Activated autologous T cells exert an anti-B-cell chronic lymphatic leukemia effect in vitro and in vivo.

BACKGROUND AIMS: The impact of chronic lymphatic leukemia (CLL) tumor burden on the autologous immune system has already been demonstrated. This study attempted to elucidate the molecular mechanisms underlying T-cell immunologic deficiencies in CLL. METHODS: Freshly isolated CD3(+) T cells from patients with a diagnosis of CLL and healthy donors were analyzed by gene expression profiling. Activated T cells from 20 patients with CLL were tested in vitro for cytotoxicity against mutated and unmutated autologous B cells and DAUDI, K562 and P815 cell lines. To investigate T-cell mediated cytotoxicity in vivo, we co-transplanted OKT3-activated T lymphocytes and autologous B-cell CLL (B-CLL) cells into NOD/SCID mice. RESULTS: Gene expression profiles of peripheral blood T cells from B-CLL patients showed 25 down-regulated, and 31 up-regulated, genes that were mainly involved in cell differentiation, proliferation, survival, apoptosis, cytoskeleton formation, vesicle trafficking and T-cell activation. After culture, the T-cell count remained unchanged, CD8 cells expanded more than CD4 and a cytotoxicity index >30% was present in 5/20 patients. Cytotoxicity against B autologous leukemic cells did not correlate with B-cell mutational status. Only activated T cells exerting cytotoxicity against autologous leukemic B cells prevented CLL in a human-mouse chimera. CONCLUSIONS: This study indicates that patients with CLL are affected by a partial immunologic defect that might be somewhat susceptible to repair. This study identifies the molecular pathways underlying T-cell deficiencies in CLL and shows that cytotoxic T-cell functions against autologous B-CLL can be rebuilt at least in part in vitro and in vivo.

Constitutively activated Notch signaling is involved in survival and apoptosis resistance of B-CLL cells.

Notch signaling is involved in tumorigenesis, but its role in B-chronic lymphocytic leukemia (B-CLL) pathogenesis is not completely defined. This study examined the expression and activation of Notch receptors in B-CLL cells and the role of Notch signaling in sustaining the survival of these cells. Our results show that B-CLL cells but not normal B cells constitutively express Notch1 and Notch2 proteins as well as their ligands Jagged1 and Jagged2. Notch signaling is constitutively activated in B-CLL cells, and its activation is further increased in B-CLL cells, which resist spontaneous apoptosis after 24-hour ex vivo culture. Notch stimulation by a soluble Jagged1 ligand increases B-CLL cell survival and is accompanied by increased nuclear factor-kappa B (NF-kappaB) activity and cellular inhibitor of apoptosis protein 2 (c-IAP2) and X-linked inhibitor of apoptosis protein (XIAP) expression. In contrast, Notch-signaling inhibition by the gamma-secretase inhibitor I (GSI; z-Leu-Leu-Nle-CHO) and the specific Notch2 down-regulation by small-interfering RNA accelerate spontaneous B-CLL cell apoptosis. Apoptotic activity of GSI is accompanied by reduction of NF-kappaB activity and c-IAP2 and XIAP expression. Overall, our findings show that Notch signaling plays a critical role in B-CLL cell survival and apoptosis resistance and suggest that it could be a novel potential therapeutic target.

Toxic epidermal necrolysis in a patient with primary myelofibrosis receiving thalidomide therapy.

Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm characterized by progressive anemia, massive splenomegaly, leukoerythroblastosis, extramedullary hematopoiesis and in about 50% of cases the presence of JAK2V617F mutation. Curative therapy in PMF is currently possible only with allogeneic haematopoietic stem cell transplantation which is, unfortunately, associated with relatively high risks of mortality and morbidity which undermine its broad applications. Non-transplant treatment modalities are used for palliative purposes. Recently, anti-angiogenic drugs such as thalidomide have been used to treat these patients on the basis of the prominent bone marrow angiogenesis. Here, we report the case of a patient suffering from JAK2V617F-positive PMF with marked bone marrow neo-angiogenesis. The patient was treated with thalidomide but after 20 days developed life-threatening toxic epidermal necrolysis (TEN). To the best of our knowledge this is the first case of TEN in a patient with PMF under thalidomide therapy.

Comparison between adenoviral and retroviral vectors for the transduction of the thymidine kinase PET reporter gene in rat mesenchymal stem cells.

