Immunotherapy for neuroblastoma using syngeneic fibroblasts transfected with IL-2 and IL-12

Cytokine-modified tumour cells have been used in clinical trials for immunotherapy of neuroblastoma, but primary tumour cells from surgical biopsies are difficult to culture. Autologous fibroblasts, however, are straightforward to manipulate in culture and easy to transfect using nonviral or viral vectors. Here we have compared the antitumour effect of fibroblasts and tumour cells transfected ex vivo to coexpress interleukin-2 (IL-2) and IL-12 in a syngeneic mouse model of neuroblastoma. Coinjection of cytokine-modified fibroblasts with Neuro-2A tumour cells abolished their in vivo tumorigenicity. Treatment of established tumours with three intratumoral doses of transfected fibroblasts showed a significant therapeutic effect with reduced growth or complete eradication of tumours in 90% of mice, associated with extensive leukocyte infiltration. Splenocytes recovered from vaccinated mice showed enhanced IL-2 production following Neuro-2A coculture, and increased cytotoxicity against Neuro-2A targets compared with controls. Furthermore, 100% of the tumour-free mice exhibited immune memory against tumour cells when rechallenged three months later. The potency of transfected fibroblasts was equivalent to that of tumour cells in all experiments. We conclude that syngeneic fibroblasts cotransfected with IL-2 and IL-12 mediate therapeutic effects against established disease, and are capable of generating immunological memory. Furthermore, as they are easier to recover and manipulate than autologous tumour cells, fibroblasts provide an attractive alternative immunotherapeutic strategy for the treatment of neuroblastoma

γ-Catenin is overexpressed in acute myeloid leukemia and promotes the stabilization and nuclear localization of β-catenin

Canonical Wnt signaling regulates the transcription of T-cell factor (TCF)-responsive genes through the stabilization and nuclear translocation of the transcriptional co-activator, β-catenin. Overexpression of β-catenin features prominently in acute myeloid leukemia (AML) and has previously been associated with poor clinical outcome. Overexpression of γ-catenin mRNA (a close homologue of β-catenin) has also been reported in AML and has been linked to the pathogenesis of this disease, however, the relative roles of these catenins in leukemia remains unclear. Here we report that overexpression and aberrant nuclear localization of γ-catenin is frequent in AML. Significantly, γ-catenin expression was associated with β-catenin stabilization and nuclear localization. Consistent with this, we found that ectopic γ-catenin expression promoted the stabilization and nuclear translocation of β-catenin in leukemia cells. β-Catenin knockdown demonstrated that both γ- and β-catenin contribute to TCF-dependent transcription in leukemia cells. These data indicate that γ-catenin expression is a significant factor in the stabilization of β-catenin in AML. We also show that although normal cells exclude nuclear translocation of both γ- and β-catenin, this level of regulation is lost in the majority of AML patients and cell lines, which allow nuclear accumulation of these catenins and inappropriate TCF-dependent transcription

Peripheral T-lymphocytes express WNT7A and its restoration in leukemia-derived lymphoblasts inhibits cell proliferation

<p>Abstract</p> <p>Background</p> <p>WNT7a, a member of the Wnt ligand family implicated in several developmental processes, has also been reported to be dysregulated in some types of tumors; however, its function and implication in oncogenesis is poorly understood. Moreover, the expression of this gene and the role that it plays in the biology of blood cells remains unclear. In addition to determining the expression of the <it>WNT7A </it>gene in blood cells, in leukemia-derived cell lines, and in samples of patients with leukemia, the aim of this study was to seek the effect of this gene in proliferation.</p> <p>Methods</p> <p>We analyzed peripheral blood mononuclear cells, sorted CD3 and CD19 cells, four leukemia-derived cell lines, and blood samples from 14 patients with Acute lymphoblastic leukemia (ALL), and 19 clinically healthy subjects. Reverse transcription followed by quantitative Real-time Polymerase chain reaction (qRT-PCR) analysis were performed to determine relative <it>WNT7A </it>expression. Restoration of WNT7a was done employing a lentiviral system and by using a recombinant human protein. Cell proliferation was measured by addition of WST-1 to cell cultures.</p> <p>Results</p> <p>WNT7a is mainly produced by CD3 T-lymphocytes, its expression decreases upon activation, and it is severely reduced in leukemia-derived cell lines, as well as in the blood samples of patients with ALL when compared with healthy controls (<it>p </it>≤0.001). By restoring <it>WNT7A </it>expression in leukemia-derived cells, we were able to demonstrate that WNT7a inhibits cell growth. A similar effect was observed when a recombinant human WNT7a protein was used. Interestingly, restoration of <it>WNT7A </it>expression in Jurkat cells did not activate the canonical Wnt/β-catenin pathway.</p> <p>Conclusions</p> <p>To our knowledge, this is the first report evidencing quantitatively decreased <it>WNT7A </it>levels in leukemia-derived cells and that <it>WNT7A </it>restoration in T-lymphocytes inhibits cell proliferation. In addition, our results also support the possible function of <it>WNT7A </it>as a tumor suppressor gene as well as a therapeutic tool.</p

