, 70, 90, 110, and 130 W. P(I-Dur) was identified by 1) a marked decrease in both respiratory exchange ratio and end-tidal partial pressure of O(2) following exercise onset [i.e., visual inspection of three independent reviewers, and the average (Avg) of the two most similar values]; or 2) the intersection (time delay, TD) of the first and second components in a biexponential nonlinear regression of the entire Vo(2)(p) response from exercise onset. P(I-Dur) did not differ among WRs (P > 0.05), regardless of the estimation method used. No differences were detected between Avg and TD (time in s) at any of the five WRs (50 W, 21 ± 6 vs. 23 ± 10 s; 70 W, 23 ± 9 vs. 23 ± 7 s; 90 W, 24 ± 3 vs. 22 ± 5 s; 110 W, 23 ± 6 vs. 22 ± 6 s; 130 W, 21 ± 6 vs. 21 ± 7 s; P > 0.05 for Avg and TD, respectively). Broad limits of agreement within Bland-Altman plots revealed relatively weak agreement among reviewers for individual estimation of P(I-Dur). A nonsignificant correlation coefficient (r = 0.13) and broad limits of agreement suggest disparity between individual Avg and TD estimates of P(I-Dur). The present data do not support a role for Mod WR in determining P(I-Dur) per se. Furthermore, this study illustrated a poor agreement of P(I-Dur) estimates derived from two different, but accepted methods.

Pulmonary oxygen uptake, heart rate (HR), and deoxyhemoglobin (HHb) kinetics were studied in a group of older adults exercising in hypoxic conditions. Fourteen healthy older adults (aged 66 ± 6 years) performed 4 exercise sessions that consisted of (i) an incremental test to exhaustion on a cycloergometer while breathing normoxic room air (fractional inspired oxygen (FiO(2)) = 20.9% O(2)); (ii) an incremental test to exhaustion on a cycloergometer while breathing hypoxic room air (FiO(2) = 15% O(2)); (iii) 3 repeated square wave cycling exercises at moderate intensity while breathing normoxic room air; and (iv) 3 repeated square wave cycling exercises at moderate intensity while breathing hypoxic room air. During all exercise sessions, pulmonary gas exchange was measured breath-by-breath; HHb was determined on the vastus lateralis muscle by near-infrared spectroscopy; and HR was collected beat-by-beat. The pulomary oxygen uptake kinetics became slower in hypoxia (31 ± 9 s) than in normoxia (27 ± 7 s) because of an increased mismatching between O(2) delivery to O(2) utilization at the level of the muscle. The HR and HHb kinetics did not change between hypoxia and normoxia.

The Growth Arrest and DNA Damage-inducible 45 (GADD45) proteins have been implicated in regulation of many cellular functions including DNA repair, cell cycle control, senescence and genotoxic stress. However, the pro-apoptotic activities have also positioned GADD45 as an essential player in oncogenesis. Emerging functional evidence implies that GADD45 proteins serve as tumor suppressors in response to diverse stimuli, connecting multiple cell signaling modules. Defects in the GADD45 pathway can be related to the initiation and progression of malignancies. Moreover, induction of GADD45 expression is an essential step for mediating anti-cancer activity of multiple chemotherapeutic drugs and the absence of GADD45 might abrogate their effects in cancer cells. In this review, we present a comprehensive discussion of the functions of GADD45 proteins, linking their regulation to effectors of cell cycle arrest, DNA repair and apoptosis. The ramifications regarding their roles as essential and central players in tumor growth suppression are also examined. We also extensively review recent literature to clarify how different chemotherapeutic drugs induce GADD45 gene expression and how its up-regulation and interaction with different molecular partners may benefit cancer chemotherapy and facilitate novel drug discovery.

Abstract: Surface waters are increasingly utilized for drinking water because groundwater sources are often polluted. Several monitoring studies have detected the presence of mutagenicity in drinking water, especially from surface sources due to the reaction of natural organic matter with disinfectant. The study aimed to investigate the genotoxic potential of the products of reaction between humic substances, which are naturally present in surface water, and three disinfectants: chlorine dioxide, sodium hypochlorite and peracetic acid. Commercial humic acids dissolved in distilled water at different total organic carbon (TOC) concentrations were studied in order to simulate natural conditions of both ground water (TOC=2.5 mg/L) and surface water (TOC=7.5 mg/L). These solutions were treated with the biocides at a 1:1 molar ratio of C:disinfectant and tested for genotoxicity using the anaphase chromosomal aberration and micronucleus tests in Allium cepa, and the Vicia faba and Tradescantia micronucleus tests. The tests were carried out after different times and with different modes of exposure, and at 1:1 and 1:10 dilutions of disinfected and undisinfected humic acid solutions. A genotoxic effect was found for sodium hypochlorite in all plant tests, at both TOCs considered, while chlorine dioxide gave positive results only with the A.cepa tests. Some positive effects were also detected for PAA (A.cepa and Tradescantia). No relevant differences were found in samples with different TOC values. The significant increase in all genotoxicity end-points induced by all tested disinfectants indicates that a genotoxic potential is exerted even in the presence of organic substances at similar concentrations to those frequently present in drinking water.