UNLABELLED: Mesenchymal stem cells (MSCs) are a promising cell line for the treatment of ischemic heart disease. To evaluate the success of their transplantation into living animals, noninvasive imaging techniques that are able to track the distribution and fate of those cells would be useful. The aim of this study was to investigate the feasibility of infecting rat MSCs with adenoviruses and retroviruses carrying the herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene; to compare the level of transgene expression induced by the 2 viral vectors; to evaluate the effects of viral transduction on cell phenotype, viability, proliferation rates, and differentiation capabilities; and to test the possibility of noninvasively imaging transduced MSCs using 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine (18F-FHBG) and small-animal PET after their transplantation into living rats. METHODS: We infected rat bone marrow MSCs with adenoviruses carrying the HSV1 mutant tk (Ad-HSV1-sr39tk) PET reporter gene (PRG) or with a retroviral construct expressing the wild-type HSV1-tk PRG. The efficacy and intensity of HSV1-sr39tk and HSV1-tk gene expression were determined by a direct comparison of [8-3H]-penciclovir ([8-3H]-PCV) cell uptake in both infected MSC populations and noninfected control MSCs. Small-animal PET studies were performed on living rats after an intramuscular injection of infected MSCs. The MSCs either have been incubated in advance with 18F-FHBG or they were administered and 18F-FHBG was thereafter intravenously administered [corrected] RESULTS: Both adenoviral and retroviral vectors can be used to introduce the tk PRG in MSCs. Neither adenovirus nor retrovirus infections significantly modify MSC phenotype, viability, proliferation, and differentiation capabilities. No significant 3H-PCV uptake was observed in noninfected MSCs. By contrast, after both adenoviral and retroviral infections, the infected MSC populations exhibited a similar, significantly higher, 3H-PCV accumulation. Small-animal PET images showed intense activity within the transplanted regions irrespective of the infected MSC population used. CONCLUSION: Our results demonstrate the feasibility of infecting MSCs with adenoviruses and retroviruses expressing the HSV1-tk PRG and suggest that infected MSCs can be noninvasively imaged with 18F-FHBG and small-animal PET after their transplantation into living animals.

Transformation by retroviral vectors of bone marrow-derived mesenchymal cells induces mitochondria-dependent cAMP-sensitive reactive oxygen species production.

Retroviral vectors are used in human gene therapy trials to stably introduce therapeutic genes in the genome of patients' cells. Their applicability, however, is frustrated by the limited viability of transformed cells and/or by risks linked to selection of oncogene-mutated clones. The reasons for these drawbacks are not yet completely understood. In this study, we show that LXSN-NeoR gene/interleukin-7-engineered mesenchymal stromal cells exhibited a marked enhancement of reactive oxygen species production compared with untransfected cells. This effect resulted to be independent on the product of the gene carried by the retroviral vehicle as it was reproducible in cells transfected with the empty vector alone. Stable transfection of mesenchymal stromal cells with the different retroviral vectors pBabe-puro and PINCO-puro and the lentiviral vector pSico PGK-puro caused similar redox imbalance, unveiling a phenomenon of more general impact. The enhanced production of reactive oxygen species over the basal level was attributable to mitochondrial dysfunction and brought back to altered activity of the NADH-CoQ oxidoreductase (complex I) of the respiratory chain. The oxidative stress in transfected mesenchymal stem cells was completely reversed by treatment with a cAMP analog, thus pointing to alteration in the protein kinase A-dependent signaling pathway of the host cell. Transfection of mesenchymal stromal cells with a PINCO-parental vector harboring the green fluorescent protein gene as selection marker in place of the puromycin-resistance gene resulted in no alteration of the redox phenotype. These novel findings provide insights and caveats to the applicability of cell- or gene-based therapies and indicate possible intervention to improve them. Disclosure of potential conflicts of interest is found at the end of this article.

Evidence of jak2 val617phe positive essential thrombocythemia with splanchnic thrombosis during estroprogestinic treatment.

The discovery of the Janus kinase 2 Val617Phe mutation has brought new insights into the development of myeloproliferative disorders; however, the pathogenesis of essential thrombocythemia and its related thrombotic complications has not been completely understood. Although the Janus kinase 2 Val617Phe mutation confirms the initially suspected clonal character of the disease, factors influencing clonal transformation and expansion in the bone marrow have not been fully detected. Furthermore, patients affected by essential thrombocythemia who are carriers of the Janus kinase 2 Val617Phe mutation show a higher incidence of venous thromboembolism both before, and at the time of diagnosis, compared with noncarriers, and recent evidence of splanchnic and cerebral vein thrombosis in carriers of the Janus kinase 2 Val617Phe mutation has been reported. The intake of oral contraceptives is a strong and independent risk factor for venous thromboembolism. In addition, in-vitro tests showed both an altered primary haemostatic plug formation and enhanced platelet aggregation in patients taking such drugs. Little is known, though, about the influence of steroid hormones on both megakaryopoiesis and platelet function in patients with the Janus kinase 2 Val617Phe mutation. Herewith, we report the case of a 30-year-old woman who took a third generation oral contraceptive for 5 months and developed an essential thrombocythemia with spleno-portal axis and superior mesenteric vein thrombosis. She was found to carry the kinase gene Janus kinase 2 mutation.