Heparan sulfate inhibits hematopoietic stem and progenitor cell migration and engraftment in mucopolysaccharidosis I.

Mucopolysaccharidosis I Hurler (MPSI-H) is a pediatric lysosomal storage disease caused by genetic deficiencies in IDUA, coding for ?-l-iduronidase. Idua?/? mice share similar clinical pathology with patients, including the accumulation of the undegraded glycosaminoglycans (GAGs) heparan sulfate (HS), and dermatan sulfate (DS), progressive neurodegeneration, and dysostosis multiplex. Hematopoietic stem cell transplantation (HSCT) is the most effective treatment for Hurler patients, but reduced intensity conditioning is a risk factor in transplantation, suggesting an underlying defect in hematopoietic cell engraftment. HS is a co-receptor in the CXCL12/CXCR4 axis of hematopoietic stem and progenitor cell (HSPC) migration to the bone marrow (BM), but the effect of HS alterations on HSPC migration, or the functional role of HS in MPSI-H are unknown. We demonstrate defective WT HSPC engraftment and migration in Idua?/? recipient BM, particularly under reduced intensity conditioning. Both intra- but especially extracellular Idua?/? BM HS was significantly increased and abnormally sulfated. Soluble heparinase-sensitive GAGs from Idua?/? BM and specifically 2-O-sulfated HS, elevated in Idua?/? BM, both inhibited CXCL12-mediated WT HSPC transwell migration, while DS had no effect. Thus we have shown that excess overly sulfated extracellular HS binds, and sequesters CXCL12, limiting hematopoietic migration and providing a potential mechanism for the limited scope of HSCT in Hurler disease

Myeloid/Microglial driven autologous hematopoietic stem cell gene therapy corrects a neuronopathic lysosomal disease

Mucopolysaccharidosis type IIIA (MPSIIIA) is a lysosomal storage disorder caused by mutations in N-sulfoglucosamine sulfohydrolase (SGSH), resulting in heparan sulfate (HS) accumulation and progressive neurodegeneration. There are no treatments. We previously demonstrated improved neuropathology in MPSIIIA mice using lentiviral vectors (LVs) overexpressing SGSH in wild-type (WT) hematopoietic stem cell (HSC) transplants (HSCTs), achieved via donor monocyte/microglial engraftment in the brain. However, neurological disease was not corrected using LVs in autologous MPSIIIA HSCTs. To improve brain expression via monocyte/microglial specificity, LVs expressing enhanced green fluorescent protein (eGFP) under ubiquitous phosphoglycerate kinase (PGK) or myeloid-specific promoters were compared in transplanted HSCs. LV-CD11b-GFP gave significantly higher monocyte/B-cell eGFP expression than LV-PGK-GFP or LV-CD18-GFP after 6 months. Subsequently, autologous MPSIIIA HSCs were transduced with either LV-PGK-coSGSH or LV-CD11b-coSGSH vectors expressing codon-optimized SGSH and transplanted into MPSIIIA mice. Eight months after HSCT, LV-PGK-coSGSH vectors produced bone marrow SGSH (576% normal activity) similar to LV-CD11b-coSGSH (473%), but LV-CD11b-coSGSH had significantly higher brain expression (11 versus 7%), demonstrating improved brain specificity. LV-CD11b-coSGSH normalized MPSIIIA behavior, brain HS, GM2 ganglioside, and neuroinflammation to WT levels, whereas LV-PGK-coSGSH partly corrected neuropathology but not behavior. We demonstrate compelling evidence of neurological disease correction using autologous myeloid driven lentiviral-HSC gene therapy in MPSIIIA mice. © The American Society of Gene & Cell Therapy

Neuroblastoma immunotherapy using a novel vector system

Available from British Library Document Supply Centre-DSC:DXN052972 / BLDSC - British Library Document Supply CentreSIGLEGBUnited Kingdo

Liver Regeneration by Hematopoietic Stem Cells: Have We Reached the End of the Road?

