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Studio dei correlati virologici, patologici e clinici in pazienti pediatrici trapiantati di rene: applicazione di nuove indagini molecolari
Full text linkThe relevance of viral infections in allograft lesion development is still unclear, although some viruses such as HCMV, EBV, VZV, HHV6, HHV8, seem to have a particular role especially during the first months after transplantation, and the polyomavirus BK (BKV), JC (JCV) and the parvovirus B19, have been implicated in the occur of kidney injury after more time following transplant.
In this study we investigated systemic and intrarenal viral infections in kidney transplant young recipients and we analysed the association of these data with the risk of acute rejection and chronic allograft injuries predictive of long-term dysfunction.
The presence of DNA sequences of human herpesviruses, polyomaviruses, and parvovirus B19 was analysed in renal allograft biopsies performed at baseline, for acute renal dysfunction and for follow-up during the first two years post transplant. We evaluated le presence of viral sequences in 69 transplanted children and young adult who underwent kidney transplant from 2000 to 2006: donor age was less than 6 year in 15 cases. These results regarding the genomic viral presence in these patients were correlated with clinical data, viral DNAemia, renal function tests and allograft histology analysed at the same time points of the follow-up. Taken as a whole, viral DNA was detectable in 46% baseline biopsies and in 70% follow-up biopsies of kidney allografts, where it generally persisted. The most frequently detected viruses were B19 and HHV-6, already present in donor kidneys, and BKV and EBV, usually involving the allograft during follow-up. Among viruses, only the intrarenal persistence of B19 DNA and B19 DNAemia was associated with the development of chronic allograft injury: these kind of data were never demonstrated before in literature. Regarding HCMV DNAemia, it was considered a risk factor for acute rejection as already suggested in many works from the literature. So, we conclude that parvovirus B19 seems to electively target the kidney and its intrarenal persistence is associated with chronic kidney allograft injury.
In the second part of this work, in order to identify new markers for a rapidly identification of viral infections occur early after transplantation and are often transmitted from the graft, we investigated whether EBV, HCMV, BKV, and parvovirus B19 genome sequences could be detected in kidney grafts, preservation and washing solutions before implantation, and whether they correlated with the occurrence of viral infections in the recipient.
The investigation of different donor graft samples (i.e., biopsy, preservation and washing solutions) that we wholly named “Kidney Unit” (KU), increased the sensitivity of viral DNA testing, but also gave clues to the mechanism of viral transmission through the kidney graft while the different samples are enriched from different cells from the donor: resident kidney cells or circulated blood cells. Viral genome sequences were frequently detected in donor renal graft units, especially in preservation and washing solutions. Overall, viral DNA was detected in at least one type of sample, including biopsy, preservation and washing solutions, in 51/75 (68%) kidney grafts and B19 was the most frequently detected virus (47%).
In agreement with their ability to establish latency in B lymphocytes and in monocytes progenitor cells, respectively, EBV and HCMV were probably carried by circulating blood cells, since viral DNA was generally detected in preservation and washing solutions, which are contaminated by blood cells, but not in kidney biopsies; whereas B19 DNA was often detected in kidney graft biopsies, besides in preservation and washing solutions, thus suggesting the virus probably infected resident kidney cells, which might be important sources of transmission to the recipient.
BKV is supposed to have tropism for the kidney and to achieve latency in renal tubular epithelial cells; however, we detected BKV DNA only in one donor kidney biopsy, whereas viral DNA was generally detectable in the allograft during follow-up, where it persisted, as we previously demonstrated.
The prevalence of EBV, HCMV, and BKV DNA was higher in preservation and washing solutions than in biopsies, indicating they were mainly carried by blood cells, whereas B19 was consistently detected in biopsies and solutions, suggesting virus was also present in resident kidney cells. Detection of viral DNA in kideny grafts was a significant risk factor for symptomatic infections in seronegative recipients in the early post-transplant period. In particular, EBV DNA-positive donor grafts were significantly associated with the risk of EBV infection in seronegative recipients, whereas the presence of B19 DNA in kidney grafts was a risk factor for B19 infection and/or DNAemia both in B19-seronegative and seropositive recipients. At variance, molecular testing for HCMV and BKV in donor graft had poor diagnostic utility.