Eubacterium plautii infection in a kidney transplant recipient: a noteworthy case of pleural effusion and fever.

We report a noteworthy case of Eubacterium plautii infection after kidney transplantation. Our 33-yr-old transplant recipient received standard care; his post-transplant course was uneventful. However, on day 44 he underwent an emergency laparotomy for perforation of the ileum. He was initially treated with ceftazidime, fluconazole and metronidazole, but his fever persisted, so he was switched to meropenem and vancocin. We could not find any cause for his infection. On day 70, his temperature normalized. On day 75, he developed severe leukopenia (280 cell/mL). His cytomegalovirus-DNA test result was negative, so all immunosuppressants, except for prednisone, were stopped; instead, antibiotic prophylaxis was started, using caspofungin, trimethoprim-sulfamethoxazole and ciprofloxacin. On day 83, he underwent percutaneous drainage of massive left pleural effusion. We repeatedly cultured the pleural liquid, but it was not till three wk later that we were finally able to identify the causative organism. We hypothesize that the microorganism - which normally resides on the surface of the intestinal lumen - entered the bloodstream via bacterial translocation, eventually colonizing the pleurae. This translocation was favored by our patient poor clinical condition, his immunosuppressive treatment and his heavy antibiotherapy. Our experience highlights the need for wiser use of antibiotics in transplant recipients.

Sustained long-term engraftment and transgene expression of peripheral blood CD34+ cells transduced with third-generation lentiviral vectors.

As mobilized peripheral blood (MPB) represents an attractive cell source for gene therapy, we investigated the ability of third-generation lentiviral vectors (LVs) to transfer the enhanced green fluorescent protein gene into MPB CD34(+) cells in culture conditions allowing expansion of transplantable human hematopoietic stem cells. To date, few studies have reported transduction of MPB cells with vesicular stomatitis virus G pseudotyped LVs. The critical issue remains whether primitive, hematopoietic repopulating cells have, indeed, been transduced. In vitro (5 weeks' culture in FLT3 ligand + thrombopoietin + stem cell factor + interleukin 6) and in vivo (serial transplantation in NOD/SCID mice) experiments show that MPB CD34(+) cells can be effectively long-term transduced by LV and maintain their proliferation, self-renewal, and multilineage differentiation potentials. We show that expansion following transduction improves the engraftment of transduced MPB CD34(+) (4.6-fold expansion of SCID repopulating cells by limiting dilution studies). We propose ex vivo expansion after transduction as an effective tool to improve gene therapy protocols with MPB. Disclosure of potential conflicts of interest is found at the end of this article.

Endothelial differentiation of hematopoietic stem and progenitor cells from patients with multiple myeloma.

PURPOSE: Vasculogenesis is a physiologic process typical of fetal development in which new blood vessels develop from undifferentiated precursors (or angioblasts). In tumors, near angiogenesis, vasculogenesis contributes to the formation of the microvascular plexus that is important for diffusion. Here, we show that hematopoietic stem and progenitor cells (HSPC) of multiple myeloma (MM) patients are able to differentiate into cells with endothelial phenotype on exposure to angiogenic cytokines. EXPERIMENTAL DESIGN: Circulating HSPCs were purified with an anti-CD133 antibody from patients with newly diagnosed MM before autologous transplantation and exposed to vascular endothelial growth factor (VEGF), fibroblast growth factor-2 and insulin-like growth factor in a 3-week culture. RESULTS: HSPCs gradually lost CD133 expression and acquired VEGF receptor-2, factor VIII-related antigen, and vascular endothelial-cadherin expression. The expression pattern overlapped with paired MM endothelial cells (MMEC). During culture, cells adhered to fibronectin, spread, and acquired an endothelial cell shape. Differentiated HSPCs also became capillarogenic in the Matrigel assay with maximal activity at the third week of culture. Bone marrow biopsies revealed HSPCs inside the neovessel wall in patients with MM but not in those with monoclonal gammopathy of undetermined significance. CONCLUSIONS: In patients with MM, but not in those with monoclonal gammopathy of undetermined significance, HSPCs contribute to the neovessel wall building together with MMECs. Therefore, besides angiogenesis, HSPC-linked vasculogenesis contributes to neovascularization in MM patients. Tentatively, we hypothesize that in HSPC cultures a multipotent cell population expressing low VEGF receptor-2 levels corresponds to the endothelial progenitor cell precursor and seems to be the MMEC precursor.

