MONOGRAFIA – Le colture cellulari in ambito biomedico-diagnostico

Indagini citofluorimetriche nella vitalità e morte cellulare. I. Necrosi, apoptosi e proliferazione cellulare

Francesca Luchetti*, Barbara Canonico, Letizia Biagiarelli, Lucia Bucci, Massimo Valentini, Stefano Pap

Dipartimento di Scienze dell’Uomo, Ambiente e Natura, Università degli Studi di Urbino “Carlo Bo”, Urbino
Biochimica Clinica: 2009;33(2):83-92 [Article in italian]

ABSTRACT
Flow cytometry assays for viability and cell death. I. Necrosis, apoptosis and cell proliferation. Today, several assays and antibodies are available for the analysis of apoptosis, cell cycle and DNA repair, cell viability and vitality, proliferation, migration, adhesion, endo- and exocytosis. Many of the assays are fluorescence- or colorimetric-based, offering sensitivity, practicability, as well as safety. These reagents have been validated on multiple instrument platforms, including microscopy and flow cytometry analysers. Here we describe some advances in application of flow cytometry particularly related to cell cycle analysis and cell proliferation studies, giving some general information and reporting protocols currently used in laboratories. Particularly, fluorescent probes have been organized into categories based on different aspects of apoptosis, necrosis and cell proliferation.

Indagini citofluorimetriche nella vitalità e morte cellulare. II. Studio di altre funzioni cellulari

Barbara Canonico*, Francesca Luchetti, Marcella Arcangeletti, Massimo Valentini, Stefano Papa

Dipartimento di Scienze dell’Uomo, Ambiente e Natura, Università degli Studi di Urbino “Carlo Bo”, Urbino

Biochimica Clinica: 2009;33(2):93-103 [Article in italian]

ABSTRACT
Flow cytometry assays for viability and cell death. II. Evalutaion of other cellular functions. In this second companion paper, we review some advances in application of flow cytometry to evaluate calcium flux, cell membrane, lysosome compartment, mitochondrial membrane mass and potential, giving some general information and reporting operative protocols.

Marcatori di danno cellulare linfocitario e loro possibili applicazioni in ambito biomedico-diagnostico

Riccardo Ientile

Dipartimento di Scienze Biochimiche, Fisiologiche e della Nutrizione, Università di Messina, Policlinico Universitario, Messina

Biochimica Clinica: 2009;33(2):104-111 [Article in italian]

ABSTRACT
Biomarkers of lymphocyte damage and their possible clinical application. Mature T cells are long-lived cells that are maintained through continuous contact with exogenous ligands and certain cytokines. However, after leaving the thymus as naïve cells, T cells can survive for prolonged periods as re-circulating resting cells. The mitogen-stimula-ted cultures of lymphocytes can be used as a model system to study biochemical changes that may be associated to alterations in proliferative phases of the cell cycle. This state of immune/proliferative activation makes cells more susceptible to mitogen-induced apoptosis. Evidence for changes in the levels of some biomarkers related to perturbations of lymphocyte proliferative homeostasis leading to programmed cell death is reported in this review. Some of these results are relevant in the chronic HIV infection and in cell response to alterations in oxidative status. Indeed, alterations in the expression of cell cycle-related proteins are consistently observed in CD4 and CD8 cells from HIV-infected patients after in vitro mitogenic stimulation. Furthermore, senescence is associated with progressive decline in immune functions, which is assigned in part to increased apoptosis. Recent results demonstrate that the nuclear factor (NF)-κB is one of the transcription factor that play an important role in the regulation of immune response genes. The activation of NF-κB pathway has been shown to inhibit apoptosis in human lymphocytes. Thereby, defects in NF-κB signaling pathway appear to be responsible for increased tumor necrosis factor-a-induced apoptosis in lymphocytes from aged humans. These findings may provide valuable information for evaluating a link between the perturbation of lymphocyte proliferative homeostasis and the activation of apoptotic machinery.

Utilizzo dei fibroblasti in coltura nella diagnostica biochimica delle malattie da accumulo liso somale

Vanna Chigorno*, Marina Pitto, Sandro Sonnino, Giancarlo Goi

Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Università di Milano, Milano

Biochimica Clinica: 2009;33(2):112-121 [Article in italian]

ABSTRACT
Use of cultured fibroblasts for the biochemical diagnosis of lysosomal metabolic diseases. This review delineates the main advantages deriving from the use of cultured fibroblasts for the biochemical diagnosis of metabolic diseases. Fibroblasts can be frozen in liquid nitrogen and successively re-plated and cultured without undergoing metabolic modifications, thus making possible the execution of comparative metabolic studies on viable cells. Furthermore, the use of these cells for the biochemical diagnosis of lysosomal diseases is described and the specific techniques for their recognition, principally using radioactive and/or fluorescent substrates, are summarized.

