Ricerca di Open Reading Frames (ORFs) nella regione 8q24 coinvolta nel rischio di tumore al colon retto e alla prostata nell'uomo

Il Cancro Colon Rettale (CRC) è una neoplasia maligna molto diffusa nei paesi occidentali che colpisce la porzione centrale (colon) e terminale (retto) dell’intestino crasso: rappresenta il risultato finale di una serie di eventi mutazionali che portano le cellule della mucosa del colon a trasformarsi da sane in neoplastiche. Si tratta di una patologia multifattoriale alla cui insorgenza contribuiscono fattori di rischio ambientali (stili di vita, familiarità…) e genetici. Da recenti studi di associazione effettuati su tutto il genoma è emersa l’esistenza di un locus di suscettibilità a livello del braccio lungo del cromosoma 8 (8q24). In questa zona sono presenti dei polimorfismi a singolo nucleotide (SNPs). Uno di questi, rs6983267, fa sì che a livello del nucleotide 128482487 la G venga sostituita da una T. Chi possiede la forma variante ha un aumento del rischio di sviluppare cancro colon-rettale ed alla prostata (con un OR compreso tra 1.17 e 1.22). Partendo dall’osservazione che la regione entro 8q24 risulta essere ultra-conservata e che all’interno di essa è presente un polimorfismo associato al rischio di sviluppare cancro del colon retto (ma anche della prostata) ma che in tale regione non sono presenti geni noti o predetti, in questa tesi ci si è posti l’obiettivo di cercare di individuare la presenza di un trascritto, possibilmente soggetto a splicing. Dopo una innumerevole serie di RT-PCR effettuate sull’RNA messaggero estratto da tessuti normali è effettivamente emersa la presenza di un amplificato che indica una attività trascrizionale di una porzione di circa 400 nucleotidi, all’interno della regione ultra-conservata. La porzione di amplificato non subisce attività di splicing e questo rende impossibile l’identificazione di strutture esoniche e introniche. Il trascritto potrebbe codificare per una Open Reading Frame senza introni, tuttavia alcune considerazioni evoluzionistiche porterebbero ad escludere questa ipotesi. Si potrebbe supporre la presenza di un RNA con funzioni regolatrici. L’RNA possiede la coda poli-A (dato che il cDNA è stato preparato con oligo-dT). Le ricerche nei vari database non hanno permesso di caratterizzare l’amplificato da un punto di vista funzionale e gli esperimenti effettuati mediante 3’ RACE non hanno permesso di estendere il frammento alla sua estremità 3’. Si conclude che il trascritto, svolge un ruolo, non ancora ben identificato. Si renderanno necessarie ulteriori indagini di tipo funzionale

Residual tumor micro-foci and overwhelming regulatory T lymphocyte infiltration are the causes of bladder cancer recurrence

Bladder cancer has an unexplained, high recurrence rate. Causes of recurrence might include the presence of sporadic tumor micro-foci in the residual urothelial tissue after surgery associated with an inverted ratio between intratumoral effector and regulatory T cell subsets. Hence, surgical specimens of both tumors and autologous, macroscopically/histologically free-of-tumor tissues were collected from 28 and 20 patients affected by bladder or renal cancer, respectively. The frequencies of effector (IFN?+ and IL17+ T cells) and regulatory (CD4+CD25hiCD127lo and CD8+CD28-CD127loCD39+ Treg) T cell subpopulations among tumor infiltrating lymphocytes were analyzed by immunofluorescence, while the gene expression of MAGE-A1 and MAGE-A2 tumor-associated antigens was studied by RT-PCR. The results show that both the T cell infiltrate and the frequency of MAGE-A1/A2 gene expression were comparable in tumors and in autologous free-of-tumor tissues in bladder cancer, while the autologous free-of-tumor renal tissues showed reduced T cell infiltrate and frequency of MAGE gene expression as compared to the autologous tumors. Importantly, the intra-tumor T effector/Treg cell ratio was consistently <1 in bladder cancer patients (n. 7) who relapsed within two years, while it was always >1 in patients (n. 6) without recurrence (regardless of tumor stage) (P = 0.0006, Odds ratio = 195). These unprecedented findings clarify the pathogenic mechanism of bladder cancer recurrence and suggest that microscopically undetectable micro-foci of tumor may predispose to recurrence when associated with an inverted intratumoral T effector/Treg cell ratio

AIRE polymorphism, melanoma antigen-specific T cell immunity, and susceptibility to melanoma

AIRE is involved in susceptibility to melanoma perhaps regulating T cell immunity against melanoma antigens (MA). To address this issue, AIRE and MAGEB2 expressions were measured by real time PCR in medullary thymic epithelial cells (mTECs) from two strains of C57BL/6 mice bearing either T or C allelic variant of the rs1800522 AIRE SNP. Moreover, the extent of apoptosis induced by mTECs in MAGEB2-specific T cells and the susceptibility to in vivo melanoma B16F10 cell challenge were compared in the two mouse strains. The C allelic variant, protective in humans against melanoma, induced lower AIRE and MAGEB2 expression in C57BL/6 mouse mTECs than the T allele. Moreover, mTECs expressing the C allelic variant induced lower extent of apoptosis in MAGEB2-specific syngeneic T cells than mTECs bearing the T allelic variant (p < 0.05). Vaccination against MAGEB2 induced higher frequency of MAGEB2-specific CTL and exerted higher protective effect against melanoma development in mice bearing the CC AIRE genotype than in those bearing the TT one (p < 0.05). These findings show that allelic variants of one AIRE SNP may differentially shape the MA-specific T cell repertoire potentially influencing susceptibility to melanoma

Residual tumor micro-foci and overwhelming regulatory T lymphocyte infiltration are the causes of bladder cancer recurrence.