Mesenchymal cells recruit and regulate T regulatory cells.

OBJECTIVE: Despite much investigation into T regulatory cells (Tregs), little is known about the mechanism controlling their recruitment and function. Because multipotent mesenchymal stromal cells (MSCs) exert an immune regulatory function and suppress T-cell proliferation, this in vitro study investigated their role in Treg recruitment and function. MATERIALS AND METHODS: Human MSCs and different T cell populations (CD3(+), CD3(+)/CD45RA(+), CD3(+)/CD45RO(+), CD4(+)/CD25(+), CD4(+)/CD25(+)/CD45RO(+), CD4(+)/CD25(+)/CD45RA(+)) from healthy donors were cocultured for up to 15 days. Harvested lymphocytes were analyzed by flow cytometry and FoxP3 and CD127 expressions were measured by real-time polymerase chain reaction. Their regulatory activity was assessed. RESULTS: We demonstrate MSC recruit Tregs from a fraction of CD3(+) and from immunoselected CD3(+)/CD45RA(+) and CD3(+)/CD45RO(+) fractions. After culture with MSCs both immunoselected fractions registered increases in the CD4(+)/CD25(bright)/FoxP3 subset and CD127 expression was downregulated. When purified Treg populations (CD4/CD25(+), CD4/CD25(+)/CD45RA(+), and CD4/CD25(+)/CD45RO(+)) are used in MSC cocultures, they maintain FoxP3 expression and CD127 expression is downregulated. Treg suppressive capacity was maintained in Treg populations that were layered on MSC for up to 15 days while control Tregs lost all suppressive activity after 5 days culture. CONCLUSIONS: In conclusion, our study demonstrates that MSCs recruit, regulate, and maintain T-regulatory phenotype and function over time.

Large-scale generation of human allodepleted anti-3rd party lymphocytes.

Although adoptive transfer of donor lymphocytes protects from infections and relapse after allogeneic hematopoietic stem cell transplantation in both mice and in men, it is associated with a high risk of graft versus host disease (GvHD) which rises with HLA mismatching and the number of T lymphocytes that are infused. Elimination/reduction of alloreactive donor T lymphocytes is an appealing approach and several strategies have been proposed. Here we describe generation of anti-3rd party T lymphocytes under conditions of IL-2 deprivation and their effects in a pre-clinical murine model. Our results clearly indicated that anti-3rd party T lymphocytes generated on a large scale by means of IL-2 deprivation maintain a broad T cell repertoire, do not proliferate in a mixed lymphocyte reaction and do not cause GvHD in NOD-SCID mice. These anti-3rd party lymphocytes contain a large adaptive T regulatory cell subset which might contribute to in vitro and in vivo immune modulation.

Sustained ventricular tachycardia in a thalidomide-treated patient with primary plasma-cell leukemia.

BACKGROUND: A 68-year-old man diagnosed with primary plasma-cell leukemia was given thalidomide maintenance treatment for his disease. He had previously failed induction therapy with cyclophosphamide, vincristine, adriamycin, and dexamethasone, but achieved complete remission after melphalan therapy. Multiple syncopal episodes started to occur during thalidomide treatment, and a Holter electrocardiogram showed multiple abnormalities, with an episode of sustained ventricular tachycardia. INVESTIGATIONS: Blood tests, peripheral blood smear, bone-marrow biopsy and aspirate, Holter electrocardiogram. DIAGNOSIS: Sustained ventricular tachycardia possibly owing to thalidomide treatment. MANAGEMENT: Thalidomide withdrawal, dexamethasone maintenance therapy, monthly oral courses of combined melphalan and prednisone, salvage therapy with bortezomib.

The hypoxia-inducible factor is stabilized in circulating hematopoietic stem cells under normoxic conditions.