This study aimed at questioning the validity of the ventilatory method to determine the anaerobic threshold (respiratory compensation point [RCP]) during an incremental roller-ski skating test to exhaustion. Nine elite crosscountry skiers were evaluated. The skiers carried out an incremental roller-ski test on a treadmill with the V2 skating technique. Ventilatory parameters were continuously collected breath by breath, thanks to a portable gas exchange measurement system. Poling signal was obtained using instrumented ski poles. For each stage, ventilatory and poling signals were synchronized and averaged. The poor coefficient of interobserver reliability for the time at RCP confirmed the great difficulty felt by the 3 blinded reviewers for the RCP determination. Moreover, the reviewer agreed with the impossibility of determining RCP in 4 of the 9 skiers. There was no significant difference between breathing frequency (Bf) and poling frequency (Pf) during the last 8 stages. However, it seems that the differences observed during the first stages arose from the use of either a strictly 1:1 or a 1:2 Bf to Pf ratio when the exercise intensity was still moderate. So, even if there were significant differences between the frequencies, the Bf was strictly subordinate to the Pf during the entire test. In the same way, the normalized tidal volume and peak poling forces curves were superposable. These findings showed that when the upper body is mainly involved in the propulsion, the determinants of the ventilation are strictly dependent on the poling pattern during an incremental test to exhaustion. Thus, during roller-ski skating, the determination of RCP must be used cautiously because too much depending on mechanical factors.

Aim: The purpose of this study is to estimate the energy cost of ski mountaineering at different speeds under laboratory conditions., Methods: By using roller skis on a motorized treadmill we have estimated the energy cost and biomechanics parameters of ski mountaineering as a function of climbing speed at the gradient of 21%., Results: The metabolic energy spent for unit mass and distance, C, shows a broad minimum of about 10.6±0.2 J kg-1m-1 at roughly 3.5 km h-1. In addition we find a size-dependent effect: tall subjects spend less metabolic energy for unit mass and distance than small subjects at the same speed., Conclusion: The value of C measured in laboratory agrees with that obtained in the field at the preferred speed. This shows that skiers self select a speed that minimizes their metabolic cost. The dependence of C on the subject's size is explained by a simple model of the skier's dynamics. In addition we have calculated the ratio between mechanical work and metabolic energy, which may give some hints on the efficiency as a function of the speed. It turns out that efficiency increases with the speed up to a maximum located at around 4.5 km/h.

Clostridium difficile, associated with a wide spectrum of diseases in humans, as well as in several animal species, is an important cause of colitis in adult horses and foals. The aim of this study was to investigate by toxin gene profile and PCR-ribotyping the molecular characteristics of 14 C. difficile strains isolated from 42 faeces of healthy horses. Both toxin genes, tcdA and tcdB, were present in only 1 isolate (7.1%). Six isolates (42.9%) demonstrated tcdA-/tcdB+ genotype, and seven isolates (50.0%) were tcdA-/tcdB-. All strains were binary toxin genes negative (cdtA-/cdtB-). The PCR-positive strains, except for the tcdA+/tcdB+ isolate, tested negative for, in vitro, A and/or B toxins production by EIA. Eleven distinct ribotypes were observed. In conclusion, C. difficile can be present in the normal intestinal flora of healthy adult horses, in addition to foals. These animals could therefore play an important role as potential reservoirs of toxigenic strains., (Published by Elsevier Ltd.)