The liver is the organ with the highest regenerative capacity in the human body. However, various insults, including viral infections, alcohol or drug abuse, and metabolic overload, may cause chronic inflammation and fibrosis, leading to irreversible liver dysfunction. Despite advances in surgery and pharmacological treatments, liver diseases remain a leading cause of death worldwide. To address the shortage of donor liver organs for orthotopic liver transplantation, cell therapy in liver disease has emerged as a promising regenerative treatment. Sources include primary hepatocytes or functional hepatocytes generated from the reprogramming of induced pluripotent stem cells (iPSC). Different types of stem cells have also been employed for transplantation to trigger regeneration, including hematopoietic stem cells (HSCs), mesenchymal stromal cells (MSCs), endothelial progenitor cells (EPCs) as well as adult and fetal liver progenitor cells. HSCs, usually defined by the expression of CD34 and CD133, and MSCs, defined by the expression of CD105, CD73, and CD90, are attractive sources due to their autologous nature, ease of isolation and cryopreservation. The present review focuses on the use of bone marrow HSCs for liver regeneration, presenting evidence for an ongoing crosstalk between the hematopoietic and the hepatic system. This relationship commences during embryogenesis when the fetal liver emerges as the crossroads between the two systems converging the presence of different origins of cells (mesoderm and endoderm) in the same organ. Ample evidence indicates that the fetal liver supports the maturation and expansion of HSCs during development but also later on in life. Moreover, the fact that the adult liver remains one of the few sites for extramedullary hematopoiesis&mdash;albeit pathological&mdash;suggests that this relationship between the two systems is ongoing. Can, however, the hematopoietic system offer similar support to the liver? The majority of clinical studies using hematopoietic cell transplantation in patients with liver disease report favourable observations. The underlying mechanism&mdash;whether paracrine, fusion or transdifferentiation or a combination of the three&mdash;remains to be confirmed

Academic social and emotional functioning of school - aged children diagnosed with attention deficit / hyperactivity disorder (AD/HD) and depression