The hypoxia-inducible factor (HIF) transcriptional system enables cell adaptation to limited O(2) availability, transducing this signal into patho-physiological responses such as angiogenesis, erythropoiesis, vasomotor control, and altered energy metabolism, as well as cell survival decisions. However, other factors beyond hypoxia are known to activate this pleiotropic transcription factor. The aim of this study was to characterize HIF in human hematopoietic stem cells (HSCs) and evidence is provided that granulocyte colony stimulating factor-mobilized CD34+- and CD133+-HSCs express a stabilized cytoplasmic form of HIF-1alpha under normoxic conditions. It is shown that HIF-1alpha stabilization correlates with down-regulation of the tumour suppressor von Hippel-Lindau protein (pVHL) and is positively controlled by NADPH-oxidase-dependent production of reactive oxygen species, indicating a specific O(2)-independent post-transcriptional control of HIF in mobilized HSCs. This novel finding is discussed in the context of the proposed role of HIF as a mediator of progenitor cell recruitment to injured ischemic tissues and/or in the control of the maintenance of the undifferentiated state.

Loss of bone mineral density and secondary hyperparathyroidism are complications of autologous stem cell transplantation.

Patients who underwent autologous stem cell transplantation (ASCT) are prone to decreased bone mineral density (BMD). We measured BMD in 180 patients who underwent ASCT for hematologic malignancies. Patients were evaluated with a median of 6.2 years after ASCT. Twenty patients who received only chemotherapy were evaluated as controls. The loss of bone mass was greater during the first year after ASCT, since majority of patients recover BMD and normalize bone turnover markers during the following years. After ASCT, over half of the patients show osteopenia or osteoporosis independent of the sex. According to the results of other groups, our results emphasize the potential usefulness of antiresorptive agents to prevent or treat post-ASCT osteopenia or osteoporosis, and the importance of the measurement of BMD as an integral component to the follow-up of ASCT.

The proteasome inhibitor bortezomib affects osteoblast differentiation in vitro and in vivo in multiple myeloma patients.

The proteasome inhibitor bortezomib may increase osteoblast-related markers in multiple myeloma (MM) patients; however, its potential osteoblastic stimulatory effect is not known. In this study, we show that bortezomib significantly induced a stimulatory effect on osteoblast markers in human mesenchymal cells without affecting the number of osteoblast progenitors in bone marrow cultures or the viability of mature osteoblasts. Consistently we found that bortezomib significantly increased the transcription factor Runx2/Cbfa1 activity in human osteoblast progenitors and osteoblasts without affecting nuclear and cytoplasmatic active beta-catenin levels. Consequently a stimulatory effect of bortezomib on bone nodule formation was also demonstrated in osteoblast progenitors. These in vitro observations were confirmed in vivo by the finding of a significant increase in the number of osteoblastic cells x mm(2) of bone tissue and in the number of Runx2/Cbfa1-positive osteoblastic cells that was observed in MM patients who responded to bortezomib. Our in vitro and in vivo observations support the hypothesis that a direct stimulatory effect on bone formation process could occur during bortezomib treatment.

Bone-marrow derived hematopoietic stem/progenitor cells express multiple isoforms of NADPH oxidase and produce constitutively reactive oxygen species.

Consolidated evidence highlights the importance of redox signalling in poising the balance between self-renewal and differentiation in adult stem cells. The present study shows that human hematopoietic stem/progenitor cells (HSCs) constitutively generate low levels of hydrogen peroxide whose production is inhibited by DPI, apocynin, catalase, and LY294002 and scarcely stimulated by PMA. Moreover, it is shown that HSCs express at the mRNA and protein levels the catalytic subunits of NOX1, NOX2, and NOX4 isoforms of the NADPH oxidase family along with the complete battery of the regulatory subunits p22, p40, p47, p67, rac1, rac2, NOXO1, and NOXA1 as well as the splicing variant NOX2s and that the three NOX isoforms are largely co-expressed in the same HSC. These findings are interpreted in terms of a positive feed-back mechanism of NOXs activation enabling a fine tuning of the ROS level to be possibly used in redox-mediated signalling for growth and differentiation of HSCs.

Role of reactive oxygen species as signal molecules in the pre-commitment phase of adult stem cells.

This mini-review summarizes evidence, provided by our group, relevant to the understanding of how redox signalling may control the fate of adult hematopoietic stem/progenitor cells (HSPCs). In particular it is shown that bone marrow-derived human HSPC are endowed with a composite panel of constitutively active NADPH-oxidases (NOXs) comprising the cell membrane-localized catalytic subunits of the NOX1, NOX2 and NOX4 isoforms. It is proposed that the coordinated activity of the NOX isoforms in HSPCs function as environmental oxygen sensor and generate low level of ROS, which likely serve as second messengers. The pro-oxidant setting, entering into play when HSPCs leave the hypoxic bone marrow niche, would enable them to be more responsive to proliferative/differentiative stimuli. Moreover it is suggested that enhanced ROS elicit mitochondrial "differentiation" in a pre-commitment phase needed to match the bioenergetic request in the oncoming proliferation/differentiation process.