Nuova ricerca

MARIA CRISTINA BASCHIERI

Interinale
Dipartimento di Scienze Mediche e Chirurgiche Materno-Infantili e dell'Adulto

Home |


Pubblicazioni

2022 - Autologous Marrow Mesenchymal Stem Cell Driving Bone Regeneration in a Rabbit Model of Femoral Head Osteonecrosis [Articolo su rivista]
Mastrolia, I.; Giorgini, A.; Murgia, A.; Loschi, P.; Petrachi, T.; Rasini, V.; Pinelli, M.; Pinto, V.; Lolli, F.; Chiavelli, C.; Grisendi, G.; Baschieri, M. C.; Santis, G. D.; Catani, F.; Dominici, M.; Veronesi, E.
abstract

Osteonecrosis of the femoral head (ONFH) is a progressive degenerative disease that ultimately requires a total hip replacement. Mesenchymal stromal/stem cells (MSCs), particularly the ones isolated from bone marrow (BM), could be promising tools to restore bone tissue in ONFH. Here, we established a rabbit model to mimic the pathogenic features of human ONFH and to challenge an autologous MSC-based treatment. ON has been originally induced by the synergic combination of surgery and steroid administration. Autologous BM-MSCs were then implanted in the FH, aiming to restore the damaged tissue. Histological analyses confirmed bone formation in the BM-MSC treated rabbit femurs but not in the controls. In addition, the model also allowed investigations on BM-MSCs isolated before (ON-BM-MSCs) and after (ON+BM-MSCs) ON induction to dissect the impact of ON damage on MSC behavior in an affected microenvironment, accounting for those clinical approaches foreseeing MSCs generally isolated from affected patients. BM-MSCs, isolated before and after ON induction, revealed similar growth rates, immunophenotypic profiles, and differentiation abilities regardless of the ON. Our data support the use of ON+BM-MSCs as a promising autologous therapeutic tool to treat ON, paving the way for a more consolidated use into the clinical settings.

2022 - Dissecting Immunotherapy Strategies for Small Cell Lung Cancer: Antibodies, Ionizing Radiation and CAR-T [Articolo su rivista]
Guaitoli, G.; Neri, G.; Cabitza, E.; Natalizio, S.; Mastrodomenico, L.; Talerico, S.; Trudu, L.; Lauro, C.; Chiavelli, C.; Baschieri, M. C.; Bruni, A.; Dominici, M.; Bertolini, F.
abstract

Small cell lung cancer (SCLC) is a highly aggressive malignancy that accounts for about 14% of all lung cancers. Platinum-based chemotherapy has been the only available treatment for a long time, until the introduction of immune checkpoint inhibitors (ICIs) recently changed first-line standard of care and shed light on the pivotal role of the immune system. Despite improved survival in a subset of patients, a lot of them still do not benefit from first-line chemo-immunotherapy, and several studies are investigating whether different combination strategies (with both systemic and local treatments, such as radiotherapy) may improve patient outcomes. Moreover, research of biomarkers that may be used to predict patients’ outcomes is ongoing. In addition to ICIs, immunotherapy offers other different strategies, including naked monoclonal antibodies targeting tumor associated antigens, conjugated antibody, bispecific antibodies and cellular therapies. In this review, we summarize the main evidence available about the use of immunotherapy in SCLC, the rationale behind combination strategies and the studies that are currently ongoing in this setting, in order to give the reader a clear and complete view of this rapidly expanding topic.

2017 - Differential gene expression patterns in HER2 positive metastatic breast cancer patients according to hormone receptor status [Poster]
Omarini, Claudia; Kaleci, Shaniko; Guaitoli, Giorgia; Bettelli, Stefania Raffaella; Cecilia, Caprera; Manfredini, Samantha; Caggia, Federica; Baschieri, MARIA CRISTINA; Luca, Moscetti; Maiorana, Antonino; Cascinu, Stefano; Piacentini, Federico
abstract

Background: HER2 positive breast cancer (HER2+ BC) is a heterogeneous disease. Presenting features, patterns of recurrence and survival of HER2+ BC can differ according to hormone receptors (HR) status. The purpose of this study is to highlight different gene profiles and molecular pathways between HR+ and HR- metastatic HER2+ BCs. Material and Methods: 34 HER2+ metastatic BC patients were included: 18 patients with HR+/HER2+ and 14 with HR-/HER2+. Data regarding tumor characteristics, treatment information and clinical outcomes were collected. The expression of 770 genes and 13 molecular pathways were evaluated by means of Nanostring PanCancer pathway panel performed on BC formalin-fixed paraffin-embedded tissues from diagnostic core biopsy or surgical resection. Results: Gene expression analysis identified 118 genes with significantly different expression in the two cohorts. All but one of these genes were over-expressed; only the gene CACNG6 was down-regulated in HR+/HER2+ group. In particular, 93 genes were over-expressed in HR-/HER2+ while 24 were overexpressed in HR+/HER2+. Most of these genes encoded growth factors, pro- or anti-inflammatory interleukins and DNA repair factors. 62% of these genes were involved in PI3K, MAPK and RAS pathways (32, 22 and 18, respectively). PI3K, MAPK and NOTCH pathways were differently expressed between HR+/HER2+ and HR-/HER2+ (p=0.003, p=0.0018, p=0.02, respectively). All these three pathways were overexpressed in HR-/HER2+ BC. In particular, all the significantly different expression genes in NOTCH pathways were overexpressed in HR-/HER2+ group. Conclusions: This genome expression analysis identified a gene expression profile able to differentiate HR+ versus HR- HER2+ metastatic BC. The overexpression of PI3K, MAPK and NOTCH pathways in HR-/HER2+ BC could justify its more aggressive behaviour. The validation of this HER2+ BC profile needs further investigation. Keywords: metastatic breast cancer, gene expression profile, HER2 positive breast cancer, PanCancer