In this study, we evaluated the NTPDases and ecto-5'-nucleotidase (CD73) expression profiles and the pattern of adenine nucleotide hydrolysis in rats submitted to the Walker 256 tumor model, 6, 10 and 15 days after the subcutaneous inoculation. Using RT-PCR analysis, we identified mRNA for all of the members of the ecto-nucleoside triphosphate diphosphohydrolase family investigated and a 5'-nucleotidase. By quantitative real-time PCR, Entpd1 (Cd39) and Entpd2 (Cd39L1) and CD73 were identified as the dominant genes expressed by the Walker 256 tumor, at all times studied. Extracellular adenine nucleotide hydrolysis by the Walker 256 tumor was estimated by HPLC analysis. Rapid hydrolysis of extracellular ATP by the tumor cells was observed, leading to the formation of adenosine and inosine in cells obtained from solid tumors at 6 and 10 days after inoculation. Cells obtained from solid tumors at 15 days of growth presented high levels of AMP and presented adenosine as a final product after 90 min of incubation. Results demonstrate that the presence of NTPDases and 5'-nucleotidase enzymes in Walker 256 tumor cells may be important for regulation of the extracellular adenine nucleotides/adenine nucleoside ratio, therefore leading to tumor growth.

Background and Aim of the Work: In recent years, the diagnosis of toxoplasmosis has been improved by Real-time PCR assays. In this study we compared the performances of two Real-time PCRs (FRET and TaqMan protocols) already described in the literature, and one nested-PCR, currently used in our laboratory for the molecular diagnosis of toxoplasmosis., Methods: We evaluated the sensitivity and the specificity of a FRET- and a TaqMan-based Real-time PCRs targeting a 529 bp repeat region and the 18S RNA gene, respectively, and a nested-PCR, targeting the B1-gene of Toxoplasma gondii. We also tested, through nested-PCR, 46 biological samples obtained during a period of 29 months from pregnant women or immunocompromised patients with suspected T. gondii infection., Results: The analytical sensitivity of nested and TaqMan PCRs was approximately 10(3) tachyzoites/ml. FRET assay showed a sensitivity of 102 tachyzoites/ml. Three out of 46 biological samples were nested-PCR-positive and these results were also confirmed by both Real-time PCRs., Conclusions: Nested- and real-time PCRs evaluated in this study resulted very sensitive and specific; in particular FRET PCR resulted more sensitive than the other assays, probably because of the greater copy number of the target sequence. Real-time PCR assays are easy-to-use, producing results faster than conventional PCR systems and reducing contamination risks.

Inflammatory and degenerative pathophysiological processes within the CNS are important causes of human disease. Astrocytes appear to modulate these reactions and are a major source of inflammatory mediators, e.g. extracellular adenine nucleotides, in nervous tissues. Actions following extracellular nucleotides binding to type 2 purinergic receptors are regulated by ectonucleotidases, including members of the CD39/ecto-nucleoside triphosphate diphosphohydrolase family. The ectonucleotidases of astrocytes expressed by rat brain rapidly convert extracellular ATP to ADP, ultimately to AMP. RT-PCR, immunocytochemistry as well as Western blotting analysis demonstrated expression of multiple ecto-nucleoside triphosphate diphosphohydrolase family members at both the mRNA and protein level. By quantitative real-time PCR, we identified Entpd2 (CD39L1) as the dominant Entpd gene expressed by rat hippocampal, cortical and cerebellar astrocytes. These data in combination with the elevated ecto-ATPase activity observed in these brain regions, suggest that NTPDase2, an ecto-enzyme that preferentially hydrolyzes ATP, is the major ecto-nucleoside triphosphate diphosphohydrolase expressed by rat astrocytes. NTPDase2 may modulate inflammatory reactions within the CNS and could represent a useful therapeutic target in human disease.

Human rotavirus (HRV) is recognized as the most common cause of severe gastroenteritis in children under 5 years of age. Due to the lack of recent reports about the surveillance of HRV infection in Italy, in this study we assessed the prevalence rate of HRV infection on 1,340 stool samples belonging to 1,264 pediatric patients hospitalized with acute gastroenteritis in the period January 2000--December 2002. The stool samples were submitted to virological investigations by electron microscopy (EM) and conventional cell culture, as well as from January 2002 by RT-PCR for norovirus detection. Reovirus-like particles observed by EM were identified by electropherotyping. Single HRV infections were detected in 302 cases (23.9%, ranging from 19.1% in 2000 to 30.2% in 2001). Mixed infections were observed in 28 cases in which HRV was found to be associated with adenovirus in 16 cases (1.3%), with picornavirus in 4 (0.3%), and with norovirus in 8 (2.1% of the 388 cases examined in 2002). The 3 major epidemic periods of HRV infections were March--May 2000 (66 cases), December 2000--May 2001 (128 cases) and September 2001--April 2002 (105 cases) with peaks in March, January and March, and January, respectively. In the periods of major incidence, single HRV infection accounted even for 52.5% of the gastroenteritis cases monthly examined. According to age distribution, 68.9% (208 cases) of HRV infected children was under 4 years (69.6%: 230/330 cases, including mixed infections) and 16.9% (51 cases) was in the 5-12-year age-group. The epidemiological aspects of HRV infection, also compared to other enteric virus infections, will contribute to assess the magnitude of the problem of HRV in different settings and to devise strategies for intervention.

