Il poliomavirus delle cellule di Merkel (MCPyV) causa un'infezione asintomatica nell’uomo. In rari casi, MCPyV induce un tumore cutaneo aggressivo con limitate opzioni terapeutiche, il carcinoma delle cellule di Merkel (CCM). Nonostante le IgG anti-MCPyV siano state rilevate con una prevalenza relativamente alta negli individui sani, dati precedenti indicano tassi di prevalenza variabili. Sono quindi necessari nuovi saggi immunologici per determinare la sierologia MCPyV nell’uomo. Lo scopo di questa tesi era di indagare l'impatto dell'infezione da MCPyV nella popolazione sana. È stato dunque sviluppato e validato un nuovo test ELISA che impiega due peptidi sintetici che mimano gli epitopi delle proteine capsidiche di MCPyV (VP1/2) per valutare la presenza di IgG anti-MCPyV in sieri di soggetti sani, compresi bambini (HC, 0-20 anni), adulti (HS, 21-65 anni) e anziani (ES, 66-100 anni). I peptidi sono stati disegnati in silico per rilevare le IgG verso MCPyV. Il test è stato valutato in termini di performance sui sieri di controllo MCPyV-positivi/-negativi, mostrando prestazioni elevate. L'ELISA è stato esteso a sieri HC (n=344), HS (n=510) ed ES (n=226) con una sierologia per MCPyV ignota. La prevalenza delle IgG anti-MCPyV era del 40.7% negli HC. È stata inoltre valutata la prevalenza di MCPyV specifica per età/genere. La coorte 0-5 anni ha evidenziato una prevalenza di MCPyV più bassa rispetto alle altre coorti. Il profilo sierologico di MCPyV ha mostrato densità ottiche (OD) più basse nella coorte 0-5 anni rispetto alle altre coorti. OD più alte sono state trovate nelle femmine rispetto ai maschi. La prevalenza delle IgM anti-MCPyV nel gruppo HC è risultata essere del 29.7%, con tassi simili negli HC stratificati per età. La coorte 0-5 anni aveva le OD più basse rispetto alle altre coorti. Le coorti 6-10/11-15 anni avevano OD più alte della coorte 16-20 anni. Sono stati valutati simultaneamente i pattern sierici di IgG e IgM anti-MCPyV negli HC. Le OD di IgG e IgM hanno mostrato una tendenza inversa, con rispettivo aumento e diminuzione, nella coorte HC più adulta. La prevalenza delle IgG sieriche di MCPyV era del 61.8% negli HS. Le femmine hanno mostrato OD più alte dei maschi. La prevalenza delle IgG anti-MCPyV negli ES era del 63.7%; una percentuale superiore al 40.7% degli HC e simile al 61.8% degli HS. La sierologia di MCPyV è stata anche studiata in HC, HS ed ES, stratificati in coorti di 5 anni. La prevalenza di MCPyV era inferiore nella coorte 0-5 anni (13%) rispetto alle altre coorti (52.3-72%). Inoltre, la coorte 0-5 anni aveva i valori di OD più bassi. OD più elevate sono state trovate nelle coorti 6-10/11-15/16-20 anni rispetto alle altre coorti. I risultati di questa tesi indicano che l'ELISA indiretto qui sviluppato è affidabile nel rilevare IgG anti-MCPyV sieriche. I nostri dati immunologici rivelano che l'infezione da MCPyV è diffusa nell'uomo. Dopo la sieroconversione in età pediatrica, MCPyV circola senza sintomi nella popolazione prepuberale, adulta e anziana con un'elevata prevalenza, senza variazioni nell'età adulta e nella senilità. Sono necessari nuovi approcci per il trattamento del CCM. In una seconda fase della tesi, gli effetti antineoplastici di ATRA e decitabina sono stati valutati in vitro in linee cellulari di CCM, per sviluppare nuove terapie antitumorali con applicazione clinica. Le attività dei due farmaci sono state analizzate in linee cellulari MCC13, MCC26, MKL-1 e Peta per valutarne il potenziale antiproliferativo. La dose IC50 di ATRA era rispettivamente di 100 µM e 75 µM in MCC13 e Peta. ATRA non ha dato effetti sulle MCC26. La dose IC50 di decitabina era di 0.5 μM sia in MCC13 che in MCC26, mentre era di 50 nM nelle MKL-1. Sia ATRA che decitabina hanno ridotto la proliferazione delle cellule CCM in vitro. Questi risultati preliminari possono aprire la strada a nuovi approcci per la terapia CCM.