2017 - Differential gene expression patterns in HER2 positive metastatic breast cancer patients according to hormone receptor status. [Abstract in Rivista]
Omarini, C; Kaleci, S; Guaitoli, G; Bettelli, S; Caprera, Cecilia; Manfredini, S; Caggia, F; Baschieri, Cristina; Moscetti, Luca; Maiorana, A; Cascinu, S; Piacentini, F
abstract

Background: HER2 positive breast cancer (HER2+ BC) is a heterogeneous disease. Presenting features, patterns of recurrence and survival of HER2+ BC can differ according to hormone receptors (HR) status. The purpose of this study is to highlight different gene profile and molecular pathways between HR+ and HR- metastatic HER2+ BCs. Materials and methods: 34 HER2+ metastatic BC patients were included: 18 patients with HR+/HER2+ and 14 with HR-/HER2+. Data regarding tumor characteristics, treatment information and clinical outcomes were collected. The expression of 770 genes and 13 molecular pathways were evaluated by means of Nanostring PanCancer pathway panel performed on BC formalin-fixed paraffin-embedded tissues from diagnostic core biopsy or surgical resection specimen. Results: Gene expression analysis identified 118 genes with significantly different expression in the two cohorts. All but one of these genes were over-expressed; only the gene CACNG6 was down-regulated in HR+/HER2+ group. In particular, 93 genes were over-expressed in HR-/HER2+ while 24 were overexpressed in HR+/HER2+. Most of these genes encoded growth factors, pro- or anti-inflammatory interleukins and DNA repair factors. 62% of these genes were involved in PI3K, MAPK and RAS pathways (32, 22 and 18, respectively). PI3K, MAPK and NOTCH pathways were differently expressed between HR+/HER2+ and HR-/HER2 + (p = 0.003, p = 0.0018, p = 0.02, respectively). All these three pathways were overexpressed in HR-/HER2+ BC. In particular, all the significantly different expression genes in NOTCH pathways were overexpressed in HR-/HER2+ group. Conclusions: This genome expression analysis identified a gene expression profile able to differentiate HR+ versus HR- HER2+ metastatic BC. The overexpression of PI3K, MAPK and NOTCH pathways in HR-/HER2+ BC could justify its more aggressive behaviour. The validation of this HER2+ BC profile needs further investigation.

2014 - An antibody reactivity-based assay for diagnosis of invasive candidiasis using protein array [Articolo su rivista]
Ardizzoni, Andrea; Posteraro, Brunella; Baschieri, MARIA CRISTINA; Bugli, Francesca; Sáez Rosòn, Arantza; Manca, Lidia; Cacaci, Margherita; Paroni Sterbini, Francesco; De Waure, Chiara; Sevilla, Maria Jesus; Peppoloni, Samuele; Sanguinetti, Maurizio; Moragues, Maria Dolores; Blasi, Elisabetta
abstract

The increased incidence of invasive candidiasis and of patients at risk requires early diagnosis and treatment to improve prognosis and survival. The aim of this study was to set up a ten-protein array-based immunoassay to assess the IgG antibody responses against ten well-known immunogenic C. albicans proteins (Bgl2, Eno1, Pgk1, Pdc11, Fba1, Adh1, Als3, Hwp1, Hsp90 and Grp2) in 51 patients with invasive candidiasis (IC) and in 38 culture-negative controls (non-IC). Antibody levels were higher against Bgl2, Eno1, Pgk1, Als3, Hwp1 and Grp2, than against Adh1, Pdc11, Fba1 and Hsp90, irrespectively of the patient group considered. Moreover, the IgG levels against Bgl2, Eno1, Pgk1 and Grp2 were significantly higher in IC than in non-IC patients. Furthermore, the ROC curves generated by the analysis of the antibody responses against Bgl2, Grp2 and Pgk1 displayed AUC values above 0.7, thus discriminating IC and non-IC patients. According to these results, the employment of the microarray immunoassay (a rapid, sensitive and multiparametric system), in parallel with conventional diagnostics, can help to spot IC patients. This ultimately will allow to initiate an early, focused and optimized antifungal therapy.

2014 - Differential efficacy of endodontic obturation procedures: an ex vivo study [Articolo su rivista]
Ardizzoni, Andrea; Generali, Luigi; Righi, Elena; Baschieri, MARIA CRISTINA; Cavani, Francesco; Manca, Lidia; Lugli, Eleonora; Migliarese, Luigi; Blasi, Elisabetta; Neglia, Rachele Giovanna
abstract

By means of a double-chamber model, different root canal filling materials and procedures were compared. Briefly, the root canals of single-rooted human teeth, extracted for periodontal reasons, were instrumented and obturated by gutta-percha/Pulp Canal Sealer EWT (PCS) or by Resilon, in association with different sealers (Real Seal, RelyX Unicem or Meta). Obturation was achieved by traditional continuous wave of condensation technique (TCWCT), a modified version of it (MCWCT), or single cone technique (SCT). The obturated roots, inserted in a double-chamber model, were sterilized by gamma irradiation. Next, Enterococcus faecalis was added to the upper chamber and the specimens were incubated at 37 °C for up to 120 days; the development of turbidity in the lower chambers' broths indicated bacterial leakage through the obturated root canals. The kinetics of leakage were analyzed in different groups by means of Kaplan-Meier statistics and compared by log-rank test. The results showed that root canals obturated with either gutta-percha/PCS using the MCWCT, Resilon/Real Seal SCT or Resilon/RelyX Unicem using the TCWCT displayed significantly better performance than the remaining groups (p < 0.01). Histological evaluation, performed to investigate microbial localization inside specimens, confirmed that this parameter varied according to the obturation procedures and materials employed. This ex vivo study indicates that gutta-percha/PCS, if used with the MCWCT, is as effective as Resilon when coupled to Real Seal with the SCT or, interestingly, to RelyX Unicem with the TCWCT. These data suggest that further improvement of the currently employed root canal filling procedures is achievable, depending on both the filling materials and the technique employed, thus encouraging clinical studies in this direction.

