Academic literature on the topic 'Trapianto di cellule staminali emopoietiche'

Since its discovery, more than a decade ago, induced pluripotent stem cells (iPS) have had a prominent relevance in the environments of biomedical research and, at the same time, their origin has been related to the search for an ethical alternative to use of the stem cells obtained from internal mass of the human embryo. In this article we intend to give an overview of its possible applications in the advancement of biomedicine and its relationship with bioethics. From its possible application to regenerate tissues, after proceeding to their differentiation; testing of drugs for different conditions; or their use in models of diseases, among which the neurological ones stand out. Also, its application in obtaining germ cells and human embryos. The situation of the first clinical trial to regenerate a tissue from the subject’s own iPS cells, and the recent allogeneic transplantation in Japan, suggest advances in the clinical translation of these cells. On the other hand, the production of germ cells from iPS cells and the new cells called extended pluripotent stem cells (EPS), obtained by genetic reprogramming through a chemical cocktail, that give rise not only to the tissues of the embryonic layers, but also extra- embryonic, are a new path to making clonation by another route.----------Fin dalla sua scoperta, per oltre un decennio, cellule staminali pluripotenti indotte (iPS) hanno un’importanza notevole nella ricerca biomedica ambienti e, allo stesso tempo, la sua origine è legata alla ricerca di un’alternativa etica all’utilizzo le cellule staminali ottenute dalla massa interna dell’embrione umano. In questo articolo diamo una panoramica delle possibili applicazioni nell’avanzamento biomedicina e la loro relazione bioetica. Dalla sua possibile applicazione di rigenerare il tessuto, quindi procedere alla differenziazione; la sperimentazione di farmaci per diversi disturbi; o il suo uso in modelli di malattie, tra cui spiccano quelle neurologiche. Così come la sua applicazione nell’ottenere cellule germinali e embrioni umani. La situazione del primo studio clinico per rigenerare un tessuto da cellule iPS e proprio trapianto recente del soggetto in Giappone rappresentano passi nella traduzione clinica di queste cellule. Inoltre la produzione di cellule germinali dalle cellule iPS e nuove cellule chiamate cellule staminali pluripotenti estesi (EPS), riprogrammando geneticamente da un cocktail chimico, causando non solo ai tessuti degli strati embrionali, ma extraembrionali anche costituire un nuovo percorso verso la clonazione di un altro itinerario.

Le osteopetrosi sono un raro gruppo eterogeneo di condizioni patologiche, caratterizzate da aumento della densità dell'osso dovuto ad un disordine del riassorbimento da parte degli osteoclasti. Esse sono più frequenti in gruppi etnici all'interno dei quali sono presenti consanguinei. Ne sono state descritte almeno cinque tipi, ma le forme più note sono la autosomica recessiva (AROP, detta anche maligna), la intermedia (IOP, anch'essa recessiva ma con insorgenza più tardiva), la forma con acidosi tubulare e la autosomica dominante (ADOP). Riportiamo un caso di AROP diagnosticato in epoca neonatale (3 settimane). Esso dimostra come alcune caratteristiche radiologiche (ma non tutte), siano già presenti alla nascita. Alla luce delle recenti acquisizioni terapeutiche (soprattutto trapianto di cellule staminali), una diagnosi precoce della malattia potrebbe, pertanto, prevenire importanti modificazioni patologiche alcune delle quali gravemente invalidanti.

In questa seconda parte, l’attenzione viene focalizzata sulle “cellule staminali non embrionali”, cioè le cellule staminali somatiche (di origine fetale o adulta) e le cellule staminali del sangue del cordone ombelicale. Queste cellule, spesso definite “cellule staminali adulte”, sono state identificate prima delle cellule staminali embrionali. Infatti, l’espressione stessa di cellula “staminale” deriva dall’identificazione delle cellule staminali emopoietiche nel midollo osseo (1961). Più tardi le ricerche hanno evidenziato la presenza di tali cellule immature, multipotenti, che si auto-rinnovano e si auto-differenziano pressoché in tutti i tessuti ed organi del feto e dell’adulto. Appena scoperte, queste cellule staminali “adulte” hanno trovato subito un impiego terapeutico con i primi trapianti di midollo osseo per il trattamento di patologie, maligne e non, del sangue e del sistema linfoide. Oggi le cellule staminali emopoietiche sono usate anche nel trattamento di malattie auto-immuni, come la sclerosi multipla o il lupus erythematosus e nella medicina rigenerativa. Una seconda fonte importante di cellule staminali “adulte” è rappresentata dalle cellule staminali mesenchimali, situate principalmente nel midollo osseo, progenitrici di vari ceppi cellulari: osso, cartilagine, muscolo, tessuto adiposo e astrociti. Queste cellule sembrano avere un ruolo-chiave nella rigenerazione dei tessuti. Sono stati isolati diversi tipi di cellule mesenchimali multipotenti, con proprietà paragonabili a quelle delle cellule staminali embrionali. Il più noto è quello delle MAPCs di Catherine Verfaillie. Queste cellule sono usate clinicamente per vari scopi, tra cui la rigenerazione del miocardio infartuato, l’angiogenesi terapeutica in pazienti con ischemia periferica acuta (specialmente la malattia di Buerger) e il bioengineering (rivestimento cellulare di legamenti o di valvole cardiache sostitutive). In questo ambito si sono registrati risultati incoraggianti nell’animale per il trattamento delle malattie neurodegenerative, dell’ictus, del trauma cerebrale e dei danni del midollo spinale. Sono stati isolati molti altri tipi di cellule staminali “adulte” le cui proprietà riparatrici sono state verificate con successo nell’animale: cellule staminali neuronali (per il morbo di Parkinson, la sclerosi multipla, il morbo di Huntington, l’ictus, il trauma cerebrale, le lesioni del midollo spinale), cellule staminali muscolari (per l’incontinenza urinaria, il danno miocardico), cellule staminali endoteliali (per l’ischemia acuta periferica), cellule staminali cardiache, cellule staminali della retina (per la degenerazione maculare), cellule staminali del limbus della cornea (per il danno corneale). Allo stato attuale, i risultati clinici più promettenti si sono ottenuti con le cellule staminali del sangue del cordone ombelicale (UCB), che hanno portato allo sviluppo di un’area di mercato caratterizzata dalla creazione di banche private di UCB. Generalmente le cellule UCB provocano, al massimo, una reazione immune piuttosto blanda quando vengono trapiantate in soggetti con donatori non compatibili. Si usano con successo laddove sia necessaria una riparazione o rigenerazione nell’organismo del ricevente. I migliori risultati con cellule staminali UCB, fino ad ora, sono stati ottenuti nel trattamento di bambini con morbo di Krabbe. Benefici si sono ottenuti anche dal trapianto