In recent years Attention Deficit/ Hyperactivity Disorder (ADHD) and depression have attracted a lot of scientific interest. Modern psychological research has proved that these disorders, each one separately, have a lot of serious implications on the psychological state of the child and therefore on his/her social and school performance. As a result, this research aimed firstly to study the prevalence of depressive symptoms in school-age children (n=61), who had been referred to public mental health centers and filled the criteria of DSM-IV for ADHD. Secondly, this research investigated the sex differences in relation to a) the symptoms of depression, b) the form of emotional and behavioral problems that the children with ADHD showed as well as the degree to which these problems were recognized by both their mothers and teachers and c) the efficiency that children with ADHD had in their academic functioning. Finally, the effect of the symptoms of depression on a) the school performance and adjustment, b) the social functioning and c) the emotional functioning of the children with ADHD was studied. The results showed that a percentage of 34, 43 % of the sample were rated as people with high levels of depressive symptoms. This research proves that the parents are quite informed on their children’s symptoms of depression and do not hesitate to mention them. According to the assessment of the three sources of information- children, parents, educators- sex is not a serious factor to affect the children with ADHD to demonstrate symptoms of depression. While, according to the estimation of the parents and educators, there is no difference between boys and girls with ADHD regarding the internalizing problems, the above carriers of evaluation tend to converge that the boys with ADHD demonstrate a higher degree of delinquent behavior rather than the girls with ADHD. As far as the academic functioning is concerned, the research showed that, according to the educators’ opinion, the girls with ADHD show significantly more gaps only in arithmetic skills. Finally, regarding the social functioning, the fact that there are depression symptoms has a strong impact on the children’s with ADHD sociability and academic functioning, according to the statement of the children themselves. Subjects that have to do with the learning difficulties, which usually accompany children with ADHD, are also studied, comparing the information obtained by both mental health professionals and educators. The findings of this research are discussed in relation to the international bibliography. Finally, some suggestions are made aiming at helping mental health professionals and educators to enable them to early diagnose and get through the difficulties that children with ADHD face.Η σύγχρονη επιστημονική έρευνα έχει δείξει, ότι η Διαταραχή Ελλειμματικής Προσοχής-Υπερκινητικότητας (ΔΕΠ-Υ) και η κατάθλιψη, η κάθε μία χωριστά, έχουν σοβαρές επιπτώσεις στη συναισθηματική κατάσταση και συνακόλουθα στην κοινωνική και σχολική λειτουργικότητα του παιδιού. Η παρούσα έρευνα είχε ως πρώτο στόχο τη διερεύνηση της καταθλιπτικής συμπτωματολογίας σε κλινικό δείγμα παιδιών σχολικής ηλικίας 9 - 11 ετών, τα οποία είχαν παραπεμφθεί σε δημόσιες παιδοψυχιατρικές υπηρεσίες και πληρούσαν τα κριτήρια για τη διάγνωση της ΔΕΠ-Υ, όπως αυτή ορίζεται στο DSM-IV. Διερευνήθηκαν οι διαφορές φύλου α) ως προς την καταθλιπτική συμπτωματολογία, β) ως προς τον τύπο των προβλημάτων συναισθήματος και συμπεριφοράς που παρουσίασαν τα παιδιά με ΔΕΠ-Υ, καθώς και το βαθμό στον οποίον αυτά τα προβλήματα έγιναν αντιληπτά από τις μητέρες και τους εκπαιδευτικούς τους και γ) ως προς την επάρκεια των παιδιών με ΔΕΠ-Υ στον τομέα της σχολικής λειτουργικότητας. Τέλος, εξετάστηκε η επίδραση της ύπαρξης καταθλιπτικής συμπτωματολογίας στη σχολική, κοινωνική και συναισθηματική λειτουργικότητα των παιδιών με ΔΕΠ-Υ. Τα αποτελέσματα έδειξαν ότι ένα σημαντικό ποσοστό (34,43%) των παιδιών με ΔΕΠ-Υ παρουσιάζει παράλληλα συμπτώματα κατάθλιψης. Στην παρούσα μελέτη φαίνεται ότι οι γονείς είναι αρκετά ενήμεροι για τα συμπτώματα κατάθλιψης των παιδιών τους και δεν διστάζουν να τα αναφέρουν. Με βάση τις εκτιμήσεις των τριών πηγών πληροφόρησης - παιδιών, γονέων και εκπαιδευτικών - ο παράγοντας φύλο δεν επιδρά σημαντικά στην εκδήλωση καταθλιπτικής συμπτωματολογίας. Ενώ, σύμφωνα με τις εκτιμήσεις των γονέων και των εκπαιδευτικών, δεν υπάρχει, ως προς την ύπαρξη προβλημάτων εσωτερίκευσης, διαφοροποίηση μεταξύ αγοριών και κοριτσιών με ΔΕΠ-Υ, οι γονείς και οι εκπαιδευτικοί συμφωνούν στο ότι τα αγόρια με ΔΕΠ-Υ εκδηλώνουν σε μεγαλύτερο βαθμό παραπτωματική συμπεριφορά από ό,τι τα κορίτσια. Ως προς τη σχολική λειτουργικότητα, οι εκπαιδευτικοί θεωρούν ότι τα κορίτσια με ΔΕΠ-Υ παρουσιάζουν σε σημαντικό βαθμό περισσότερα ελλείμματα από τα αγόρια μόνο στον τομέα των αριθμητικών ικανοτήτων. Τέλος, όσον αφορά στον τομέα της σχολικής και κοινωνικής λειτουργικότητας, η ύπαρξη καταθλιπτικής συμπτωματολογίας έχει σημαντική επίπτωση στην κοινωνικότητα καθώς και στη σχολική επίδοση και προσαρμογή των παιδιών με ΔΕΠ-Υ, όπως αυτές ομολογήθηκαν από τα ίδια τα παιδιά. Τα ευρήματα της παρούσας έρευνας συζητούνται με βάση τα ευρήματα από τη διεθνή βιβλιογραφία. Τέλος, διατυπώνονται προτάσεις για τον τρόπο που οι επαγγελματίες ψυχικής υγείας και οι εκπαιδευτικοί μπορούν να βοηθήσουν στην έγκαιρη αναγνώριση και αντιμετώπιση των δυσκολιών που αντιμετωπίζουν τα παιδιά με ΔΕΠ-Υ