2013 - DIAGNOSI SIEROLOGICA, MEDIANTE MYCOARRAY, DI UN CASO DI ISTOPLASMOSI DA IMPORTAZIONE IN UN TURISTA ITALIANO DI RITORNO DAL BRASILE [Poster]
Ardizzoni, Andrea; Baschieri, MARIA CRISTINA; Manca, Lidia; Salvadori, Caterina; Marinacci, Ginevra; Farina, Claudio; Viale, Pierluigi; Blasi, Elisabetta
abstract

Introduzione L’istoplasmosi, infezione granulomatosa provocata dal fungo dimorfo Histoplasma capsulatum, viene contratta per inalazione dei conidi in aree dove tale fungo è endemico. Date le manifestazioni cliniche aspecifiche, la diagnosi di laboratorio è estremamente importante per confermare il sospetto di malattia, soprattutto in aree come la nostra, in cui i casi, sebbene rari, possono essere sia da importazione che autoctoni. Recentemente, è stato messo a punto il “mycoarray”, un saggio multiparametrico rapido, in grado di rilevare anticorpi specifici nei confronti di Histoplasma ed altri funghi dimorfi (1). Metodi Estratti antigenici da funghi dimorfi (H. capsulatum, C. immitis, B. dermatitidis, P. brasiliensis) sono stati deposti su vetrini microarray mediante un sistema robotizzato ad alta precisione. I vetrini sono stati cimentati con il siero del paziente e la formazione degli immunocomplessi è stata rivelata utilizzando anticorpi secondari marcati in fluorescenza. Dopo lettura, mediante un apposito scanner, il segnale è stato quantificato ed analizzato. Risultati Il paziente (maschio italiano di 30 anni, di ritorno da un viaggio “on-the-road” in Brasile), era stato ricoverato per una febbre persistente che non rispondeva a terapia antibiotica empirica. A seguito della presenza di infiltrati e di linfoadenopatia mediastinica, rivelati mediante TAC del torace, è stata condotta una biopsia linfonodale suggestiva di infezione fungina da dimorfi. L’analisi del siero mediante mycoarray ha mostrato una elevata sieroreattività, IgG e IgM, nei confronti degli antigeni di H. capsulatum. Nessuna reattività è invece stata evidenziata nei confronti degli antigeni da altri funghi dimorfi (2). Conclusioni Grazie alle sue peculiarità (miniaturizzazione, multiparametricità e rapidità di esecuzione) il mycoarray si rivela uno strumento prezioso per facilitare la diagnosi rapida di micosi primitive, specialmente in paesi non endemici. Ringraziamenti Lavoro in parte supportato da MIUR, PRIN-200985J87J Bibliografia (1) Ardizzoni et al., (2011) New Microbiol, 34:307-16. (2) Ardizzoni et al., (2013) JTM, 20:336-9

2013 - Diagnostic utility of a microarray based on 11 proteins of Candida albicans for the diagnosis of invasive candidiasis [Abstract in Rivista]
Sáez Rosón, A; Ardizzoni, Andrea; Baschieri, MARIA CRISTINA; Bugli, F; Paroni Sterbini, F; Orsi, Carlotta Francesca; Manca, Lidia; Sanguinetti, M; Posteraro, B; Cuétara, Ms; Olazabal, I; García Ruiz, Jc; Peppoloni, Samuele; Blasi, Elisabetta; Moragues, Md
abstract

Objective. The purpose of this study was to set up a protein microarray test based on 11 proteins of C. albicans for the detection of IgG antibodies in sera from patients with invasive candidiasis (IC) and determine its diagnostic utility. Methods and patients. The microarray was set up with 10 C. albicans recombinant proteins: Eno1, Als3-N, Hwp1-N, Eno1-RM, Bgl2, Grp, Pgk1, Fba, Pdc, Adh1, and an enolase purified from a dithiothreitol-extract of cell walls from C. albicans mycelial phase (CW-Eno). Antigens, IgG standard curve, and controls were printed onto glass slides using computer controlled high speed robotics. The arrays were processed with sera from IC and control patients and then fluorescence labeled secondary antibodies were added. The signal captured by each spot was detected by a laser scan reader and quantified. When possible, the cut-off values werecalculated as mean mass of antibody detected in the control group sera plus 2 times the standard deviation. Twenty three sera from 15 patients with proven IC due to C. albicans and 33 sera from 28 patients at risk for IC but without clinical or microbiological data confirming a fungal infection (control group) were studied. Results. The performance of the microarray assay for each antigen is shown in Table 1. The best results were obtained with CW-Eno, showing a sensitivity and a specificity of 80 and 96.43%, respectively. Pgk1, Grp and Eno1 exhibited lower sensitivity values (33-47%), but their specificity reached values equal or greater than 93% Also, for all the other antigens, the specificity was very high with values above 96%. Furthermore, we clustered those antigens which separately had returned the best sensitivity. As shown in Table 2, the clustering raised the sensitivity up to 100%, while the specificity ranged between 85 and dropped to 89% . Conclusions. The detection of serum IgG antibodies against proteins of C. albicans by a protein microarray exhibited moderate diagnostic utility values when assessed independently, being CW-Eno the main exception. However, when considering the microarray results either as a whole or as clusters of selected proteins (CW-Eno, Pgk1, Eno1 and Grp; or CW-eno and Pgk1), the system proved to be an efficient diagnostic tool for IC. Further studies with a larger number of patients are needed to confirm the results of the present study.