I fibroblasti in coltura nella diagnostica delle malattie mitocondriali su base Genetica

Barbara Garavaglia, Valeria Tiranti

Centro per lo Studio delle Malattie Mitocondriali Pediatriche, U.O. Neurogenetica Molecolare, Fondazione IRCCS Istituto Neurologico “C. Besta”, Milano

Biochimica Clinica: 2009;33(2):122-128 [Article in italian]

ABSTRACT
Cultured fibroblasts in the diagnosis of genetic mitochondrial diseases. Defects of respiratory chain carrying out oxidative phosphorylation (OXPHOS) are the biochemical hallmarks of human mitochondrial disorders. From the genetic point of view, these diseases can be caused by mutations in the mitochondrial DNA (mtDNA) or in the vast repertoire of nuclear genes, which products are functionally related to the OXPHOS. mtDNA encodes only 13 of the ~85 polypeptides comprising the OXPHOS complexes, plus two rRNA and 22 tRNA required for the partially autonomous mitochondrial translational apparatus. While the gene products encoded by mtDNA are essential, they comprise only a tiny fraction of the total number of proteins involved in the functional respiratory chain. All other mitochondrial proteins, including those required for mtDNA maintenance and expression, OXPHOS assembly, and biosynthesis of cofactors, are nuclear−encoded and imported to mitochondria. We have worked out cellular systems in which the nuclear or mitochondrial origin of an OXPHOS defect can be persuasively demonstrated. These systems are based on the creation of cybrids and hybrids that are also used to characterize, by biochemical and molecular-genetic approaches, the pathogenicity of mutations affecting mtDNA.

Colture di cellule staminali neurali come modello per lo studio di patologie del sistema nervoso, studi farmacologici e di neurotossicità

Emanuele Cacci, Stefano Biagioni

Dipartimento di Biologia Cellulare e dello Sviluppo, Università La Sapienza, Roma

Biochimica Clinica: 2009;33(2):129-136 [Article in italian]

ABSTRACT
Neural stem cell coltures as model to study neurological diseases and evaluate drug neurotoxicity. New effective therapies are required to treat chronic and acute brain pathologies. Clinical intervention strategies aimed to increase mobilization of neural stem cells (NSC) located in the adult mammalian brain and to strengthen neurogenesis have been proposed as possible regenerative therapy in several brain pathologies. Though in vivo studies are mandatory, human and rodents NSC cultures constitute an important complementary model to identify the effects of endogenous (e.g. pro-inflammatory cytokines produced in the lesioned brain) and exogenous molecules (e.g. drugs) on neurogenesis. In this review we illustrate the use of NSC as a cellular model to clarify mechanisms implicated in brain disease and to screen the efficacy of new drugs. We will also present some studies that highlight the rising interest around embryonic inducible pluripotent cells (iPS) as cellular models for studying brain pathologies in vitro.

Characterization of 30 low cryocrit cryoglobulins in patients with antibodies against hepatitis C virus

Umberto Basile, Monica Di Nunno, Francesca Gulli, Roberta Gilestri

Laboratorio di Immunologia, Istituto di Patologia Generale, Università Cattolica del Sacro Cuore, Roma

Biochimica Clinica: 2009;33(2):137-140 [Article in english]

ABSTRACT

Mixed cryoglobulinemia, i.e. the presence of IgG and/or IgM precipitating at temperature <37 °C and dissolving in serum on reheating, is strongly associated with hepatitis C virus (HCV) infection. We studied by immunofixation electrophoresis (IFE) monoclonal and/or polyclonal patterns of cryoglobulins in 30 patients with low cryocrit and positive anti-HCV test. Ten out of 30 IFEs presented an unexpected pattern, characterized by tiny monoclonal components and more than one polyclonal immunoglobulin of different isotype. Two patients showed two monoclonal components of different immunoglobulin classes and in a single case, a monoclonal IgM was associated with 3 different monoclonal IgG and, therefore, these cryoglobulins could be classified as type II, subtype IIb. Twelve cases showed polyclonal immunoglobulins (type III) and 5 cryoprecipitates were classified as type IIa, consisting of polyclonal IgG and monoclonal IgM. The results show the IFE role to accurately identify cryoglobulin patterns, mainly some specific cryoglobulin subtypes, improving thus the patient management and outcome.

Confronto tra metodo FPIA (“fluorescent polarized immunoassay”) e metodo CEDIA (“cloned enzyme donor immunoassay”) per la determinazione della ciclosporina A

Antonio Colatutto, Laura De Luca, Luca Isola, Nadia Pradal, Rodolfo Zaglia, Alice Lerussi, Barbara Marcon, Pierguido Sala

Dipartimento di Diagnostica di Laboratorio, Azienda Ospedaliero-Universitaria S. Maria della Misericordia, Udine

Biochimica Clinica: 2009;33(2):141-143 [Article in italian]

ABSTRACT

Comparison between FPIA and CEDIA techniques for cyclosporine A determination. Cyclosporin A (CsA) is a powerful immunosuppressant agent characterized by a narrow therapeutic range. Therefore, therapeutic drug monitoring is a crucial step for CsA treatment. Even if HPLC has been considered the standard technique for CsA determination, immunometric methods have replaced HPLC in clinical laboratory. The purpose of this study is to compare CEDIA and FPIA methods for CsA measurement. We assayed 97 whole blood samples, collected with K2EDTA as anticoagulant, from patients undergoing immunosuppressive therapy based on CsA. We found a good correlation between methods, but significant systematic and proportional biases, which denote a lack of result comparability.

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