locale di cellule UCB in pazienti con danni al midollo spinale. ---------- In this second part of the article, the attention is focused on “non embryonic stem cells”, that is somatic stem cells (from fetus or adult organisms), and umbilical cord blood stem cells. These stem cells, sometimes referred to as “adult stem cells”, were known and recognized as such before the embryonic ones. In fact the mere expression “stem” cells to designate this particular type of immature cell, from which derive all the others, more differentiated cells, came from the identification of the hematopoietic stem cells, in bone marrow (1961). Later investigations have shown that there are such cells, immature, multipotent, self-renewing, and self-differentiating ones in almost all tissues and organs of fetus or adult organism. As soon as they were discovered, these “adult”, autologous stem cells were immediately put in the service of patients, with the first transplantations of bone marrow performed either for the treatment of malignancies, or for the treatment of hematologic disorders. Today, autologous hematopoietic stem cells are also used for the treatment of auto-immune diseases, such as multiple sclerosis or lupus erythematosus and for regenerative medicine. A second, important source of “adult” stem cells are the mesenchymal stem cells, found mainly in bone marrow, but also in blood, progenitors of multiple cell lineages, including bone, cartilage, muscle, adipose tissue and astrocytes, and which seem to hold the key to tissue regeneration. Different types of multipotent mesenchymal stem cells, with properties comparable to those of embryonic stem cells, have been isolated, the best known being the multipotent adult progenitor cells (MAPCs). These cells are used clinically mainly for the healing of the heart after myocardial infarction, with positive statistically significant results, for therapeutic angiogenesis in patients suffering of peripheric ischemic disease (especially Buerger’s disease), and for bioengineering (cellular coating of artificial ligaments or of prosthetic heart valves). They have given promising results in animals for the treatment of neurodegenerative diseases, ictus, brain trauma and spinal cord injuries. Many other types of “adult” stem cells have been isolated and their healing properties assessed with success in animals, such as neural stem cells (for Parkinson’s disease, multiple sclerosis, Huntington’s disease, ictus, brain trauma, spinal cord injury), muscle stem cells (for urinary incontinence, myocardial infarction), endothelial stem cells (for critical limb ischemia), cardiac stem cells, retinal stem cells (for macular degeneration), limbal stem cells (for damaged cornea). At the moment, the more promising results in patients have been obtained with umbilical cord blood stem cells (UCB), prompting the birth of a commercial trade based on private banks. Umbilical cord blood stem cells offer indeed the advantage of their immaturity: as such, they rarely trigger more than a mild immune reaction when transplanted in unrelated recipient organisms. They are used with profit wherever a healing or regenerative process is necessary in a given patient. Up to now, best results with the UCB cells have been obtained in the treatment of children with Krabbe’s disease. Some patients with injured spinal cords have also experienced benefits from UCB cells grafts.

Gli Autori intendono svolgere alcune riflessioni riguardo a informazione, consenso e comunicazione nella relazione paziente/donatore – medici nell’ambito della raccolta di cellule staminali emopoietiche da sangue periferico. Lo studio, prospettico ha coinvolto 73 pazienti/donatori “candidati” per la raccolta aferetica di cellule staminali emopoietiche da sangue periferico e si è sviluppato attraverso la somministrazione di due questionari (paziente/donatore – medico). I dati raccolti rivelano l’instaurazione di una relazione di confidenzialità tra i soggetti coinvolti nello studio e i medici; i pazienti e i donatori risultano aver ricevuto un’informazione chiara e aver avuto l’opportunità di rivolgere ai medici ulteriori domande, ad esempio in relazione alle modalità della raccolta, alle finalità del trattamento stesso, alla conservazione delle cellule staminali, e alla riservatezza dei dati personali Gli Autori prendono inoltre in considerazione gli aspetti etici e medico – legali correlati alla raccolta di cellule staminali emopoietiche da sangue periferico in caso di paziente minore. ---------- The Authors intend to make some considerations about information, consent and communication in patient/donor-physicians relationship, related to peripheral blood stem cell collection. The prospective study was carried on 73 patients interested in peripheral blood stem cell collection and it developed through the administration of two different questionnaires (physician-patient/donor). Data obtained show the establishment of a confidence relationship among the subject involved in the study and physicians; patient and donors received a clear information and had the opportunity to ask further questions to the physicians, in particular related to the terms of stem cell collection, to the purpose of the treatment itself, to the preservation of stem cells, and to the protection of personal data. The Authors paid attention to ethical and medico-legal aspects of haematopoietic stem cell collection when the patient is a minor.

Il trapianto di cellule staminali emopoietiche rappresenta la terapia di scelta per numerose patologie ematologiche. Tuttavia, la mortalità da trapianto (non relapse mortality-NRM), ha limitato per lungo tempo il suo utilizzo in pazienti di età >65 anni. L’età non può più essere considerata una controindicazione assoluta al trapianto e il suo utilizzo in fasce di età un tempo ritenute non idonee è in sensibile aumento. La NRM è legata a tre ordini di complicanze: immunologiche (malattia del trapianto contro l’ospite, Graft versus-Host Disease -GVHD-), infettive e tossiche. La tossicità d’organo è direttamente correlata alla intensità del condizionamento che quindi viene ridotta in caso di comorbidità e nel paziente anziano. Tuttavia, ridurre l’intensità del condizionamento significa anche aumentare il rischio di ripresa della malattia ematologica di base e quindi tale aggiustamento deve essere fatto in funzione di indici di invecchiamento e di comorbidità, al fine di non ridurre la potenzialità curativa del trapianto. Per valutare le comorbidità abbiamo uno score altamente predittivo (Hematopoietic Cell Transplant-Comorbidity Index, di Sorror), mentre per valutare l’invecchiamento c’è una grande necessità clinica di marcatori innovativi di età biologica. Il presente lavoro ha l’obiettivo di valutare, nei pazienti sottoposti a trapianto allogenico di cellule staminali emopoietiche per tutte le indicazioni ematologiche, lo stato di metilazione del DNA, indice di età biologica. Lo scopo è di correlare l’epigenoma al rischio trapiantologico del singolo individuo.
Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established therapy for several life-threatening hematological disorders, mostly malignancies. However, non-relapse mortality (NRM) has limited its use in patients aged over 65 years. Nowadays age cannot be considered an absolute contraindication for this treatment and the use of transplantation, in patient groups once considered unsuitable, is increasing significantly. NRM is due to three types of complications: immunological (graft versus host disease), infectious and toxic. Organ toxicity is directly related to the intensity of conditioning which is therefore reduced in case of comorbidities and in elderly patients. However, reducing the intensity of chemotherapy also means increasing the risk of relapse of the underlying haematological disease and therefore this adjustment must consider aging and comorbidity indexes in order not to reduce the curative potential of the transplant. To evaluate comorbidities we have a highly predictive score (the Comorbidity Index Score by Sorror, HCT-CI), while to evaluate aging there is a great clinical need to apply innovative biological age markers. The present work aims to evaluate the state of DNA methylation, a biological age index, in the setting of patients undergoing allogeneic haematopoietic stem cell transplantation for all haematological indication. The aim is also to correlate the epigenome with the transplant risk of the single individual.

Allogenic stem cell transplantation represents an important therapeutic option for the treatment of a number of haematological diseases. Particularly in the context of onco-haematological diseases this treatment has been shown to improve outcome thanks to the graft versus tumour (GVT) effect. Although recent improvements in transplantation procedures, still some limitations to the applicability of these treatment persist. First, not all patients may have available an HLA (human leukocyte antigens) identical donor and secondly, conditioning regimens before transplantation may be too toxic for elderly patients or for patients with other co-morbidities. To overcome this limitations in last few years many researchers have been exploiting new approaches. The introduction of not fully matched transplantations (mismatched or haploidentical) and reduced intensity regiments have partially overcome the limitations. In the onco-haematological context the most common complications of allogenic stem cell transplantation are relapse, opportunistic infections and activation of transplanted immune system against the normal tissues of the host (graft versus host disease – GVHD). All of these represent alterations of the normal functions of the immune system and demonstrate the importance of monitoring immunity in allo-transplanted patients. Studies in these field are mostly limited to the evaluation of the first year after transplantation and data from longer follow up are limited. In this study we monitored patients undergoing haematopoietic stem cell transplantation from an alternative donor after reduced intensity regimen over one year after transplantation (up to 4-5 years after transplantation). Particularly we evaluated 6 patients (age at transplantation 34; range 15-49) undergoing haploidentical stem cell transplantataion associated with T cell depletion in vitro (immunomagnetic selection of CD34 stem cells) and in vivo (anti CD52 antibody – Alemtuzumab) followed by infusion of CD8 depleted donor lymphocytes and 18 patients (age at transplantation 40; range 22-60) undergoing transplantation from a match unrelated donor (MUD) followed by in vivo T cell depletion (anti thymocyte globulins – ATG). All the patients have been analysed at the times of clinical remission and not under pharmacological treatment. In order to compare data obtained by the single cohorts to a normal situation we also evaluated 10 healthy donors (age 37; range 24-55). The evaluation of the immune recovery has been carried out through 4 different techniques: - analysis of chimerism through amplification of 9 different short tandem repeats (STR); - evaluation of the lymphoid sub population B, T and NK (and their maturation stages) through flow cytometry immunophenotype; - analysis of the thymic productivity trough quantification of the episomal DNA sjTREC (signal joint T-cell receptor excision circle); - evaluation of the receptorial complexity of the T and B cell compartment trough analysis of the CDR3 (complementary determinating region 3) of the β chain of the T cell receptor and of the heavy chain of the immunoglobulins. Our results show no significant differences between the two groups of patients analysed in the long term immune reconstitution, neither respectively the counts of the lymphoid sub population nor regarding the complexity values for the B and T cell receptors comparing the data to the ones from healthy subjects. We show a reduction in the thymic productivity that persist over 3 years post transplantation although there are no difference comparing the two cohorts of patients. Even though the counts of the main populations normalize between 1 and 2 years after transplantation the analysis of the maturation of the B and T cell compartments highlights the persistence of alterations in the normal maturation process in all the follow up points. Particularly both patients undergoing haploidentical or MUD transplantation present an increase in the B naïve subpopulation and a decrease in the B memory subsets demonstrating an alteration in the normal B cell development probably due to an alteration in the normal function of the germinal center. Concerning the T cell compartment it has been seen a decrease in the production of naïve T cells, that reflects the low thymic production, and an increase in the effector and terminal memory compartment. These might be a mechanism involved in the control of the oncological disease . In conclusion, patients that do not relapse and do not experience other clinical problems are able to recover immunity in the long term although thymic production remains low. No significant difference are found between the two types of transplantation highlighting that haploidentical transplantation is a good alternative to HLA identical transplantation implying less problems for donor recruitment. Analysis of the maturation steps of the B and T cell compartment demonstrated the persistence of alterations long term after transplantation indicating that this evaluation should be further studied in order to elucidate the mechanism at the basis of the immune recovery. Moreover this could be a good marker for monitoring the clinical course of the patients.