2013 - IL MYCOARRAY: UN TEST RAPIDO PER LA DIAGNOSI SIEROLOGICA DI MICOSI ENDEMICHE [Poster]
Ardizzoni, Andrea; Baschieri, MARIA CRISTINA; Manca, Lidia; Meacci, Marisa; Venturelli, Claudia; Farina, Claudio; Blasi, Elisabetta
abstract

Introduzione Le micosi da funghi dimorfi, estremamente rare in Europa, sono rappresentate da casi di importazione, con l’eccezione della istoplasmosi per la quale sono stati segnalati anche casi autoctoni. La bassa frequenza, in combinazione con l’aspecificità delle manifestazioni cliniche, rende difficile una diagnosi rapida. Scopo del presente studio è la valutazione dell’utilizzo del saggio mycoarray (1) come strumento multiparametrico e rapido, a supporto della sierologia convenzionale, nella diagnosi di laboratorio delle micosi endemiche. Metodi Antigeni fungini (H. capsulatum, C. immitis, B. dermatitidis e P. brasiliensis), diluizioni scalari di anticorpi IgG e IgM e controlli sono stati deposti su vetrini microarray per mezzo di un sistema robotizzato ad alta precisione. I vetrini così ottenuti sono stati cimentati col siero, opportunamente diluito, e successivamente con anticorpi secondari marcati con fluorofori per rivelare l’avvenuta formazione degli immunocomplessi. Il segnale è stato acquisito mediante uno scanner, quantificato ed analizzato per mezzo di un apposito software. Risultati Il mycoarray ha mostrato elevate sensibilità e specificità: un primo studio retrospettivo, condotto su sieri da pazienti con diagnosi certa di istoplasmosi o di coccidioidomicosi, ha fornito risultati consistenti con i dati clinici e di laboratorio, acquisiti con la diagnostica di routine. Indagini su ulteriori campioni da casi clinici “sospetti”, analizzati con il mycoarray, hanno fornito risultati originali utili per la diagnosi definitiva di micosi endemica. Conclusioni Il mycoarray presenta una serie di peculiarità (miniaturizzazione, multiparametricità e rapidità di esecuzione) che lo rendono estremamente utile come strumento di laboratorio a sussidio del percorso convenzionale nella diagnosi di micosi primitive, specialmente in zone a bassa endemicità. Ringraziamenti Lavoro in parte supportato da MIUR, PRIN-200985J87J Bibliografia (1) Ardizzoni et al., (2011) New Microbiol, 34:307-16.

2013 - The Mycoarray as an aid for diagnosis of an imported case of Histoplasmosis in an Italian traveler returning from Brasil [Articolo su rivista]
Ardizzoni, Andrea; Baschieri, Maria Cristina; Manca, Lidia; Salvadori, Caterina; Marinacci, Ginevra; Farina, Claudio; Viale, Pierluigi; Blasi, Elisabetta
abstract

We describe an imported case of Histoplasmosis, whose serological profile was established by means of a protein-based microarray platform, the recently described mycoarray. Because of its peculiarities, such a novel tool greatly facilitates the rapid and multiparametric assessment of patients' serological status and lends itself to be employed as an aid in the diagnosis of primary mycoses, especially in non endemic countries.

2012 - IL MICROARRAY PROTEICO NELL’ANALISI DEI LIQUIDI AMNIOTICI: RICERCA DI MARKER PRECOCI DI INFIAMMAZIONE E/O INFEZIONE CORRELABILI AL PARTO PRETERMINE [Abstract in Atti di Convegno]
Manca, Lidia; Ardizzoni, Andrea; Baschieri, MARIA CRISTINA; Capodanno, Francesco; Soncini, Emanuele; Peppoloni, Samuele; LA SALA, Giovanni Battista; Blasi, Elisabetta
abstract

40° Congresso della SIM, Riccione 7-10 ottobre, 2012

2012 - PROFILO DELLA RISPOSTA ANTICORPALE A CANDIDA ALBICANS IN DIFFERENTI CATEGORIE DI PAZIENTI A RISCHIO DI INFEZIONI FUNGINE INVASIVE [Poster]
Baschieri, MARIA CRISTINA; Ardizzoni, Andrea; Manca, Lidia; Saez Roson, Arantza; Bugli, Francesca; Paroni Sterbini, Francesco; Orsi, Carlotta Francesca; Sanguinetti, Maurizio; Posteraro, Brunella; Moragues, Maria Dolores; Blasi, Elisabetta
abstract

L’aumento continuo nel numero e nella tipologia dei pazienti immunocompromessi comporta un continuo incremento dei casi di infezioni fungine invasive (IFI), il cui esito infausto è spesso ascrivibile al ritardo nella diagnosi e conseguentemente all’impossibilità di instaurare una precoce ed appropriata terapia antifungina. La candidosi invasiva (IC), particolarmente frequente, riveste un ruolo di primaria importanza tra le IFI, visto anche l’elevato tasso di mortalità (30-50%). I metodi comunemente impiegati nella diagnosi di IFI sono spesso caratterizzati da lunghi tempi di indagine oltre che da scarsa sensibilità e specificità. Grazie all’impiego di nuove tecnologie particolarmente versatili e molto sensibili (es: microarray proteici), studi recenti hanno riproposto la sierologia come percorso diagnostico rilevante nella identificazione rapida e precoce di IC. In particolare, sono state fornite le prime evidenze circa la presenza di specifici marker immunologici in grado di portare alla distinzione tra pazienti affetti da IC e soggetti di controllo. Alla luce di questi dati, abbiamo messo a punto un microarray proteico per la rilevazione dei titoli anticorpali IgG nei confronti di 10 diversi antigeni ricombinanti di C. albicans (Adh, Als-3, Bgl-2, Fba, Grp, Hwp-1, Pdc, Pgk e due diverse forme ricombinanti di Eno-1) e di 1 antigene purificato (Eno-1) dalla parete. Mediante questo chip, vengono saggiati sieri da diverse categorie di soggetti (pazienti affetti da IC, pazienti ospedalizzati per patologie non IFI e soggetti sani di controllo). I dati finora ottenuti mostrano come i livelli di anticorpi nei confronti di alcuni antigeni, in particolare Eno-1, Grp e Pgk, siano più alti nei pazienti con IC rispetto alle altre categorie di soggetti considerati. Una volta acquisiti sufficienti dati, verranno fatte valutazioni di tipo statistico e sarà quindi possibile stabilire l’efficacia di questo nuovo percorso di indagine, sia nell’individuazione che nel monitoraggio di soggetti con e/o a rischio di IFI.