Merkel cell polyomavirus (MCPyV) which causes an almost ubiquitous, asymptomatic infection, rarely causes an aggressive skin tumor with limited therapeutic options, i.e., Merkel cell carcinoma (MCC). Despite serum anti-MCPyV IgGs have been detected with a relatively high prevalence in healthy individuals, previous studies reported variable rates. Innovative immunoassays are needed to determine the MCPyV serology in humans. The main aim of the present thesis was to investigate the impact of oncogenic MCPyV infection in the healthy population. To this end, a new indirect ELISA assay using two synthetic peptides mimicking epitopes of MCPyV capsid proteins 1 and 2 (VP1/VP2) was developed and validated to analyze the presence of anti-MCPyV IgGs in sera from healthy individuals, including children (HC, 0-20 yrs), adults (HS, 21-65 yrs), and elderly (ES, 66-100 yrs). Synthetic peptides were initially computationally designed to detect IgGs to MCPyV mimotopes. The assay performance in detecting anti-MCPyV IgGs was afterwards determined on MCPyV-positive/-negative control sera. The assay showed a high performance. Then, the ELISA was extended to HC (n=344), HS (n=510) and ES (n=226) sera with unknown MCPyV serology. Age-/gender-specific MCPyV seroprevalences were examined. The overall prevalence of serum anti-MCPyV IgGs was 40.7% in HC. HC age stratification revealed a significantly lower MCPyV seroprevalence in the 0-5 yrs cohort (13%) compared with the older cohorts. MCPyV serological profile showed that the 0-5 yrs cohort had the lowest Optical densities (ODs) compared to the older cohorts. Higher ODs were found in females than males. The prevalence of anti-MCPyV IgMs was 29.7% in HC, with similar rates in age-stratified HC. The 0-5 yrs cohort had the lowest IgM ODs compared to the older cohorts. The 6-10/11-15 yrs cohorts had the highest ODs compared to the 16-20 yrs cohort. A higher seroprevalence of anti-MCPyV IgG and IgM were found in females than in males. The anti-MCPyV IgG and IgM reactivity patterns in HC were also simultaneously evaluated. IgG and IgM ODs showed an inverse trend, as being increased and decreased, respectively, in the older HC cohort. The prevalence of serum anti-MCPyV IgGs was 61.8% in HS group. Higher ODs were found in females than in males. MCPyV-seroprevalence of ES sera was 63.7%, a proportion higher than the 40.7% observed in HC, and similar to the 61.8% detected in the HS. MCPyV-serology was then examined in HC, HS, and ES, which were stratified in 5-yrs range cohorts. MCPyV seroprevalence was lower in the 0-5 yrs cohort (13%) than in the older cohorts (52.3-72%). Moreover, the 0-5 yrs cohort had the lowest OD values. Higher ODs were found in 6-10, 11-15 and 16-20 yrs cohorts compared to the other cohorts. The results of the present thesis indicate that the assay developed herein can efficiently detect serum anti-MCPyV IgGs. Our data indicate that MCPyV infection is widespread in humans. After seroconversion in pediatric age, MCPyV circulates asymptomatically in the prepuberal, adult and elder populations at a high prevalence, without substantial age-related variations in adulthood and senility. New approaches for MCC treatment are needed. In the second phase of this thesis, the antineoplastic effects of ATRA and decitabine drugs were evaluated in vitro in MCC cell lines, to develop new antitumor therapies with clinical application. The activities of the two drugs were assayed as single agents in MCC cell lines MCC13, MCC26, MKL-1 and Peta to verify their antiproliferative potential. The ATRA IC50-dose was 100 µM and 75 µM in MCC13 and Peta, respectively. ATRA showed no antiproliferative effects on MCC26. The decitabine IC50-dose was 0.5 μM in both MCC13 and MCC26, while it was 50 nM in MKL-1 cells. Both ATRA and decitabine effectively reduced the proliferation of MCC cells in vitro. These preliminary results may improve the development of new approaches for MCC therapy.

