Laboratorio Of Biotecnologie

Pavia, Italy

Laboratorio Of Biotecnologie

Pavia, Italy
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Borghini S.,CNR Institute of Molecular Genetics | Borghini S.,University of Genoa | Borghini S.,Instituto G Gaslini | Borghini S.,Laboratorio Of Biotecnologie | And 60 more authors.
Journal of Rheumatology | Year: 2011

Objective. Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is an autosomal-dominant multisystemic autoinflammatory condition. Patients display different mutations of the TNF receptor superfamily 1A gene (TNFRSF1A), coding for a nearly ubiquitous TNF receptor (TNFR1). No TNFRSF1A mutation has been identified in a proportion of patients with TRAPS-like phenotype. Methods. We investigated mechanisms downregulating the TNF-induced inflammatory response such as (1) receptor shedding, producing a secreted form acting as a TNF inhibitor; (2) receptor internalization with subsequent induction of apoptosis; and (3) negative regulation of nuclear factor-κB (NF-κB) transcription. We analyzed the sequence of genes known to play a pivotal role in these pathways, in 5 patients with TRAPS symptoms and showing shedding and/or apoptosis defects, but without mutations of the TNFRSF1A gene. Results. Sequence analysis of 3 genes involved in TNFR1 shedding (ERAP1, NUCB2, RBMX) and 3 genes involved in negative regulation of NF-κB signaling (TNFAIP3, CARP-2) or NF-κB transcription (ZFP36) revealed only a few unreported variants, apparently neutral. Conclusion. Our study rules out any involvement in the pathogenesis of TRAPS of some of the genes known to regulate TNFR1 shedding and TNF-induced NF-κB signaling and transcription. Gene(s) responsible for TRAPS-like syndrome remain to be investigated among currently unidentified genes likely involved in these pathways, or by applying the genome-wide function-free sequencing approach. The Journal of Rheumatology Copyright © 2011. All rights reserved.

Ghidini C.,Biotechnology Laboratory | Zanotti C.,Biotechnology Laboratory | Boccacci S.,Spedali Civili | Lanfranchi A.,Spedali Civili | And 3 more authors.
Diagnostic Molecular Pathology | Year: 2011

The presence of myxovirus resistance protein A (MxA) RNA was studied in 55 febrile children with primary immunodeficiency, 27 of whom underwent hematopoietic cell transplantation, and in 28 age-matched controls. The level of MxA RNA was above the cutoff, established as the 95th percentile found in controls, with primary immunodeficiency either undergoing transplantation or not in febrile patients, and with a documented diagnosis of infection by adenovirus, cytomegalovirus, Epstein-Barr virus, respiratory syncytial virus, and rotavirus. The presence of rare viral infections, unrecognized among those that more frequently occur in patients with primary immunodeficiency and in patients undergoing transplantation, may explain the high MxA RNA levels observed in some patients with fever but undetectable genomes or antibodies for the more common viruses. The level of MxA in febrile patients with acute graft versus host disease was below the cutoff, with a median level comparable with that observed in patients with primary immunodeficiency, who did not undergo transplantation and were without fever and infections, but significantly lower compared with controls. The level of MxA was well correlated with viral infections in follow-up samples. These data indicate that the measurement of MxA RNA is simple and useful to detect viral infections and in distinguishing them from acute graft versus host disease after allogeneic cell transplantation. Copyright © 2011 by Lippincott Williams & Wilkins.

Gervasoni A.,Fondazione Poliambulanza Instituto Ospedaliero | Sandri M.T.,Italian National Cancer Institute | Nascimbeni R.,University of Brescia | Zorzino L.,Italian National Cancer Institute | And 6 more authors.
Oncology Reports | Year: 2011