2012 - Protein microarrays on midtrimester amniotic fluids: a novel approach for the diagnosis of early intrauterine inflammation related to preterm delivery [Articolo su rivista]
La Sala, Giovanni Battista; Ardizzoni, Andrea; Capodanno, F; Manca, Lidia; Baschieri, Maria Cristina; Soncini, E; Peppoloni, Samuele; Blasi, Elisabetta
abstract

Novel technologies that allow simultaneous assessment of multiple biomarkers provide new and promising diagnostic/prognostic approaches. By protein microarrays, here we analyzed amniotic fluids (AF) from 50 women with preterm delivery (PTD) and 50 control women, who delivered at term. In detail, cytokines, chemokines, matrix metalloproteinases and antigen-specific antibodies were assessed. The AF analysis showed significant differences between women with preterm and term delivery in the levels of IL-1alpha, IL-1beta, IL-4, IL-6, IL-8, MCP-1, IFN-gamma and anti-HSV2 IgG. No significant differences were observed in the levels of TNF-alpha, MMP-2, MMP-9 and specific IgG for seven vertically transmitted pathogens. In conclusion, we demonstrated the feasibility of protein microarrays in the diagnosis of early intrauterine inflammation. The significant association between the increased levels of certain cytokines and preterm delivery argues on their relevance as early pathogenetic markers for identification of risk patients.

2011 - DETECTION OF ANTIGEN-SPECIFIC ANTIBODIES IN SERUM AND FOLLICULAR FLUIDS BY PROTEIN MICROARRAYS. [Poster]
Ardizzoni, Andrea; Baschieri, MARIA CRISTINA; Manca, Lidia; Capodanno, Francesco; LA SALA, Giovanni Battista; Blasi, Elisabetta
abstract

Background. Wide spectrum serological screening of women prior to or during pregnancy may greatly help in preventing vertically transmitted infections (VTI) and their severe consequences to the foetus/newborn. Protein microarrays, made up by spotting many antigens onto a restricted area of a microscope slide, allow to detect, in one shot, specific antibodies against a wide range of antigenic specificities. Objectives. To detect, in paired serum and follicular fluid (FF) samples, antigen-specific antibodies against vertically transmitted pathogens by protein microarrays; to investigate the potential relation between antibody profiles and pregnancy outcome. Methods. Serum and FF paired samples were collected from 102 women undergoing in vitro fertilization (IVF). Antigens, human antibodies and controls were spotted in an orderly manner by high speed robotics. Microarrays were processed with serum or FF and the occurred immunocomplexes were revealed by fluorescently-labelled secondary antibodies. The fluorescent signals were read by a laser scanner and quantified by a dedicated software. Conclusions. Antigen-specific antibodies can be effectively detected in serum and FF by microarray. The presence or absence of certain antigen-specific antibodies is significantly related to clinical parameters such as the number of inseminated good quality oocytes or the number of successful embryo transfers. These results encourage protein microarrays employment for wide spectrum investigations in diagnosing VTI; in particular, the use of biological matrices other than serum may help addressing yet unravelled questions.

2011 - Detection of follicular fluid and serum antibodies by protein microarrays in women undergoing in vitro fertilization treatment. [Articolo su rivista]
Ardizzoni, Andrea; Manca, Lidia; Capodanno, F; Baschieri, MARIA CRISTINA; Rondini, I; Peppoloni, Samuele; Righi, Elena; LA SALA, Giovanni Battista; Blasi, Elisabetta
abstract

A protein microarray serological assay was used to assess the antibody profile of 102women subjected to in vitro fertilization treatment. The studies were conducted on pairs of serum and follicular fluid samples, collected from each woman on the same day at the time of oocyte recovery. The samples, stored as frozen aliquotes, were assessed by both microarray and ELISA. Follicular fluids and sera were screened to detect the presence of specific IgG and IgM antibodies against seven vertically transmitted pathogens. The IgG reactivityof follicular fluids closely mirrored that of serum in all the patients and for all the antigens, with an agreement of more than 85%. IgM antibodies were undetectable in follicular fluids. The antibody patterns were subsequently related to the biological and clinical outcomesof in vitro fertilization cycles. The results showed that varicella zoster virus (VZV) IgG positive women and cytomegalovirus (CMV) IgG negative women had on average a higher number of inseminated, good quality oocytes compared to VZV IgG negative and CMV IgG positive women. In addition, the rate of successful embryo transfers was significantly higher in Toxoplasma gondii IgG negative women than in their positive counterparts. Overall, the microarray was proven to be a suitable tool for detecting analytes in follicular fluids, therefore supporting its application in a wide spectrum of investigations.

2011 - I MICROARRAY PROTEICI: NUOVI STRUMENTI DI INDAGINE NELLA DIAGNOSTICA DELLE INFEZIONI DA PATOGENI DEL COMPLESSO TORCH. [Poster]
Ardizzoni, Andrea; Manca, Lidia; Baschieri, MARIA CRISTINA; Capodanno, Francesco; LA SALA, Giovanni Battista; Blasi, Elisabetta
abstract