L'outcome dei pazienti sottoposti a trapianto allogenico di cellule staminali emopoietiche è fortemente influenzato da graft versus leukemia (GvL) e graft versus host disease (GvHD) che sono mediate, almeno in parte, dagli antigeni minori di istocompatibilità (mHAgs). In letteratura sono stati identificati 26 mHAgs che sono stati correlati a GvHD/GvL con risultati incompleti e in alcuni casi contrastanti; inoltre manca una metodica che sia in grado di genotipizzare contemporaneamente un pannello così ampio. Il lavoro è stato finalizzato alla preparazione di un protocollo di laboratorio che permetta di studiare in modo efficace i 26 mHAgs identificati, per poi correlarli con GvHD/GvL all’interno di uno specifico gruppo di trapiantati. Utilizzando la metodica IPlex Gold Mass Array Sequenom e tecniche di biologia molecolare convenzionale sono stati genotipizzati 26 antigeni minori di istocompatibilità per 46 coppie full-matched. Tutti i pazienti inclusi nel progetto di studio erano stati sottoposti a trapianto allogenico di cellule staminali emopoietiche da donatore familiare o volontario full-compatibile per leucemia mieloide cronica (n=46) o leucemia acuta linfoblastica Philadelphia positiva (LAL-Ph+, n=24). Il progetto ha confermato l'efficienza (98.6%) e la fattibilità delle metodiche proposte. Dal lavoro è inoltre emerso che, le differenze tra donatore e ricevente a libello mHAgs ACC-1, ACC-4, ACC-5, LB-MTHFD1-1Q, UGT2B17, DPH1, LRH1 potrebbero essere fattori predittivi di GvHD (p<0.05). La seconda evidenza è legata a un trend secondo cui il mismatch per LB-ADIR1 protegge dalla recidiva di malattia, in particolare nei confronti della LAL-Ph+ che è scarsamente responsiva all'allo-immunoterapia. Questo lavoro pilota, la cui casistica deve quindi essere ampliata, ha dimostrato l’efficacia della genotipizzazione con IPlex Gold Sequenom e l’elevato potenziale degli mHAgs sia come fattori predittivi di GvHD che come driver di GvL.
The outcome of allogeneic stem cell transplantation (Allo-SCT) is closely related to graft versus host disease (GvHD) and graft versus leukemia (GvL) effects which, in part, are mediated by mHAgs. Twenty-six mHAgs have been identified and reported to be differently and variably correlated with GVHD or GVL, but a simultaneous method to genotype a so large panel of mHAgs has never been employed. The aim of this work has been to develop a feasible method to genotype all the 26 mHAgs described so far and to test them for their correlation with GVHD and GVL in a group of donor/recipient pairs submitted to allo-SCT. For a multi-genotyping of 23 mHAgs we used iPlex Gold Mass Array technology (3 multiplex). For the other three mHAgs we designed other three assays based on conventional molecular biology. By these methods, we tested the 26 mHAgs in 46 donor/recipient pairs full-matched that underwent allo-SCT (sibling or MUD) because of Philadelphia positive CML (n=46) or ALL-Ph+ (n=24). Maldi-Tof IPlex Gold technology proved a high degree of efficiency (98.6%). As expected, sibling pairs showed most identity of MUD pairs. Notably, donor/recipient mismatch on ACC-1, ACC-4, ACC-5, LB-MTHFD1-1Q, UGT2B17, DPH1, LRH1 can drive GvHD effect (p<0.01). Next we identified that LB-ADIR1 can enhance (p=ns, but there is a trend) GvL effect specially on ALL-Ph+ that is otherwise un-responsible to allo-immunotherapy. Our data generated by a multi-genotype technique confirm the role of mHAgs in addressing GvL (in some cases without GvHD) and suggest that a study of mHAgs could be perfomed before transplant in order to better investigate the role of the known and new mHAgs involved in GvHD and GvL effects.

L'outcome dei pazienti sottoposti a trapianto allogenico di cellule staminali emopoietiche è fortemente influenzato da graft versus leukemia (GvL) e graft versus host disease (GvHD) che sono mediate, almeno in parte, dagli antigeni minori di istocompatibilità (mHAgs). In letteratura sono stati identificati 26 mHAgs che sono stati correlati a GvHD/GvL con risultati incompleti e in alcuni casi contrastanti; inoltre manca una metodica che sia in grado di genotipizzare contemporaneamente un pannello così ampio. Il lavoro è stato finalizzato alla preparazione di un protocollo di laboratorio che permetta di studiare in modo efficace i 26 mHAgs identificati, per poi correlarli con GvHD/GvL all’interno di uno specifico gruppo di trapiantati. Utilizzando la metodica IPlex Gold Mass Array Sequenom e tecniche di biologia molecolare convenzionale sono stati genotipizzati 26 antigeni minori di istocompatibilità per 46 coppie full-matched. Tutti i pazienti inclusi nel progetto di studio erano stati sottoposti a trapianto allogenico di cellule staminali emopoietiche da donatore familiare o volontario full-compatibile per leucemia mieloide cronica (n=46) o leucemia acuta linfoblastica Philadelphia positiva (LAL-Ph+, n=24). Il progetto ha confermato l'efficienza (98.6%) e la fattibilità delle metodiche proposte. Dal lavoro è inoltre emerso che, le differenze tra donatore e ricevente a libello mHAgs ACC-1, ACC-4, ACC-5, LB-MTHFD1-1Q, UGT2B17, DPH1, LRH1 potrebbero essere fattori predittivi di GvHD (p<0.05). La seconda evidenza è legata a un trend secondo cui il mismatch per LB-ADIR1 protegge dalla recidiva di malattia, in particolare nei confronti della LAL-Ph+ che è scarsamente responsiva all'allo-immunoterapia. Questo lavoro pilota, la cui casistica deve quindi essere ampliata, ha dimostrato l’efficacia della genotipizzazione con IPlex Gold Sequenom e l’elevato potenziale degli mHAgs sia come fattori predittivi di GvHD che come driver di GvL.
The outcome of allogeneic stem cell transplantation (Allo-SCT) is closely related to graft versus host disease (GvHD) and graft versus leukemia (GvL) effects which, in part, are mediated by mHAgs. Twenty-six mHAgs have been identified and reported to be differently and variably correlated with GVHD or GVL, but a simultaneous method to genotype a so large panel of mHAgs has never been employed. The aim of this work has been to develop a feasible method to genotype all the 26 mHAgs described so far and to test them for their correlation with GVHD and GVL in a group of donor/recipient pairs submitted to allo-SCT. For a multi-genotyping of 23 mHAgs we used iPlex Gold Mass Array technology (3 multiplex). For the other three mHAgs we designed other three assays based on conventional molecular biology. By these methods, we tested the 26 mHAgs in 46 donor/recipient pairs full-matched that underwent allo-SCT (sibling or MUD) because of Philadelphia positive CML (n=46) or ALL-Ph+ (n=24). Maldi-Tof IPlex Gold technology proved a high degree of efficiency (98.6%). As expected, sibling pairs showed most identity of MUD pairs. Notably, donor/recipient mismatch on ACC-1, ACC-4, ACC-5, LB-MTHFD1-1Q, UGT2B17, DPH1, LRH1 can drive GvHD effect (p<0.01). Next we identified that LB-ADIR1 can enhance (p=ns, but there is a trend) GvL effect specially on ALL-Ph+ that is otherwise un-responsible to allo-immunotherapy. Our data generated by a multi-genotype technique confirm the role of mHAgs in addressing GvL (in some cases without GvHD) and suggest that a study of mHAgs could be perfomed before transplant in order to better investigate the role of the known and new mHAgs involved in GvHD and GvL effects.