The detection of circulating tumor cells (CTCs) has considerable utility in the clinical management of patients with solid cancers. However, the phenotypic heterogeneity of CTCs and their low numbers in the bloodstream of patients means that no standardized detection method currently exists for these cells. This, together with differences in pre-analytical sample processing, has led to the collection and accumulation of inconsistent data among independent studies. Here, we compare the ability of three methods to detect CTCs in the blood of colorectal cancer patients. Specifically, different aliquots of the same blood sample were screened for the presence of CTCs by a multimarker RT-PCR assay, the standardized CellSearch assay and dHPLC-based gene mutation analysis. In the population tested, none of the blood samples analysed appeared to be positive by all three methods. Of the samples, 75% were positive for the presence of CTCs by the RT-PCR method. Only 20% were positive by the CellSearch assay, while 14.3% of samples displayed gene mutations consistent with the presence of CTCs when the dHPLC method was applied. The samples which were positive for CTCs by the CellSearch assay did not overlap with those that were positive by dHPLC. Interestingly, however, all of these samples were positive when assessed by RT-PCR. Conversely, of the samples that resulted negative by RT-PCR analysis, none appeared to be positive by either of the other methods. These data, therefore, indicate that of the three methods tested, the multimarker RT-PCR assay provides maximal probability of CTC detection. Here, we present the preliminary results of an ongoing clinical study. Future follow-up involving detection of CTCs in the blood of colorectal cancer patients using these three distinct methods will allow us to verify whether either a single method, or a combination of different assays, is necessary to uncover further prognostic significance of circulating tumor cells. Copyright © 2011 Spandidos Publications Ltd. All rights reserved.

Mercati F.,Sezione di Anatomia Veterinaria | Dall'Aglio C.,Sezione di Anatomia Veterinaria | Pascucci L.,Sezione di Anatomia Veterinaria | Boiti C.,Laboratorio Of Biotecnologie | Ceccarelli P.,Sezione di Anatomia Veterinaria
Acta Histochemica | Year: 2012

In veterinary medicine, there is an increasing interest in the study of the endo-cannabinoid system and the possible use of the cannabinoids for the treatment of several diseases. Cannabinoid receptors (CB) are widely distributed in human and laboratory animal tissues, justifying the involvement of the endo-cannabinoid system in a great number of metabolic ways. Since there are no data regarding cannabinoid receptors in hair follicles of domestic animals, we investigated the presence and localization of CB1 receptor in dog hair follicles. By using a goat anti-CB1 polyclonal antibody, we observed CB1 receptor in the proximal part of both primary and secondary hair follicles. Staining was localized in the inner root sheath cells. We suppose that the endo-cannabinoid system is involved in the molecular mechanisms regulating hair follicle activity in dog. The identification of CB1 receptor at the level of the inner root sheath may help in the understanding of hair follicle biology and the possibility that cannabinoid molecules could be considered as suitable therapeutic tools in dog. © 2011 Elsevier GmbH.

Chiarini M.,Laboratorio Of Biotecnologie | Sottini A.,Laboratorio Of Biotecnologie | Ghidini C.,Laboratorio Of Biotecnologie | Zanotti C.,Laboratorio Of Biotecnologie | And 7 more authors.
Multiple Sclerosis | Year: 2010

The immunomodulating activity of glatiramer acetate on T-cells of multiple sclerosis patients has only been partially clarified. The objective of this work was to investigate whether glatiramer acetate modifies thymic release of newly produced T-cells and the peripheral composition of the T-cell repertoire. T-cell receptor excision circles, thymic naive (CD4+CD45RA +CCR7+CD31+) T helper cells, and central (CD4+CD45RA-CCR7+) and effector (CD4 +CD45RA-CCR7-) memory T-cells were evaluated in 89 untreated patients, 84 patients treated for at least 1 year, and 31 patients beginning treatment at the time of inclusion in the study and then followed-up for 12 months; controls were 81 healthy donors. The T-cell repertoire was analysed in selected samples. The percentage of thymicnaive T helper cells was diminished in untreated patients, but rose to control values in treated subjects; a decrease in central memory T-cells was also observed in treated patients. Follow-up patients could be divided into two subgroups, one showing unmodified thymicnaive T helper cells and T-cell diversity, the other in which the increased release of new T-cells was accompanied by modifications of the T-cell repertoire. Glatiramer acetate modifies the peripheral T-cell pool by activating a thymopoietic pathway of T-cell release that leads to a different setting of T-cell diversity and, likely, to a dilution of autoreactive T-cells.