Objective: To investigate the expression of the novel Ets transcription factor ESE-1 in rheumatoid synovium and in cells derived from joint tissues, and to analyze the role of nuclear factor kappaB (NF-kappaB) as one of the central downstream targets in mediating the induction of ESE-1 by proinflammatory cytokines., Methods: ESE-1 protein expression was analyzed by immunohistochemistry using antibodies in synovial tissues from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). ESE-1 messenger RNA (mRNA) levels were analyzed by reverse transcriptase-polymerase chain reaction or Northern blotting in human chondrocytes, synovial fibroblasts, osteoblasts, and macrophages, before and after exposure to interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), or lipopolysaccharide (LPS) with or without prior infection with an adenovirus encoding the inhibitor of nuclear factor kappaB (IkappaB). The wild-type ESE-1 promoter and the ESE-1 promoter mutated in the NF-kappaB site were cloned into a luciferase reporter vector and analyzed in transient transfections. Electrophoretic mobility shift assays (EMSAs) and supershift assays with antibodies against members of the NF-kappaB family were conducted using the NF-kappaB site from the ESE-1 promoter as a probe., Results: Immunohistochemical analysis showed specific expression of ESE-1 in cells of the synovial lining layer and in some mononuclear and endothelial cells in RA and OA synovial tissues. ESE-1 mRNA expression could be induced by IL-1beta and TNFalpha in cells such as synovial fibroblasts, chondrocytes, osteoblasts, and monocytes. Transient transfection experiments and EMSAs showed that induction of ESE-1 gene expression by IL-1beta requires activation of NF-kappaB and binding of p50 and p65 family members to the NF-kappaB site in the ESE-1 promoter. Overexpression of IkappaB using an adenoviral vector blocked IL-1beta-induced ESE-1 mRNA expression. Chromatin immunoprecipitation further confirmed that NF-kappaB binds to the ESE-1 promoter in vivo., Conclusion: ESE-1 is expressed in synovial tissues in RA and, to a variable extent, in OA, and is specifically induced in synovial fibroblasts, chondrocytes, osteoblasts, and monocyte/macrophages by IL-1beta, TNFalpha, or LPS. This induction relies on the translocation of the NF-kappaB family members p50 and p65 to the nucleus and transactivation of the ESE-1 promoter via a high-affinity NF-kappaB binding site. ESE-1 may play a role in mediating some effects of proinflammatory stimuli in cells at sites of inflammation.

Adenoviruses have been used for gene therapy or immunization due to their ability to efficiently infect a broad range of cells and tissues. These applications as well as specificity could be improved further by redirecting binding of the virus to specific cell types. In this regard, modification of viral genes encoding coat proteins is an option to achieve improvement in retargeting. In this report, we describe a substitution in the adenovirus type 2 fiber globular region by the 44 amino acid C4 domain of human immunodeficiency virus type 1 gp120. In vitro translation analysis and immunoprecipitation assays show that the incorporation of the C4 domain into the fiber protein does not ablate its trimerization property and demonstrates the availability of the C4 epitope for interaction with monoclonal anti-C4 antibody. The recombinant adenovirus containing this modified fiber was also characterized by immunoprecipitation with the same antibody, showing the viability of such kind of modification.

Three GST fusion recombinant antigen of Treponema pallidum, described as GST-rTp47, GST-rTp17 and GST-rTp15 were analyzed by Western blotting techniques. We have tested 53 serum samples: 25 from patients at different clinical stages of syphilis, all of them presenting anti-treponemal antibody, 25 from healthy blood donors and three from patients with sexually transmitted disease (STD) other than syphilis. Almost all samples from patients with syphilis presented a strong reactivity with GST-rTp17 antigen. Some samples were non-reactive or showed a weak reaction with GST-rTp47 and/or GST-rTp15, and apparently there was no correlation with the stage of disease. There was no seropositivity among blood donors. No sample reacted with purified GST. We concluded that due to their specificity these recombinant antigens can be used as GST fusion protein for development of syphilis diagnostic assays.