Background.Hematopoietic stem cell transplantation (HSCT) is now the therapeutic treatment for malignant and nonmalignant hematologic diseases and could represent a challenge to pediatric patients and their families. Advances in HSCT procedure have significantly increased the number of HSCT performed each year and have improved long term survival rates, so studies have shifted their focus from survival time to Quality of Life (QoL). Childhood HSCT survivors have been shown to be prone to develop psychological, cognitive, social and familiar, as well as medical, adverse outcomes. (Khera et al., 2012; Syrjala et al., 2012). The aim of the present study is to evaluate psychosocial and behavioral features and QoL of childhood HSCT survivors and their families (including siblings). Secondary aims are to evaluate the level of agreement between survivor-parents reports as for psychopathological areas and QoL; to evaluate parental stress and finally to identify risk and protective factors for mental health. Methods. Paediatric HSCT survivors (at least 5 years post transplantation) and their parent each completed a questionnaire package that included Achenbach test (Child Behaviour Checklist-CBCL; Youth Self Report-YSR), Pediatric Quality of Life Inventory 4.0 (PedsQL) and a brief unstructured text. Parents were also asked to complete an anamnestic interview and two other test: Short Form-36 (SF-36) and Parenting Stress Index scale (PSI). Siblings were asked to complete YSR and a brief unstructured text. Data were analysed using SPSS software. Results From 2012, 37 pediatric HSCT survivors (62% male) were enrolled in the study. The age at HSCT was 10 yrs on average (range 0,7-11,2 yrs). Fifty four percent of survivors were treated with allogenic unrelated HSCT. Seventy-four parent and 38 siblings were also tested. YSR scores exceeded the normative cut off for total competences (22%), social competences (13%) and somatic complains (8%), while CBCL scores were mainly pathological for total competences (49%), activities (22%) and somatic symptoms (11%). Symptoms reported to CBCL were more if: the survivor is female, there were a allogenic related HSCT, there were medical toxicities and if timing since communication of therapeutic choice and HSCT were longer. CBCL and YSR present a good level of agreement only for total competences scale (K=0.06, p= 0.002), somatic symptoms (K=0.21, p= 0.003) and attention problems (k=0.13; p=0.02). As for PedsQL, there was a poor level of agreement between survivors and parents only for physical area (K=0.05; p=0.31) and emotional area (K=0.09; p=0.06). In SF-36 mothers, perceive a worse QoL for “role limitation” scale when they have only one child or more than three children, if timing since communication and HSCT was brief and if survivor was treated with allogenic related HSCT. Fathers perceive a worse mental health if they have one child or more than two children, and if they were married. Siblings who were closer to survivor appear to present a worse QoL according to parents. Conclusion. Pediatric HSCT survivors present psychological distress in many areas (internalizing, externalizing, attention and social areas), with a prevalence of somatic symptoms.As for QoL, parents report better emotional function and lower physical function of the survivor. The first sibling appears to perceive a worse QoL. Mother and father perceive different kind of distress, but having two children appears to be homogeneously protective. Studies are needed to explore discordant perception and finally to improve outcomes and care for pediatric HSCT patients and their families.
Premesse. Il trapianto di cellule staminali ematopoietiche (HSCT) rappresenta ad oggi la terapia per alcune patologie ematologiche maligne e non maligne e costituisce una sfida per iI paziente in età pediatrica e per la sua famiglia. Il progresso medico relativo alla procedura di HSCT ha significativamente aumentato il numero di trapianti eseguiti ogni anno ed ha migliorato le percentuali di sopravvivenza a lungo termine, pertanto gli studi scientifici hanno spostato il loro focus dalla valutazione dei tassi di sopravvivenza all’esplorazione della Qualità di Vita (QoL). I sopravvissuti a HSCT avvenuto in età pediatrica sono stati dimostrati essere una popolazione più suscettibile allo sviluppo di sequele psicologiche, cognitive, sociali e familiari, oltre che mediche (Khera et al., 2012; Syrjala et al., 2012). L’obiettivo dello studio è la valutazione degli aspetti psicosociali e comportamentali oltre che della QoL dei soggetti pediatrici sopravvissuti ad HSCT e delle loro famiglie (includendo i fratelli). Obiettivi secondari sono: la valutazione del livello di accordo tra survivor e genitori relativamente agli aspetti psicopatologici ed alla QoL, la valutazione dello stress genitoriale e infine l’identificazione di fattori di rischio e fattori protettivi per la salute mentale di questi soggetti. Metodo. Sono stati testati soggetti pediatrici sopravvissuti ad HSCT (almeno 5 anni dopo il trapianto) e i loro genitori. Ad ognuno è stato richiesto di completare una batteria di test che includeva i questionari di Achenbach (Child BehaviourChecklist-CBCL; Youth Self Report-YSR) e il PediatricQuality of Life Inventory 4.0 (PedsQL) e di produrre un breve testo libero. Ai genitori è stata richiesto inoltre di completare una intervista anamnestica ed ulteriori due test: il Short Form-36 (SF-36) e il Parenting Stress Index (PSI). Ai fratelli del sopravvissuto è stato chiesto di completare la YSR e di produrre un breve testo libero. I dati sono stati elaborati con l’utilizzo del software SPSS. Risultati. Dal 2012 sono stati reclutati 37 soggetti sopravvissuti ad HSCT (62% maschi). L’età media al momento dell’HSCT era 10 anni (range 0,7-11,2 anni). Cinquantaquattro percento dei sopravvissuti era stato sottoposto a trapianto allogenico non familiare. Sono stati inoltre testati 74 genitori e 38 fratelli. I punteggi dello YSR superano i cut off per competenze totali (22%), competenze sociali (13%) e lamentele somatiche (8%), mentre i punteggi alle CBCL risultano patologici principalmente nelle aree di competenze totali (49%), attività (22%) e sintomi somatici (11%). I sintomi riportati alla CBCL appaiono più significativi se: il sopravvissuto è femmina, in caso di trapianto allogenico familiare, presenza di tossicità mediche e se il tempo intercorso tra la comunicazione della scelta terapeutica e l’HSCT era più lungo. CBCL e YSR presentano un buon grado di accordo solo relativamente alle scale competenze totali (K=0.06, p= 0.002), sintomi somatici (K=0.21, p= 0.003) e problemi di attenzione (k=0.13; p=0.02). Rispetto alla PedsQL genitori e survivor non appaiono in accordo per l’area fisica (K=0.05; p=0.31) ed emotiva (K=0.09; p=0.06). Nella SF-36 le madri percepiscono una peggiore QoL nella scala relativa alla “limitazione del ruolo” quando hanno un solo figlio o più di tre figli, se il tempo intercorso tra la comunicazione della scelta terapeutica e l’HSCT è breve e in caso di trapianto allogenico familiare. I padri percepiscono una salute mentale peggiore se hanno un unico figlio o più di due e se sono sposati. I fratelli più vicini al sopravvissuto per ordine di genitura sembrano presentare una peggiore QoL secondo i genitori. Conclusioni.I soggetti sopravvissuti a HSCT in età pediatrica presentano livelli di stress psicologico in diverse aree (internalizzante, esternalizzante, dell’attenzione e della socializzazione), con una prevalenza di sintomi somatici. Rispetto alla QoL, i genitori riportano un funzionamento emotivo del sopravvissuto migliore a fronte di un peggiore funzionamento fisico. Il primo fratello sembra percepire una QoL peggiore. Madri e padri percepiscono diversamente il proprio distress, ma il fatto di avere due figli appare rappresentare in modo omogeneo un fattore protettivo. Saranno necessari ulteriori studi per approfondire la discordanza tra le percezioni e infine migliorare l’outcome e la cura dei pazienti pediatrici sottoposti ad HSCT e delle loro famiglie.