In conclusion, this study demonstrates that detection of viral nucleic acids in preservation and washing solutions of a solid organ, i.e., the kidney, before implantation could be a useful test to identify recipients with increased risk of infections, especially symptomatic infections, in the early post-transplant period. The sensitivity and specificity of the test depends on viral tropism for cells and tissues of the graft.Il ruolo dell’infezione virale nell’insorgenza di lesioni nel rene trapiantato non è stato ancora del tutto precisato sebbene alcuni virus come HCMV, EBV, VZV, HHV6, HHV8, in una fase più precoce, e il poliomavirus BK (BKV) e JC (JCV) e al parvovirus B19, dopo più tempo dal trapianto, sembrano avere una precisa funzione nel determinare danni a carico del rene trapiantato. Infatti, tutti questi virus sono stati già descritti come importanti patogeni con tropismo renale.
Nel presente studio sono state investigate le infezioni virali, intrarenali e sistemiche, in una casistica di bambini e giovani adulti che sono stati sottoposti a trapianto di rene dal 2000 al 2006. Più esattamente sono stati analizzati i dati della prevalenza delle sequenze genomiche virali intrarenali e delle infezioni sistemiche (DNAemia) in associazione con il rischio di insorgenza di rigetto acuto e/o di lesioni croniche del rene trapiantato.
La presenza delle sequenze genomiche virali dei virus erpetici umani, dei poliomavirus e del parvovirus B19 è stata analizzata a livello della biopsie di rene eseguite al momento del trapianto, biopsie baseline, in presenza di disfunzioni renali acute e durante i primi due anni dal trapianto seguendo i tempi del protocollo di follow-up cioè a 6, 12 e 24 mesi post trapianto.
Sono stati studiati 69 riceventi pediatrici, bambini e giovani adulti, con un’età media pari a 13 anni che avevano ricevuto il rene da donatore deceduto in 65 casi e in 4 casi da famigliare vivente: l’età dei donatori era inferiore a 6 anni in 15 casi.
I risultati di questa prima parte dello studio, relativi alla prevalenza del DNA virale intrarenale sono stati correlati con i dati clinici, i dati di viremia (DNAemia), di funzionalità del rene trapiantato e con le valutazioni istologiche dello stesso momento del follow-up.
Globalmente, il DNA virale è stato ritrovato nel 46% delle biopsie baseline e nel 70% delle biopsie di follow-up, dove generalmente persiste nelle biopsie successive. I virus più frequentemente identificati sono il parvovirus B19 e l’herpesvirus HHV6, già presenti a livello delle biopsie di rene del donatore. Mentre la presenza delle sequenze genomiche dei virus EBV e BKV è stata associata alla comparsa di lesioni acute nel rene trapiantato. Tra tutti i virus studiati e ritrovati a livello del rene del ricevente, soltanto il DNA del parvovirus B19 e le relativa DNAemia sono state associate con lo sviluppo di lesioni croniche del rene trapiantato: tale dato non era mai stato dimostrato in precedenti studi della letteratura. Per quanto riguarda il HCMV, la relativa DNAemia è stata considerata un fattore di rischio per la comparsa di episodi di rigetto acuto: dato, questo, già dimostrato e confermato con il nostro studio. Quindi è possibile concludere che il parvovirus B19 sembra preferire, in modo particolare, il rene come possibile bersaglio da infettare e la sua persistenza intrarenale è associata con la comparsa di lesioni croniche del rene trapiantato.
Nella seconda parte del presente studio, con l’intento di identificare nuovi marcatori del rischio di infezione del ricevente trapiantato di rene, è stata valutata la presenza delle sequenze genomiche virali di EBV, HCMV, BKV, e del parvovirus B19 nel rene del donatore prima dell’impianto; più precisamente sono state analizzate le biopsie, le soluzioni di conservazione e di lavaggio dell’organo prima che questo venga trapiantato. È stato osservato poi se la presenza del DNA virale nell’unità rene (ovvero l’insieme dei diversi campioni derivati dal donatore: biopsia, soluzione di conservazione e di lavaggio) correlava con la comparsa dell’infezione virale nel ricevente. L’ indagine condotta a livello dei diversi campioni dell’unità rene del donatore, consente di aumentare la sensibilità del test molecolare, ma anche da maggiori indicazioni relative al meccanismo di trasmissione dell’infezione virale mediante il rene trapiantato dal momento che i diversi campioni dell’unità rene sono arricchiti di più frazioni cellulari del donatore: sono presenti sia le cellule residenti del rene a livello della biopsia, ma anche le cellule del sangue circolante soprattutto nel liquido di lavaggio.