Lo screening sierologico su larga scala, effettuato prima o durante la gravidanza, fornisce un mezzo efficace per prevenire le infezioni a trasmissione verticale (ITV) e le loro gravi conseguenze per il prodotto del concepimento. Utilizzando un microarray proteico da noi messo a punto (1), abbiamo voluto determinare i livelli di anticorpi antigene-specifici nei confronti di patogeni a trasmissione verticale nel siero, nei liquidi follicolari (LF) e nei liquidi amniotici (LA) di pazienti gravide sottoposte a IVF, al fine di correlare profili anticorpali specifici con l’esito della gravidanza. Sieri e LF sono stati prelevati da 102 pazienti sottoposte a trattamenti di procreazione medicalmente assistita. LA sono stati ottenuti da 100 pazienti di cui 50 con parto pretermine e 50 con parto a termine. I microarray, allestiti con antigeni, anticorpi e controlli di segnale, come precedentemente descritto (1), sono stati processati con siero, LF o LA e la formazione degli immunocomplessi è stata rivelata mediante anticorpi secondari marcati in fluorescenza. I segnali sono stati letti con uno scanner contenente laser a differenti lunghezze d’onda e successivamente quantificati ed analizzati da un software dedicato. Il saggio ha consentito di rivelare in maniera efficace IgG antigene-specifiche in tutte le matrici saggiate, mentre anticorpi IgM sono stati rilevati solamente nel siero. In particolare, la presenza o assenza di determinati anticorpi IgG nelle coppie di campioni siero/LF provenienti dalla stessa paziente è risultata significativamente correlata a parametri clinici, quali il numero di oociti inseminati di buona qualità o il numero di trasferimenti embrionali avvenuti con successo (2). L’analisi dei profili anticorpali sui LA nel secondo gruppo di 100 pazienti è tuttora in corso. Su questo gruppo di pazienti, inoltre, si sta impiegando un microarray commerciale per valutare la presenza di citochine e/o chemochine da correlare eventualmente con il quadro clinico della paziente e con l’esito della gravidanza (parto a termine o pretermine). Risultati preliminari hanno consentito di dimostrare l’efficacia dell’approccio, evidenziando la presenza di livelli significativi di alcune citochine nei fluidi amniotici. Sono in corso indagini per determinare il contenuto di citochine in tutti i LA oggetto di studio. I dati finora ottenuti sostengono fortemente l’utilizzo del microarray proteico in indagini ad ampio spettro nella diagnosi di ITV; inoltre, l’utilizzo di matrici biologiche differenti dal siero può fornire un valido contributo per la risoluzione di quesiti diagnostici ancora irrisolti. (1) Ardizzoni et al., Eur J Clin Microbiol Infect Dis, 2009. (2) Ardizzoni et al., J Reprod Immunol, 2011.

2011 - I MICROARRAY PROTEICI: POSSIBILI SCENARI NELLA DIAGNOSTICA DELLE MICOSI INVASIVE [Poster]
Blasi, Elisabetta; Baschieri, MARIA CRISTINA; Ardizzoni, Andrea
abstract

I microarray proteici sono comunemente distinguibili in due tipi, uno dedicato a stabilire la presenza e la quantità di una proteina o di un anticorpo specifico presenti in una determinata matrice biologica (“abundance-based microarrays”) e l’altro a valutarne la funzione (“function-based microarrays”) (1). Per quanto attiene alla prima categoria, sono stati descritti sistemi a cattura molto simili a quelli utilizzati nei saggi ELISA che prevedono l’immobilizzazione su un supporto solido di molecole di cattura. Tali molecole possono essere anticorpi, soprattutto monoclonali, per la rilevazione quali-quantitativa di analiti specifici, oppure antigeni purificati/ricombinati per la determinazione di specifici titoli anticorpali. Ad oggi, questo tipo di microarray ha trovato un grosso impiego nel campo della proteomica, nella ricerca di nuovi farmaci e nella identificazione di marcatori di malattia, principalmente nell’ambito delle infezioni virali e dei tumori. Le caratteristiche dei saggi diagnostici su piattaforma microarray, quali l’elevata sensibilità, la multiparametricità, la miniaturizzazione e la possibilità di automazione, hanno portato alla messa a punto di piattaforme innovative, particolarmente utili anche per la loro duttilità. L’utilizzo dei microarray proteici in diagnostica ha portato inoltre a riconsiderare la sierologia come strumento di indagine potenzialmente utile nella diagnosi di micosi opportunistiche invasive e nella valutazione dell’efficacia di terapie antifungine. In un lavoro recentemente pubblicato, è stato identificato un gruppo di 13 antigeni di Candida albicans che consente di discriminare pazienti con candidemia, da soggetti sani o colonizzati; sempre gli stessi autori hanno descritto un altro gruppo di 33 antigeni, che permette di distinguere sierologicamente pazienti in fase acuta di malattia da pazienti convalescenti (2). Dall’altra parte, nella diagnosi di micosi primitive da patogeni quali Histoplasma capsulatum e Coccidioides immitis, in cui la sierologia riveste un ruolo di primo piano, l’impiego del microarray proteico ha portato ad accorpare in un unico chip antigeni dei diversi patogeni (3). In particolare, il saggio messo a punto consente di identificare simultaneamente soggetti positivi per uno o più patogeni, sulla base dei livelli di IgM e/o IgG specifiche. Similmente, sono stati riconosciuti come marcatori di polmonite gli alti livelli di anticorpi sierici verso la proteina Msg1 di Pneumocystis jirovecii (4), mentre la comparsa di citochine proinfiammatorie e della proteina C-reattiva sono risultate preziosi marcatori sierologici precoci di aspergillosi invasiva (5). Nel complesso, visto l’incremento nel numero sia di micosi opportunistiche in soggetti particolarmente difficili sia di micosi primitive (in passato inusuali, ora più frequenti dato l’aumento dei flussi turistici e migratori), i contributi forniti dalle nuove tecnologie saranno fondamentali per comprendere meglio il quadro clinico associato alla micosi invasiva, grazie ad un metodo diagnostico veloce e multiparametrico. Questo aspetto risulterà particolarmente interessante in quanto consentirà di valutare contemporaneamente tipi diversi di parametri che includono non solo il patogeno ed i suoi prodotti, ma anche l’ospite con la sua peculiare reattività antimicrobica, sia innata che adattativa. (1) LaBaer and Ramachandran, Curr Opin Chem Biol, 2005 (2) Mochon et al., Plos Pathogens, 2010 (3) Ardizzoni et al., New Microbiol, 2011 (4) Djawe et al., Plos One, 2010 (5) Chai et al., J. Infect Dis, 2010