Background. The term metabolic syndrome (MS) identifies a cluster of cardiovascular risk factors having insulin resistance as a key pathogenetic clue. A high prevalence of a MS-like pattern has been reported in cancer chemotherapy survivors, notably among patients affected by oncohematologic diseases. However, MS in cancer patients clinically differs from its “spontaneous” counterpart, bearing a resemblance to other MS-like pictures observed in solid transplant recipients and in HIV survivors, attributed to immunosuppressive and anti-retroviral drugs. These observations enabled to coin the term “secondary MS” characterized by possible pathogenetic differences with spontaneous MS. Disturbances in adipokine homeostasis have been sometimes claimed as possible alternative pathogenetic clues for “secondary MS”. Hematopoietic stem cell transplantation (HSCT) is a particularly interesting field of investigation, due to the delivery of high dose chemotherapy in autologous and of immunosuppression combined with chemotherapy in allogeneic HSCT. Over the last 15 years, an increased prevalence of MS has been recognized in HSCT recipients as a post-transplant long term effect. Nevertheless, most of the available study have a retrospective design and mainly refer to allogeneic HSCT, overlooking the contribution of high dose chemotherapy alone. Moreover, the inclusion criteria are widely different among the reports and do not consider possible pathogenetic clues. In a previous retrospective study, our group reported an increased prevalence of MS among HSCT long term survivors in continuous CR and off therapy, associated with an altered pattern of adipokine expression. In a subsequent study we proved that post-transplant MS differ from spontaneous MS by distribution of the single MS features and by adipokine profile. Aim of the study The above considerations enabled us to plan a prospective study aimed at evaluate in a series of HSCT recipients, at fixed intervals, the prevalence of MS features and the evolution of biochemical and biological parameters possibly involved in MS pathogenesis. Materials and methods After approval by the local Ethic Commission, patients referred either to autologous or allogeneic HSCT, were enrolled to the study over a two years period. For each patient, an anonymous record including anamnestic and clinical data, laboratory and adipokine profile was made. Data were collected at fixed interval: before transplant, after a month, after 100 days, after a year and subsequently every each year. According to the ATPIII criteria a diagnosis of MS was established and each of its components was defined. At each time, patients with MS were compared with those without. In order to better assess the risk of developing MS, the series was also split into three groups: patient with baseline MS, patients developing MS and patients never developing MS. Baseline values were also compared with those of 27 post-transplant MS patients, 27 healthy controls and 27 patients with spontaneous MS of a subsequent comparative study. The following variables were considered as possibly linked to the development of MS or each of its component: age, sex, baseline disease, type of transplant (autologous or allogeneic), blood glucose, insulin, insulin resistance index (HOMA IR = fasting insulin mcU/mL x fasting glucose mmol/L), HbA1c, triglycerides, total cholesterol, HDL cholesterol, LDL cholesterol according to Friedewald formula, uric acid, microalbuminuria, CRP, fibrinogen, TSH, FSH, LH, testosterone, 17-beta estradiol, familiar history for diabetes or cardiovascular diseases, leptin, alpha-TNF, resistin, adiponectin, hypogonadism (testosterone < 231 ng/100 mL in men, 17- -estradiol < 20 pg/mL in women), GVHD and smoking. Relationships between the selected variables and MS or its single features were assessed at each time point with a univariate analysis, through the Student t test for differences in median values and through the chi-square test for differences in incidences, and with a multivariate analysis through a Cox regression model. Results Ninety-nine patients undergoing HSCT (M/F 58/41, autologous 52, allogeneic 47, median age 49 years, range 20-69) were enrolled in the study and had completed a year of follow up. Relying on basal values, a diagnosis of MS could be established 24 patients, 11 referred to autologous and 13 to allogeneic HSCT. Eleven additional patients had double dyslipidemia, 6 referred to autologous and 5 to allogeneic HSCT. Patients with MS had significantly higher leptin (p<0.01) and fibrinogen (p<0.01) and lower adiponectin (p<0.01) levels in comparison with patients without MS. Patients with double dyslipidemia (DD) had significantly higher leptin (p<0.01) and fibrinogen (p<0.05) and lower adiponectin (p<0.01) levels in comparison with patients without DD. Patients referred for HSCT differed from a local reference population because of higher leptin (p<0,01) and lower adiponectin (p<0,05) levels; conversely, their leptin levels were comparable with those of patients with spontaneous MS and lower than those of post-transplant MS. Adiponectin levels were comparable with those of patients with post-transplant MS and significantly higher than those of patients with “spontaneous” MS ( p<0,05). After one month 43 HSCT patients, 13 autologous and 30 allogeneic HSCT patients, had MS. Eight additional patients had isolated double dyslipidemia, 5 after autologous and 3 after allogeneic HSCT. After 100 days, 9 patients had died, 6 of them having MS; a diagnosis of MS could be established in 34 patients. Isolated double dyslipidemia was observed in 9 patients, one autologous and 8 allogeneic HSCT recipients. After one year, 77 patients were still alive and in follow up. Of 16 deceased patients, 12 had MS. A diagnosis of MS could be established in 21 patients, whereas in three additional patients a previous diagnosis of MS could not be more confirmed. Isolated DD was observed in 8 patients, one autologous and 7 allogeneic HSCT recipients. At each interval, leptin levels were significantly reduced and adiponectin levels significantly increased in comparison to basal values. Nevertheless, patients