Le sequenze genomiche virali sono frequentemente identificate nell’unità rene del donatore, soprattutto nelle soluzioni di conservazione e di lavaggio. Globalmente, il DNA virale è stato identificato, in almeno un tipo di campione dell’unità rene, in 51 su 75 reni donati (68%) e il virus più ritrovato è il B19 (47%). In accordo con la loro capacità di definire uno stato di latenza dei linfociti B e nei monociti, il DNA dei virus EBV, nel primo caso, e HCMV nel secondo, sono stati identificati principalmente nelle soluzioni di lavaggio e di conservazione, poiché tali virus sono probabilmente veicolati dalle cellule del sangue periferico. Mentre, nel caso del parvovirus B19, il DNA virale è stato trovato spesso nelle biopsie del rene del donatore: questo suggerisce che il virus probabilmente infetta le cellule residenti del rene, le quali potrebbero essere un importante sorgente di trasmissione dell’infezione al ricevente. Il poliomavirus BK si pensa abbia un particolare tropismo per il rene e che vada in latenza nelle cellule epiteliali tubulari del rene: ciononostante nel presente studio il DNA di BKV è stato identificato solo in una biopsia di rene del donatore mentre è stato più volte ritrovato, anche in maniera persistente, nelle biopsie di follow-up. In generale, è stato possibile constatare che la presenza del DNA virale nel rene del ricevente è un importante fattore di rischio di infezione sistemica per il ricevente sieronegativo nel primo periodo successivo al trapianto. In particolare, la presenza del DNA di EBV nell’ unità rene donata comporta un più elevato rischio di infezione da EBV nel ricevente sieronegativo, mentre la persistenza di B19 nel rene del ricevente è un fattore di rischio di infezione e/o di DNAemia da B19 per il ricevente sia sieropositivo che sieronegativo. Al contrario questo tipo di indagine molecolare dell’unità rene del donatore, condotta per il HCMV e per BKV non mostra una valida utilità diagnostica. Concludendo, con questo studio è stato possibile dimostrare che l’identificazione di acidi nucleici virali a livello delle soluzioni di lavaggio e di conservazione del rene da trapiantare potrebbe essere un test molecolare particolarmente utile per riconoscere i riceventi con un maggior rischio di infezione, soprattutto sistemica. La sensibilità e la specificità di tale test molecolare dipende però dal tropismo del virus per le cellule o per il tessuto dell’organo da trapiantare
West Nile virus and kidney disease
No full textWest Nile virus (WNV), the causative agent of West Nile fever and West Nile neuroinvasive disease in humans, has become endemic in many countries in all continents. Concerns on long-term mobility from WNV have arisen from recent studies that reported chronic kidney disease in patients who recovered from WNV infection, supported by data from animal models that showed prolonged excretion of the virus with urine. The purpose of this review is to summarize and discuss the results of studies in the literature that investigated WNV infection of the kidney in humans and in animal models and WNV excretion with urine, the potential damage to the kidney caused by WNV infection, the risk of WNV disease in kidney transplant recipients, the significance of detecting WNV in urine and its use in the diagnosis of WNV infection, and kidney involvement by other mosquito-borne flaviviruses
Gene therapy for thyroid cancer
No full textThyroid carcinomas are suitable targets for gene therapy because they can be highly lethal on one hand, while being susceptible to specific tumour targeting on the other hand. Several gene therapy modalities have been evaluated so far in experimental models of thyroid cancer, including tumour suppressor gene replacement, oncogene inhibition, suicide gene therapy, immunotherapy, antiangiogenesis, and viral oncolysis. All of these strategies have shown promising results, but clinical studies are lacking. Based on the clinical experience achieved in a pilot study in patients with advanced thyroid cancer and on clinical results in other types of solid cancer, it is suggested that combined gene therapy approaches, as well as multimodality therapeutic regimens, including gene therapy and conventional treatments, should be pursued to achieve clinically significant results
Evaluation of circulating thyroid-specific transcripts as markers of thyroid cancer relapse
No full textCirculating thyroid-specific transcripts have been suggested as potential molecular markers of residual or recurrent thyroid cancer. We assessed the accuracy of real-time RT-PCR-based detection of a panel of thyroid-specific markers, including TG, TPO, TSHR, NIS and PDS, in comparison with serum TG measurements in a series of 55 patients operated for differentiated thyroid cancer (DTC). Serum TG levels were higher in patients with residual thyroid tissue or metastatic cancer than in disease-free patients during thyroid hormone suppressive therapy (THST) and after stimulation with rhTSH (P < 0.05). Recombinant hTSH increased serum TG values in patients with tumor relapse (P < 0.05), but not in disease-free patients. This assay showed high specificity and good sensitivity in detecting tumor relapse (accuracy under THST = 81.4%; after rhTSH stimulation = 90.9%). TPO and TSHR mRNA, either under THST or after rhTSH, showed a significant correlation with disease status for molecular assays. Qualitative analysis of baseline and stimulated TG, NIS and PDS mRNA showed high sensitivity but low specificity in the prediction of thyroid cancer recurrence or metastases (accuracy under THST = 51%, 43% and 54%, respectively), whereas TPO and TSHR mRNA assays had higher specificity but low sensitivity, with accuracy under THST of 67% and 61%, respectively, that improved when these tests were combined. Our findings indicate that serum TG assay after TSH stimulation is the most accurate test for monitoring DTC. Combined measurements of TPO and TSHR mRNA levels during THST may represent a specific test for early detection of DTC relapse
Versatility of gene therapy vectors through viruses.