2011 - Influence of hyaluronic acid on bacterial and fungal species, including clinically relevant opportunistic pathogens. [Articolo su rivista]
Ardizzoni, Andrea; Neglia, Rachele Giovanna; Baschieri, MARIA CRISTINA; Cermelli, Claudio; Caratozzolo, M; Righi, Elena; Palmieri, Beniamino; Blasi, Elisabetta
abstract

Hyaluronic acid (HA) has several clinical applications (aesthetic surgery, dermatology, orthopaedics and ophtalmology). Following recent evidence, suggesting antimicrobial and antiviral properties for HA, we investigated its effects on 15 ATCC strains, representative ofclinically relevant bacterial and fungal species. The in vitro system employed allowed to assess optical density of broth cultures as a measure of microbial load in a time-dependent manner. The results showed that different microbial species and, sometimes, different strains belonging to the same species, are differently affected by HA. In particular, staphylococci, enterococci, Streptococcus mutans, twoEscherichia coli strains, Pseudomonas aeruginosa, Candida glabrata and C. parapsilosis displayed a HA dosedependent growth inhibition; no HA effects were detected in E. coli ATCC 13768 and C. albicans; S. sanguinis was favoured by the highest HA dose. Therefore, the influence of HA on bacteria and fungi warrants further studies aimedat better establishing its relevance in clinical applications.

2011 - The protein “mycoarray”: a novel serological assay for the laboratory diagnosis of primitive endemic mycoses [Articolo su rivista]
Ardizzoni, Andrea; Baschieri, MARIA CRISTINA; Manca, Lidia; Orsi, Carlotta Francesca; C., Venturelli; M., Meacci; Peppoloni, Samuele; C., Farina; Blasi, Elisabetta
abstract

A protein microarray containing fungal antigens (the “mycoarray”) has been set up to provide rapid and appropriateserodiagnosis of primitive endemic mycoses, an important cause of morbidity and mortality in an increasingly highnumber of patients. The mycoarray consists of three antigen extracts (histoplasmin, coccidioidin and Coccidioides “TP”)and antibody dilution curves were spotted on microarray slides. The arrays were processed with coccidioidomycosisand histoplasmosis patients’ sera or with control sera and the occurring immunocomplexes were detected by indirectimmunofluorescence. In agreement with clinical and microbiological diagnosis, the results distinguished betweenhistoplasmosis and coccidioidomycosis patients. In addition, the assay could clearly discriminate between IgM andIgG antibody reactivity. No reactivity was ever observed in the arrays processed with negative control sera. Therefore,this pilot study demonstrates that the “mycoarray” is sensitive and specific enough to discriminate between healthyindividuals and patients with histoplasmosis or coccidioidomycosis. Because of miniaturization and multiparametricity,the new assay cuts costs and processing time. Thus, once clinically validated and implemented as a large-scale array,the “mycoarray” will be ready to be applied to the daily clinical practice.

2011 - VALUTAZIONE DEI PROFILI IgG E IgM NEI CONFRONTI DI PATOGENI A TRASMISSIONE VERTICALE IN DIVERSE MATRICI BIOLOGICHE MEDIANTE MICROARRAY PROTEICO [Poster]
Ardizzoni, Andrea; Manca, Lidia; Baschieri, MARIA CRISTINA; Capodanno, Francesco; LA SALA, Giovanni Battista; Blasi, Elisabetta
abstract

Lo screening sierologico su larga scala, effettuato prima o durante la gravidanza, fornisce un mezzo efficace per prevenire infezioni a trasmissione verticale (ITV) e le loro gravi conseguenze per il prodotto del concepimento. Utilizzando un microarray proteico da noi messo a punto (1), abbiamo voluto determinare i livelli di anticorpi antigene-specifici nei confronti di patogeni a trasmissione verticale nel siero, nei fluidi follicolari (FF) e nei fluidi amniotici (FA) di pazienti gravide, al fine di correlare profili anticorpali specifici con l’esito della gravidanza. Siero e FF sono stati prelevati da 102 pazienti sottoposte a trattamenti di procreazione medicalmente assistita. FA sono stati ottenuti da 100 pazienti di cui 50 con parto pretermine e 50 con gravidanza a termine. I microarray, allestiti con antigeni, anticorpi e controlli di segnale, come precedentemente descritto (1) sono stati processati con siero, FF o FA e la formazione degli immunocomplessi è stata rivelata mediante anticorpi secondari marcati in fluorescenza. I segnali sono stati letti con uno scanner contenente laser a differente lunghezza d’onda e successivamente quantificati ed analizzati da un software dedicato. Il saggio ha consentito di rivelare in maniera efficace anticorpi antigene-specifici in tutte le matrici saggiate. In particolare, la presenza o assenza di determinati anticorpi nelle coppie di campioni siero/FF provenienti dalla stessa paziente è risultata significativamente correlata a determinati parametri clinici, quali il numero di oociti inseminati di buona qualità o il numero di trasferimenti embrionali avvenuti con successo. L’analisi dei profili anticorpali sui FA del secondo gruppo di 100 pazienti è tuttora in corso. I dati ottenuti incoraggiano l’utilizzo del microarray proteico per indagini ad ampio spettro nella diagnosi di ITV; inoltre, l’utilizzo di matrici biologiche differenti dal siero può fornire un valido contributo per la risoluzione di problemi ancora aperti. (1) Ardizzoni et al., Eur J Clin Microbiol Infect Dis, 2009.

