Alteration of glyoxalase genes expression in response to testosterone in LNCaP and PC3 human prostate cancer cells.

Glyoxalase system, a ubiquitous detoxification pathway protecting against cellular damage caused by potent cytotoxic metabolites, is involved in the regulation of cellular growth. Aberrations in the expression of glyoxalase genes in several human cancers have been reported. Recently, we described a possible regulatory effect by estrogens on glyoxalase genes in human breast cancer cell lines. This result, along with those ones regarding changes in glyoxalases activity and expression in other human hormone-regulated cancers, such as prostate cancer, has prompted us to investigate whether also androgens, whose functional role in prostate cancer pathogenesis is well known, could modulate glyoxalases gene expression. Therefore, we treated LNCaP androgen-responsive and PC3 androgen-independent human prostate cancer cell lines with testosterone at the concentrations of 1 nM and 100 nM. After a two days treatment, glyoxalases mRNA levels as well as cell proliferation were evaluated by real-time RT-PCR analysis and [3H]thymidine incorporation, respectively. Results pointed out that testosterone affects the expression of glyoxalase system genes and cell proliferation in a different manner in the two cell lines. The possibility that modulation of glyoxalase genes expression by testosterone is due to glyoxalases-mediated intracellular response mechanisms to the androgen-induced oxidative stress or to the presence of androgen response elements (ARE) in glyoxalase promoters are discussed. Knowledge regarding the regulation of glyoxalases by testosterone may provide insights into the importance of these enzymes in human prostate carcinomas in vivo

Serum IgE Reactivity Profiling in an Asthma Affected Cohort

BACKGROUND: Epidemiological evidence indicates that atopic asthma correlates with high serum IgE levels though the contribution of allergen specific IgE to the pathogenesis and the severity of the disease is still unclear. METHODS: We developed a microarray immunoassay containing 103 allergens to study the IgE reactivity profiles of 485 asthmatic and 342 non-asthmatic individuals belonging to families whose members have a documented history of asthma and atopy. We employed k-means clustering, to investigate whether a particular IgE reactivity profile correlated with asthma and other atopic conditions such as rhinitis, conjunctivitis and eczema. RESULTS: Both case-control and parent-to-siblings analyses demonstrated that while the presence of specific IgE against individual allergens correlated poorly with pathological conditions, particular reactivity profiles were significantly associated with asthma (p<10E-09). An artificial neural network (ANN)-based algorithm, calibrated with the profile reactivity data, correctly classified as asthmatic or non-asthmatic 78% of the individual examined. Multivariate statistical analysis demonstrated that the familiar relationships of the study population did not affect the observed correlations. CONCLUSIONS: These findings indicate that asthma is a higher-order phenomenon related to patterns of IgE reactivity rather than to single antibody reactions. This notion sheds new light on the pathogenesis of the disease and can be readily employed to distinguish asthmatic and non-asthmatic individuals on the basis of their serum reactivity profile

An antigen microarray immunoassay for multiplex screening of mouse monoclonal antibodies

The mouse monoclonal antibody (mAb) technology still represents a key source of reagents for research and clinical diagnosis, although it is relatively inefficient and expensive and therefore unsuitable for high-throughput production against a vast repertoire of antigens. In this article, we describe a protocol that combines the immunization of individual mice with complex mixtures of influenza virus strains and a microarray-based immunoassay procedure to perform a parallel screening against the viral antigens. The protocol involves testing the supernatants of somatic cell hybrids against a capture substratum containing an array of different antigens. For each fusion experiment, we carried out more than 25,000 antigen-antibody reactivity tests in less than a week, a throughput that is two orders of magnitude higher than that of traditional antibody detection assays such as enzyme-linked immunosorbent assays and immunofluorescence. Using a limited number of mice, we can develop a vast repertoire of mAbs directed against nuclear and surface proteins of several human and avian influenza virus strains

APPLICAZIONE DEI MICROARRAY PROTEICI NELLA DIAGNOSTICA AVANZATA DI PATOLOGIEMICROBICHE E VIRALI

L’immobilizzazione di matrici microscopiche (microarray) di acidi nucleici su substrato solido ha rappresentato un passo avanticruciale per lo sviluppo di saggi multiparametrici che consentissero l’analisi dei livelli di espressione genica di un organismo.Tuttavia l’analisi dell’espressione genica non fornisce indicazioni sull’abbondanza e funzione di biomolecole fondamentali nelladefinizione di un fenotipo e/o nell’evoluzione di un processo patogenetico. Pertanto sono stati messi a punto microarray contenentiproteine, anticorpi, lipidi, carboidrati con applicazioni in ricerca e in diagnostica. In questa ottica nel presente lavoro, ci siè proposti di mettere a punto un sistema di diagnostica avanzata, basato sulla tecnologia del microarray proteico, per la determinazionesimultanea e multiparametrica di IgG e IgM specifiche nei confronti di antigeni microbici importanti nella diagnosticaprenatale. Microarray di IgG e IgM umane ed antigeni microbici vengono deposti su vetrini da microscopio con superficiechimicamente reattiva per mezzo di un sistema robotizzato ad alta precisione. I microarray cosí prodotti vengono incubati consiero e successivamente con anticorpi marcati con fluorofori per la rilevazione del segnale. I vetrini vengono infine analizzaticon uno strumento che combina microscopia laser confocale e ricostruzione digitale dell’immagine. L’intensitá del segnale incorrispondenza degli antigeni viene quantificata utilizzando come riferimento le curve di calibrazione generate depositando sulvetrino quantitá decrescenti di IgG e IgM. Esperimenti di validazione hanno messo in evidenza come il saggio immunologicoper la rilevazione delle IgG dirette contro gli antigeni del complesso ToRCH avesse sensibilitá di 0.5 μg/mL e una precisionetra 1,7% e 14,6% per tutti gli antigeni analizzati. Utilizzando campioni di siero provenienti da pazienti, si è ottenuta una eccellenteconcordanza tra microarray ed ELISA che sottolinea l’efficacia del sistema microarray. Considerati i notevoli vantaggi intermini di costo e convenienza, riteniamo che la tecnologia del microarray proteico possa essere, in un prossimo futuro, introdottacome sistema di routine nei laboratori di analisi