No full textAbstract: Several viruses have been engineered for gene therapy applications, and the specific properties of each viral vector have been exploited to target a variety of inherited and acquired diseases. Preclinical and clinical studies demonstrated that viral vectors are highly versatile tools capable of efficient transfer of foreign genetic information into almost all cell types and tissues. Gene therapy applications depend on vector characteristics, such as host range, cell- or tissue-specific targeting, genome integration, efficiency and duration of transgene expression, packaging capacity, and suitability for scale-up production. This review discusses the advances in the development of viral vectors, with particular emphasis on how knowledge of virus biology has been exploited to design a variety of vectors with improved safety characteristics and efficiency, potentially suitable for a large number of gene therapy applications
Investigation on Human Adrenocortical Cell Response to Adenovirus and Adenoviral Vector Infection
No full textAfter systemic administration, adenoviral vectors (AdVs) are sequestered in the liver and adrenal glands. Adenoviral vector transduction has been shown to cause cytopathic effects on human hepatocytes and to induce an inflammatory response, whereas the effect of AdVs on human adrenocortical cells has never been investigated. In this study, human adrenocortical carcinoma cell lines and primary cell cultures were used to assess the effects of wild-type adenovirus (Ad5WT) and E1/E3-deleted AdVs on cell proliferation and steroidogenesis. Ad5WT could efficiently replicate in adrenocortical cells, leading to S phase induction, followed by cell death, whereas high titer AdVs transduction had only mild effects on adrenocortical cell proliferation, with accumulation of cells in G2/M. Both AdVs and Ad5WT induced expression of inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-alpha, but, most importantly, they led to a marked and dose-dependent increase of cortisol and other steroid hormone production and consistently modulated expression of key steroidogenic enzymes and regulators of steroidogenesis. This effect, which was already apparent at 6 h post-infection, probably represented a response to adenoviral entry and/or early phases of infection. The result of this study contribute to the understanding of host response to adenoviral vector administration
Combined HSV-TK/IL-2 gene therapy in patients with recurrent glioblastoma multiforme: biological and clinical results
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Following our pilot clinical study of combined IL-2/HSV-TK gene therapy for recurrent glioblastoma multiforme (GBM), we extended the protocol to a larger population of patients and evaluated safety, feasibility, and biological activity of treatment. A total of 12 patients received intratumor injection of retroviral vector-producing cells (RVPCs), followed by intravenous ganciclovir (GCV). Treatment was well tolerated with only minor adverse events. Transduction of tumor cells was demonstrated in tumor biopsies. A marked and persistent increase of intratumor and plasma Th1 cytokine levels was demonstrated after RVPC injection. At magnetic resonance imaging evaluation, two patients had a partial response (including a patient showing disappearance of a distant noninjected tumor mass), four had a minor response, four had stable disease, and two had progressive disease. The 6- and 12-month progression-free survival rates were 47 and 14%, respectively. The 6- and 12-month overall survival rates were 58 and 25%, respectively. In conclusion, the results of our clinical protocol of gene therapy for recurrent GBM, based on combined delivery of a suicide and a cytokine gene, demonstrate that intratumor injection of RVPCs was safe, provided effective transduction of the therapeutic genes to target tumor cells, and activated a systemic cytokine cascade, with tumor responses in 50% of cases
A pilot study of combined suicide/cytokine gene therapy in two patients with end-stage anaplastic thyroid carcinoma
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This study represents the first report of gene therapy for anaplastic thyroid carcinoma, one of the most aggressive solid tumors in humans. Two patients with end-stage anaplastic thyroid carcinoma were treated by direct intratumor injection of retroviral vector producer cells followed by ganciclovir. The retroviral vector carried the human IL-2 gene and the suicide gene thymidine kinase of herpes simplex virus type 1. Treatment was safe and associated with only mild adverse events. Transduction of tumor cells and production of T helper type 1 cytokines was demonstrated in tumor biopsies. Gene therapy led also to a marked increase in T helper type 1 cytokine expression in peripheral blood mononuclear cells. Radiological evaluation of injected tumor masses demonstrated local tumor necrosis
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