Merkel cell polyomavirus, a small DNA tumour virus, in humans

MAZZIOTTA, CHIARA
2022-06-14T00:00:00+02:00

Abstract

Merkel cell polyomavirus (MCPyV) which causes an almost ubiquitous, asymptomatic infection, rarely causes an aggressive skin tumor with limited therapeutic options, i.e., Merkel cell carcinoma (MCC). Despite serum anti-MCPyV IgGs have been detected with a relatively high prevalence in healthy individuals, previous studies reported variable rates. Innovative immunoassays are needed to determine the MCPyV serology in humans. The main aim of the present thesis was to investigate the impact of oncogenic MCPyV infection in the healthy population. To this end, a new indirect ELISA assay using two synthetic peptides mimicking epitopes of MCPyV capsid proteins 1 and 2 (VP1/VP2) was developed and validated to analyze the presence of anti-MCPyV IgGs in sera from healthy individuals, including children (HC, 0-20 yrs), adults (HS, 21-65 yrs), and elderly (ES, 66-100 yrs). Synthetic peptides were initially computationally designed to detect IgGs to MCPyV mimotopes. The assay performance in detecting anti-MCPyV IgGs was afterwards determined on MCPyV-positive/-negative control sera. The assay showed a high performance. Then, the ELISA was extended to HC (n=344), HS (n=510) and ES (n=226) sera with unknown MCPyV serology. Age-/gender-specific MCPyV seroprevalences were examined. The overall prevalence of serum anti-MCPyV IgGs was 40.7% in HC. HC age stratification revealed a significantly lower MCPyV seroprevalence in the 0-5 yrs cohort (13%) compared with the older cohorts. MCPyV serological profile showed that the 0-5 yrs cohort had the lowest Optical densities (ODs) compared to the older cohorts. Higher ODs were found in females than males. The prevalence of anti-MCPyV IgMs was 29.7% in HC, with similar rates in age-stratified HC. The 0-5 yrs cohort had the lowest IgM ODs compared to the older cohorts. The 6-10/11-15 yrs cohorts had the highest ODs compared to the 16-20 yrs cohort. A higher seroprevalence of anti-MCPyV IgG and IgM were found in females than in males. The anti-MCPyV IgG and IgM reactivity patterns in HC were also simultaneously evaluated. IgG and IgM ODs showed an inverse trend, as being increased and decreased, respectively, in the older HC cohort. The prevalence of serum anti-MCPyV IgGs was 61.8% in HS group. Higher ODs were found in females than in males. MCPyV-seroprevalence of ES sera was 63.7%, a proportion higher than the 40.7% observed in HC, and similar to the 61.8% detected in the HS. MCPyV-serology was then examined in HC, HS, and ES, which were stratified in 5-yrs range cohorts. MCPyV seroprevalence was lower in the 0-5 yrs cohort (13%) than in the older cohorts (52.3-72%). Moreover, the 0-5 yrs cohort had the lowest OD values. Higher ODs were found in 6-10, 11-15 and 16-20 yrs cohorts compared to the other cohorts. The results of the present thesis indicate that the assay developed herein can efficiently detect serum anti-MCPyV IgGs. Our data indicate that MCPyV infection is widespread in humans. After seroconversion in pediatric age, MCPyV circulates asymptomatically in the prepuberal, adult and elder populations at a high prevalence, without substantial age-related variations in adulthood and senility. New approaches for MCC treatment are needed. In the second phase of this thesis, the antineoplastic effects of ATRA and decitabine drugs were evaluated in vitro in MCC cell lines, to develop new antitumor therapies with clinical application. The activities of the two drugs were assayed as single agents in MCC cell lines MCC13, MCC26, MKL-1 and Peta to verify their antiproliferative potential. The ATRA IC50-dose was 100 µM and 75 µM in MCC13 and Peta, respectively. ATRA showed no antiproliferative effects on MCC26. The decitabine IC50-dose was 0.5 μM in both MCC13 and MCC26, while it was 50 nM in MKL-1 cells. Both ATRA and decitabine effectively reduced the proliferation of MCC cells in vitro. These preliminary results may improve the development of new approaches for MCC therapy.
MARTINI, Fernanda
ROTONDO, John Charles
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Descrizione: Ph.D. thesis_Mazziotta Chiara
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11392/2489304
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