Bladder cancer has an unexplained, high recurrence rate. Causes of recurrence might include the presence of sporadic tumor micro-foci in the residual urothelial tissue after surgery associated with an inverted ratio between intratumoral effector and regulatory T cell subsets. Hence, surgical specimens of both tumors and autologous, macroscopically/histologically free-of-tumor tissues were collected from 28 and 20 patients affected by bladder or renal cancer, respectively. The frequencies of effector (IFNγ+ and IL17+ T cells) and regulatory (CD4+CD25hiCD127lo and CD8+CD28-CD127loCD39+ Treg) T cell subpopulations among tumor infiltrating lymphocytes were analyzed by immunofluorescence, while the gene expression of MAGE-A1 and MAGE-A2 tumor-associated antigens was studied by RT-PCR. The results show that both the T cell infiltrate and the frequency of MAGE-A1/A2 gene expression were comparable in tumors and in autologous free-of-tumor tissues in bladder cancer, while the autologous free-of-tumor renal tissues showed reduced T cell infiltrate and frequency of MAGE gene expression as compared to the autologous tumors. Importantly, the intra-tumor T effector/Treg cell ratio was consistently 1 in patients (n. 6) without recurrence (regardless of tumor stage) (P = 0.0006, Odds ratio = 195). These unprecedented findings clarify the pathogenic mechanism of bladder cancer recurrence and suggest that microscopically undetectable micro-foci of tumor may predispose to recurrence when associated with an inverted intratumoral T effector/Treg cell ratio

Immunogenicity of GX301 cancer vaccine: Four (telomerase peptides) are better than one

Peptide540-548, peptide611-626, peptide672-686 and peptide766-780, which are derived from human telomerase, constitute the immunogenic component of the GX301 cancer vaccine. The relative immunogenicity of these peptides is unknown, thus it is unsure whether their combined use offers real advantages over single peptide stimulation. Hence, this study compared the number of specific immune responses and responders to each peptide, as well as to their mixture (meaning the co-presence of the 4 peptides in the same culture well), achieved after ex vivo stimulation of PBMC from 21, HLA-A2+ (n.11) or HLA-A2- (n.10), healthy donors. The study was performed on freshly collected PBMC (T0) and on PBMC stimulated for 10 d with single peptides or their mixture (T1). Peptide-specific immune responses were analyzed by Elispot and cytokine intracellular staining by flow cytometry. The results showed that each peptide induced specific immune responses in some subjects, with different panels of responders among the peptides. Moreover, the numbers of responses and responders to the single peptides or their mixture were comparable. Importantly, the overall number of responders to the 4 peptides was higher than to each single peptide, or to their mixture, both at T0 and T1. These data demonstrate the immunogenicity of each of the 4 GX301 telomerase peptides. Moreover, they show the advantage of multi-peptide over single peptide stimulation, providing a clear support to their combined administration in vaccination protocols. However, the data pose a warning against peptide administration as a mixture due to possible interference phenomena during antigen presentation processes

Early and repeated IgG1Fc-pCons chimera vaccinations (GX101) improve the outcome in SLE-prone mice

A previous study showed that a tolerogenic gene vaccine based on a IgG1Fc-pCons chimera (here named GX101) protects NZB/NZW mice from SLE development. The present study was aimed at identifying the most effective schedule of immunization and the possible involvement of CD4(+)&nbsp;Foxp3(+)&nbsp;Treg in the mechanism of action, in view of its eventual translation to the human studies. NZB/NZW mice were vaccinated with B lymphocytes made transgenic by spontaneous transgenesis with a gene coding for a chimeric IgG1Fc-pCons construct. Different schedules of vaccination were set in relation to the timing and number of administrations. Survival, proteinuria levels, and CD4(+) Foxp3(+) Treg frequency were monitored during the full experiments. GX101-treated mice showed delayed disease onset and delayed mortality than controls. GX101 effects were implemented by early as well as repeated vaccine administrations. GX101 vaccination was associated with increased frequencies of CD4(+)&nbsp;CD25(+)&nbsp;Foxp3(+)&nbsp;Treg with respect to controls. This study demonstrates that early and repeated immunizations with GX101 vaccine provide a better outcome than late or single vaccine administration regarding onset/development in SLE-prone mice, acting as a possible disease-modifying approach. Vaccine effects are likely related to CD4(+)&nbsp;Foxp3(+)&nbsp;Treg cell expansion

Impaired immune response to Candida albicans in cells from Fanconi anemia patients

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and cancer predisposi-tion. Several studies show alterations of the immunological status of FA patients including defects inperipheral blood lymphocyte subsets, serum immunoglobulin levels, and inflammatory cytokines.However scanty information is available on the response of FA cells to specific infectious antigens. In thiswork we examined the response of FA cells to different immunological stimuli and found a defectiveresponse of IL-1b, TNF-aand IL-17 toCandida albicansstimulation thus pointing to a potentially impairedresponse to fungal infections of FA patients