with MS still retained a significantly altered adipokine pattern in comparison to those without. The excess in mortality of MS patients in comparison to those without, was statistically significant (p<0.04). At univariate analysis, basal adiponectin, resistin, leptin and fibrinogen levels were risk factors for developing MS. At subsequent times, leptin and resistin still resulted as risk factors for developing MS in the whole series and in autologous HSCT, but not in allogeneic HSCT; similar results were obtained for HOMA and insulin. At the different time intervals, leptin, resistin and HOMA proved to be risk factors more frequently related to the development of the single HSCT features. Moreover, patients with likely reduced previous chemotherapy exposition (multiple myeloma and autoimmune diseases) had a significantly lower risk of developing MS (p<0.02). At multivariate analysis, baseline HOMA (p<0.002), microalbuminuria (p<0.04), leptin (p<0.001) and type of transplant (p<0.002) were significant linked to the presence MS. At +30 days, HOMA (p<0.05), HBA1c (p<0.003) and type of transplant (p<0.03) were significantly linked to the presence of MS. Moreover, the presence of one of the MS features (waist circumference p<0.0001, hypertension <0.01, blood glucose p<0.03 and triglycerides p<0.0001) at baseline evaluation were predictor of developing +30 days MS. Conclusions The study bears no resemblance to the other available due to the prospective design, the inclusion of autologous HSCT patients and the evaluation of insulin resistance and adipokines. The major limitation is the relatively small size of the series. Baseline data show that patient referred to HSCT, are a population with a high prevalence of metabolic syndrome. This finding is in keeping with multiple papers dealing with MS among cancer chemotherapy survivors. Moreover, we confirm our and other previous findings that MS both after cancer chemotherapy and HSCT differ by clinical features because of higher prevalence of dyslipidemia and a lower rate of hypertension, so suggesting a somewhat different pathogenesis. Univariate and multivariate analysis underscore the major role of an altered adipokine profile, notably hyperleptinemia. As in the only comparable retrospective study, we found a burst of MS at +30 day after HSCT. The major determinants were insulin resistance and immunosuppression. The effect of previous chemotherapy cannot be underestimated, since patients with a presumably lower load of previous chemotherapy had a significantly lower risk of developing MS. As suggested by other investigations, double dyslipidemia patients built up a major reservoir of new MS cases. More generally, multivariate analysis reveals that patients with one baseline MS feature had an increased risk of subsequently developing MS. An apparent decrease in MS prevalence at +100 days and at one year, is significantly related to an higher short term mortality among patients developing post-transplant MS. Since both autologous and allogeneic HSCT recipients are involved, both TRM and early relapse are related to MS. The presented data help defining a model encompassing post chemotherapy and post-transplant MS. Cancer chemotherapy patients are a population characterized by an increased prevalence of MS with some particular clinical features pathogenetically related to adipokine profile disturbances. In the early post-transplant, a burst of MS is registered, mainly related to insulin resistance and immunosuppression and mainly deriving from patients with one baseline MS feature but not full blown MS. A medium term decrease in MS is related to an increase in mortality among patients developing post-transplant MS. A subsequent progressive increase in MS rate is attributable to an adipokine profile derangement among long term HSCT survivors, as suggested in our previous retrospective study.

L’impatto del microbiota intestinale(MI) sulla mortalità correlata al trapianto allogenico di cellule staminali emopoietiche(TCSE) è stato recentemente dimostrato. Questa osservazione corrobora l’idea di un ruolo significativo del MI nella ricostruzione immunologica successiva al TCSE e nella genesi della Graft-versus-Host-Disease acuta(GvHD). Abbiamo pertanto condotto il primo studio longitudinale sul ruolo del MI nella genesi di GvHD in pazienti pediatrici sottoposti a TCSE. Sono stati arruolati 10pazienti, di cui 5 con GvHD. Per ogni paziente sono stati raccolti campioni fecali seriati ogni 10-15 giorni fino a 100 giorni dopo il TCSE. Il profilo filogenetico del MI è stato caratterizzato mediante pyrosequencing 454 della regione ipervariabile V4 della subunità 16S dell’rRNA. Il profilo funzionale è stato valutato mediante l’analisi degli acidi-grassi-a-corta-catena utilizzando la gas cromatografia-spettroscopia di massa. Dopo il TCSE è stata osservata una profonda distruzione strutturale e funzionale del normale assetto mutualistico dell’ecosistema intestinale. La traiettoria di ricostruzione del MI dopo il TCSE è risultata essere significativamente differente tra i pazienti con e senza GvHD. In particolare, nei pazienti senza GvHD è stata evidenziata prima del TCSE una precisa signature del MI, caratterizzata da un’elevata concentrazione di Bacteroidetes e Parabacteoidetes(p<0.05, Fig. 1). Parallelamente nei pazienti senza GVHD è stato osservato un aumento significativo degli acidi-grassi-a-corta-catena e di propionato in particolare(p<0.05). Questa caratteristica signature si è proiettata dopo il TCSE, persistendo alla distruzione dell’ecosistema intestinale e dimostrando l’elevata adattabilità di questi germi. I nostri dati indicano che le dinamiche dell’ecosistema microbico intestinale possono essere un fattore in grado di influenzare l’insorgenza di GvHD. In particolare, la presenza di un profilo mutualistico pre-TCSE del MI, caratterizzato dalla presenza di germi produttori di acidi-grassi-a-corta-catena con riconosciute proprietà immunomodulatorie, sembra mitigare il rischio di sviluppare GVHD. Questi risultati aprono quindi nuove prospettive sulla possibilità di manipolare il MI pre-TCSE per modulare la ricostruzione del sistema immunitario.