2010 - The protein “mycoarray”: a novel immunoassay for the serological diagnosis of primitive invasive mycoses [Poster]
Ardizzoni, Andrea; Baschieri, MARIA CRISTINA; Manca, Lidia; Farina, Claudio; Cermelli, Claudio; Meacci, Marisa; Venturelli, Claudia; Blasi, Elisabetta
abstract

Objectives. Invasive fungal infections are an important cause of morbidity and mortality in an increasingly higher number of patients, also because of difficulties in providing a rapid and appropriate diagnosis. In some cases, detection of a specific antibody response is a crucial diagnostic tool; however, the available serological assays often provide qualitative results only, their sensitivity and specificity are poor and long time procedures are required. In addition, patients who suffer from an invasive mycosis may have multiple infections likely underestimated by conventional diagnostic approaches. In order to couple the serology of primitive invasive mycoses to the protein microarray technology, a “mycoarray” assay has been designed and set up.Methods. Four antigen extracts (histoplasmin, coccidioidin, Coccidioides “TP” antigen and aspergillin) and the appropriate controls were spotted in various conditions onto a restricted area of a microscope slide. The printed slides were then incubated with immune sera produced in goat against each single antigen or, subsequently, with human sera (6 from patients affected by primitive invasive mycoses and 7 from healthy individuals). The occurring immunocomplexes were detected by indirect immunofluorescence.Results. The pilot experiments, conducted using the goat immune sera, allowed to establish the optimal spotting conditions for each antigen in terms of both spotting buffer and extracts’ dilution. The “mycoarrays”, obtained by spotting all the fungal antigens with the best condition, were then processed with sera either from patients or control subjects. The reactivity observed in the arrays processed with the patients’ samples was in agreement with the clinical and microbiological diagnosis; no reactivity was ever observed in the arrays processed with the negative control sera.Conclusions. The “protein mycoarray” is sensitive enough to discriminate between healthy individuals and patients affected by histoplasmosis or coccidioidomycosis. This novel diagnostic tool, because of its intrinsic features, miniaturization and multiparametricity, can contribute to cut out costs and to shorten times-to-results, with the potentiality to be included in the daily clinical practice in the near future.

2009 - A protein microarray immunoassay for the serological evaluation of the antibody response in vertically transmitted infections. [Articolo su rivista]
Ardizzoni, Andrea; B., Capuccini; Baschieri, MARIA CRISTINA; Orsi, Carlotta Francesca; F., Rumpianesi; Peppoloni, Samuele; Cermelli, Claudio; M., Meacci; A., Crisanti; P., Steensgaard; Blasi, Elisabetta
abstract

The detection of specific serum antibodies is mainly achieved by enzyme-linked immunosorbent assay (ELISA). Here, we describe the setting up of a microarray-based serological assay to screen for IgG and IgM against vertically transmitted pathogens (Toxoplasma gondii, rubella virus, cytomegalovirus, herpes simplex virus types 1 and 2, varicella zoster virus, Chlamydia trachomatis). The test, accommodated onto a restricted area of a microscope slide, consists of: (a) the immobilization of antigens and human IgG and IgM antibody dilution curves, laid down in an orderly manner; (b) addition of serum samples; (c) detection of antigen–serum antibodies complexes by indirect immunofluorescence. The IgG and IgM curves provide an internal calibration system for the interpolation of the signals from the single antigens. The test was optimized in terms of spotting conditions and processing protocol. The detection limit was 400 fg for the IgG assay and 40 fg for the IgM assay; the analytical specificity was >98%. The clinical sensitivity returned an average value of 78%, the clinical specificity was >96%, the predictive values were >73%, and the efficiency was >88%. The results obtained make this test a promising tool, suitable for introduction in the clinical diagnostic routine of vertically transmitted infections, in parallel (and in future as an alternative) to ELISA.

2009 - Il microarray proteico nella sierodiagnosi delle infezioni a trasmissione verticale [Relazione in Atti di Convegno]
Ardizzoni, Andrea; Baschieri, MARIA CRISTINA; Manca, Lidia; Blasi, Elisabetta
abstract

2009 - Sierodiagnosi di infezioni a trasmissione verticale mediante microarray proteico [Poster]
Ardizzoni, Andrea; Baschieri, MARIA CRISTINA; Manca, Lidia; Cuoghi, Alessandro; Cermelli, Claudio; Peppoloni, Samuele; Blasi, Elisabetta
abstract

2008 - Microarray proteici: applicazioni nella diagnostica prenatale [Relazione in Atti di Convegno]
Ardizzoni, Andrea; Capuccini, Barbara; Baschieri, MARIA CRISTINA; Crisanti, Andrea; Gallucci, Giuseppina; Manzoni, Angelo; Meacci, Marisa; Rumpianesi, Fabio; Blasi, Elisabetta
abstract

2007 - NF-kB activation and p38 phosphorilation in microglial cells infected with Leptospira or exposed to partially purified leptospiral lipoproteins [Articolo su rivista]
Blasi, Elisabetta; Ardizzoni, Andrea; Colombari, Bruna; Neglia, Rachele Giovanna; Baschieri, MARIA CRISTINA; Peppoloni, Samuele; M., Cinco
abstract

Recently, we have shown a differential susceptibility of non-pathogenic vs. pathogenic leptospires to phagocytosis and killing by microglial cells. Although all ingested to some extent, only the pathogenic strains survived intracellularly while the non-pathogenic ones were killed in a time-dependent manner. By the same infection model, here we demonstrate that microglial cells respond to Leptospira infection with a time- and dose-dependent induction of molecular signals (p38 phosphorilation and NF-kB activation) and the production of soluble factors (cytokines and nitric oxide). Such bio-molecular response is predominantly observed against the pathogenic Leptospira; the phenomenon is reproduced by leptospiral lipoproteins and, to a lower extent, by leptospiral-derived LPS. These data provide initial evidence that Leptospira affects microglial cell response in a different manner depending upon the virulence of the infecting strain; specific bacterial components happen to be involved in the induction of such pathogen-induced immune response.