Crimi G.,Fondazione Instituto Of Ricovero E Cura A Carattere Scientifico Irccs | Pica S.,Fondazione Instituto Of Ricovero E Cura A Carattere Scientifico Irccs | Raineri C.,Fondazione Instituto Of Ricovero E Cura A Carattere Scientifico Irccs | Bramucci E.,Fondazione Instituto Of Ricovero E Cura A Carattere Scientifico Irccs | And 13 more authors.
JACC: Cardiovascular Interventions | Year: 2013

Objectives This study sought to evaluate whether remote ischemic post-conditioning (RIPC) could reduce enzymatic infarct size in patients with anterior ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention (pPCI). Background Myocardial reperfusion injury may attenuate the benefit of pPCI. In animal models, RIPC mitigates myocardial reperfusion injury. Methods One hundred patients with anterior ST-segment elevation myocardial infarction and occluded left anterior descending artery were randomized to pPCI + RIPC (n = 50) or conventional pPCI (n = 50). RIPC consisted of 3 cycles of 5 min/5 min ischemia/reperfusion by cuff inflation/deflation of the lower limb. The primary endpoint was infarct size assessed by the area under the curve of creatinine kinase-myocardial band release (CK-MB). Secondary endpoints included the following: infarct size assessed by cardiac magnetic resonance delayed enhancement volume; T 2-weighted edema volume; ST-segment resolution >50%; TIMI (Thrombolysis In Myocardial Infarction) frame count; and myocardial blush grading. Results Four patients (2 RIPC, 2 controls) were excluded due to missing samples of CK-MB. A total of 96 patients were analyzed; median area under the curve CK-MB was 8,814 (interquartile range [IQR]: 5,567 to 11,325) arbitrary units in the RIPC group and 10,065 (IQR: 7,465 to 14,004) arbitrary units in control subjects (relative reduction: 20%, 95% confidence interval: 0.2% to 28.7%; p = 0.043). Seventy-seven patients underwent a cardiac magnetic resonance scan 3 to 5 days after randomization, and 66 patients repeated a second scan after 4 months. T2-weighted edema volume was 37 ± 16 cc in RIPC patients and 47 ± 22 cc in control subjects (p = 0.049). ST-segment resolution >50% was 66% in RIPC and 37% in control subjects (p = 0.015). We observed no significant differences in TIMI frame count, myocardial blush grading, and delayed enhancement volume. Conclusions In patients with anterior ST-segment elevation myocardial infarction, RIPC at the time of pPCI reduced enzymatic infarct size and was also associated with an improvement of T 2-weighted edema volume and ST-segment resolution >50%. (Remote Postconditioning in Patients With Acute Myocardial Infarction Treated by Primary Percutaneous Coronary Intervention [PCI] [RemPostCon]; NCT00865722) © 2013 by the American College of Cardiology Foundation.

Bertanza G.,University of Brescia | Pedrazzani R.,University of Brescia | Papa M.,University of Brescia | Mazzoleni G.,University of Brescia | And 4 more authors.
Ozone: Science and Engineering | Year: 2010

Removal potential of many EDCs by conventional WWTPs is recognized. Nevertheless, in order to reach very low concentrations, further treatment (e.g., chemical oxidation) might be required. In this work, two estrogen-like substances were considered: nonylphenol (NP) (and its parent compounds) and bisphenol A (BPA). The experimental work was conducted at Verona (Northern Italy) WWTP (370,000 p.e.): the effluent was submitted to ozonation in a pilot scale plant. Beside chemical and microbiological analyses, estrogenic activity measurements were also carried out by means of breast cancer MCF-7 cells transfected with the luminescence luciferase gene. Economic feasibility of tertiary ozonation is finally discussed. © 2010 International Ozone Association.

Serana F.,University of Brescia | Airo P.,University of Brescia | Chiarini M.,Laboratorio Of Biotecnologie | Zanotti C.,Laboratorio Of Biotecnologie | And 6 more authors.
Journal of Clinical Immunology | Year: 2011

Objective The study aims to obtain more information about the immune deficit of common variable immunodeficiency (CVID) patients. Materials and Methods A new real-time PCR assay was used to quantify T and B lymphocyte mobilization from the production and maturation sites through the detection of T cell receptor excision circles (TRECs) and kappa-deleting recombination circles (KRECs) and to allow the estimation of the average number of B cell divisions. T and B lymphocyte subsets were analyzed by flow cytometry. Results The number of TREC + lymphocytes, which depends on age and gender, was significantly reduced in CVID patients. Similarly, KREC concentration was lower than in controls. Classification of patients according to the percentage of memory switched B cells showed that patients belonging to MB2 group and therefore with conserved B cell maturation have the lowest new B cell output but increased average peripheral divisions, leading to the highest B cell number. Conclusions TREC and KREC quantification can be helpful for a more complete and informative understanding of a heterogeneous disease such as CVID. © Springer Science+Business Media, LLC 2011.

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