The impact of the gut microbiota(MI) on allogeneic hematopoietic stem cell transplantation(HSCT) has been recently demonstrated. This observation supported the idea of a significant role of MI in the immunological reconstruction after HSCT and in the genesis of acute Graft-versus-Host-Disease(GvHD). We therefore conducted the first longitudinal study on the role of MI in the onset of GvHD in pediatric patients undergoing HSCT. 10 patients were enrolled, including 5 with GvHD. For each patient were collected seriated fecal samples every 10-15 days up to 100 days after HSCT. The phylogenetic profile of MI was characterized by pyrosequencing 454 of the hypervariable V4 region of the 16S rRNA subunit. The functional profile was evaluated by analysis of fatty acid-fat-to-short-chain using the gas chromatography-mass spectroscopy. After HSCT a profound structural and functional destruction of the normal position mutualistic gut ecosystem was observed. The reconstruction of the trajectory of the MI after HSCT was significantly different between patients with and without GVHD. In particular, in patients without GVHD, a precise of the MI signature before the HSCT was highlighted, characterized by a high concentration of Bacteroidetes and Parabacteoidetes(p <0.05, Fig. 1). In parallel in patients without GVHD it was observed a significant increase of the fatty-acid-to-short-chain and in particular propionate(p <0.05). This characteristic signature was projected after HSCT, persisting to the intestinal ecosystem destruction and demonstrating the high adaptability of these germs. Our data indicate that the intestinal microbial ecosystem dynamics can be a factor influencing the onset of GvHD. In particular, the presence of a mutualistic profile pre-HSCT of the MI, characterized by the presence of germs producers of acid-fat-to-short-chain recognized with immunomodulatory, seems to mitigate the risk of developing GVHD. These results therefore open new perspectives on the possibility of manipulating the MI pre-HSCT to modulate the reconstruction of the immune system.

L’impatto del microbiota intestinale(MI) sulla mortalità correlata al trapianto allogenico di cellule staminali emopoietiche(TCSE) è stato recentemente dimostrato. Questa osservazione corrobora l’idea di un ruolo significativo del MI nella ricostruzione immunologica successiva al TCSE e nella genesi della Graft-versus-Host-Disease acuta(GvHD). Abbiamo pertanto condotto il primo studio longitudinale sul ruolo del MI nella genesi di GvHD in pazienti pediatrici sottoposti a TCSE. Sono stati arruolati 10pazienti, di cui 5 con GvHD. Per ogni paziente sono stati raccolti campioni fecali seriati ogni 10-15 giorni fino a 100 giorni dopo il TCSE. Il profilo filogenetico del MI è stato caratterizzato mediante pyrosequencing 454 della regione ipervariabile V4 della subunità 16S dell’rRNA. Il profilo funzionale è stato valutato mediante l’analisi degli acidi-grassi-a-corta-catena utilizzando la gas cromatografia-spettroscopia di massa. Dopo il TCSE è stata osservata una profonda distruzione strutturale e funzionale del normale assetto mutualistico dell’ecosistema intestinale. La traiettoria di ricostruzione del MI dopo il TCSE è risultata essere significativamente differente tra i pazienti con e senza GvHD. In particolare, nei pazienti senza GvHD è stata evidenziata prima del TCSE una precisa signature del MI, caratterizzata da un’elevata concentrazione di Bacteroidetes e Parabacteoidetes(p<0.05, Fig. 1). Parallelamente nei pazienti senza GVHD è stato osservato un aumento significativo degli acidi-grassi-a-corta-catena e di propionato in particolare(p<0.05). Questa caratteristica signature si è proiettata dopo il TCSE, persistendo alla distruzione dell’ecosistema intestinale e dimostrando l’elevata adattabilità di questi germi. I nostri dati indicano che le dinamiche dell’ecosistema microbico intestinale possono essere un fattore in grado di influenzare l’insorgenza di GvHD. In particolare, la presenza di un profilo mutualistico pre-TCSE del MI, caratterizzato dalla presenza di germi produttori di acidi-grassi-a-corta-catena con riconosciute proprietà immunomodulatorie, sembra mitigare il rischio di sviluppare GVHD. Questi risultati aprono quindi nuove prospettive sulla possibilità di manipolare il MI pre-TCSE per modulare la ricostruzione del sistema immunitario.
The impact of the gut microbiota(MI) on allogeneic hematopoietic stem cell transplantation(HSCT) has been recently demonstrated. This observation supported the idea of a significant role of MI in the immunological reconstruction after HSCT and in the genesis of acute Graft-versus-Host-Disease(GvHD). We therefore conducted the first longitudinal study on the role of MI in the onset of GvHD in pediatric patients undergoing HSCT. 10 patients were enrolled, including 5 with GvHD. For each patient were collected seriated fecal samples every 10-15 days up to 100 days after HSCT. The phylogenetic profile of MI was characterized by pyrosequencing 454 of the hypervariable V4 region of the 16S rRNA subunit. The functional profile was evaluated by analysis of fatty acid-fat-to-short-chain using the gas chromatography-mass spectroscopy. After HSCT a profound structural and functional destruction of the normal position mutualistic gut ecosystem was observed. The reconstruction of the trajectory of the MI after HSCT was significantly different between patients with and without GVHD. In particular, in patients without GVHD, a precise of the MI signature before the HSCT was highlighted, characterized by a high concentration of Bacteroidetes and Parabacteoidetes(p <0.05, Fig. 1). In parallel in patients without GVHD it was observed a significant increase of the fatty-acid-to-short-chain and in particular propionate(p <0.05). This characteristic signature was projected after HSCT, persisting to the intestinal ecosystem destruction and demonstrating the high adaptability of these germs. Our data indicate that the intestinal microbial ecosystem dynamics can be a factor influencing the onset of GvHD. In particular, the presence of a mutualistic profile pre-HSCT of the MI, characterized by the presence of germs producers of acid-fat-to-short-chain recognized with immunomodulatory, seems to mitigate the risk of developing GVHD. These results therefore open new perspectives on the possibility of manipulating the MI pre-HSCT to modulate the reconstruction of the immune system.