Cytokine-induced killer cells eradicate bone and soft-tissue sarcomas.

Unresectable metastatic bone sarcoma and soft-tissue sarcomas (STS) are incurable due to the inability to eradicate chemoresistant cancer stem-like cells (sCSC) that are likely responsible for relapses and drug resistance. In this study, we investigated the preclinical activity of patient-derived cytokine-induced killer (CIK) cells against autologous bone sarcoma and STS, including against putative sCSCs. Tumor killing was evaluated both in vitro and within an immunodeficient mouse model of autologous sarcoma.

Ex vivo allogeneic stimulation significantly improves expansion of cytokine-induced killer cells without increasing their alloreactivity across HLA barriers.

Cytokine-induced killer cells (CIKs) are ex vivo expanded T-NK lymphocytes capable of HLA-unrestricted antitumor activity. CIKs are promising candidates for adoptive cancer immunotherapies; they can be generated and infused in autologous settings of cancer patients, or from donors, after allogeneic hematopoietic cell transplant. Ex vivo expansion rates of CIKs are greatly variable among patients, with consequent potential clinical limitations for "poor expanders.

FGF-2 expands murine hematopoietic stem and progenitor cells via proliferation of stromal cells, c-Kit activation, and CXCL12 down-regulation.

Cytokine-induced expansion of hematopoietic stem and progenitor cells (HSPCs) is not fully understood. In the present study, we show that whereas steady-state hematopoiesis is normal in basic fibroblast growth factor (FGF-2)-knockout mice, parathyroid hormone stimulation and myeloablative treatments failed to induce normal HSPC proliferation and recovery. In vivo FGF-2 treatment expanded stromal cells, including perivascular Nestin(+) supportive stromal cells, which may facilitate HSPC expansion by increasing SCF and reducing CXCL12 via mir-31 up-regulation.

Cytokine-induced killer (CIK) cells as feasible and effective adoptive immunotherapy for the treatment of solid tumors.

Introduction: Cytokine-induced killer (CIK) cells are heterogeneous ex vivo-expanded T lymphocytes with mixed T-NK phenotype and endowed with a wide MHC-unrestricted antitumor activity. CIK cells can be expanded from peripheral blood mononuclear cells (PBMC) cultured with the timed addition of IFN-γ, Ab anti-CD3 and IL2. A consistent subset of mature CIK cells presents a CD3(+)CD56(+) phenotype.

Purinergic signaling inhibits human acute myeloblastic leukemia cell proliferation, migration, and engraftment in immunodeficient mice.

Extracellular ATP and UTP nucleotides increase the proliferation and engraftment potential of normal human hematopoietic stem cells via the engagement of purinergic receptors (P2Rs). In the present study, we show that ATP and UTP have strikingly opposite effects on human acute myeloblastic leukemia (AML) cells. Leukemic cells express P2Rs.

Transient proteasome inhibition as a strategy to enhance lentiviral transduction of hematopoietic CD34(+) cells and T lymphocytes: implications for the use of low viral doses and large-size vectors.

The proteasome system restricts lentiviral transduction of stem cells. We exploited proteasome inhibition as a strategy to enhance transduction of both hematopoietic stem cells (HSC) and T lymphocytes with low dose or large-size lentiviral vectors (LV). HSC showed higher transduction efficiency if transiently exposed to proteasome inhibitor MG132 (41.

Gene-modified T lymphocytes in the setting of hematopoietic cell transplantation: potential benefits and possible risks.

Introduction: Allogeneic hematopoietic cell transplantation (HCT) is a consolidated treatment for several hematologic malignancies. Donor T lymphocytes can mediate a graft versus tumor (GVT) effect and control opportunistic infections but can also cause severe graft versus host disease (GVHD). Gene-transfer strategies are appealing tools to modulate T cell functions when infused after HCT.

Purinergic stimulation of human mesenchymal stem cells potentiates their chemotactic response to CXCL12 and increases the homing capacity and production of proinflammatory cytokines.

Objective: Extracellular adenosine triphosphate (ATP) is a well-recognized mediator of cell-to-cell communication. Here we show ATP effects on bone marrow (BM)-derived human mesenchymal stem cell (hMSCs) functions.

Materials And Methods: ATP-induced modification of hMSCs gene expression profile was assessed by Affymetrix technology.

Inhibition of erythropoiesis in malaria anemia: role of hemozoin and hemozoin-generated 4-hydroxynonenal.

Severe malaria anemia is characterized by inhibited/altered erythropoiesis and presence of hemozoin-(HZ)-laden bone-marrow macrophages. HZ mediates peroxidation of unsaturated fatty acids and production of bioactive aldehydes such as 4-hydroxynonenal (HNE). HZ-laden human monocytes inhibited growth of cocultivated human erythroid cells and produced HNE that diffused to adjacent cells generating HNE-protein adducts.

Enhanced c-Met activity promotes G-CSF-induced mobilization of hematopoietic progenitor cells via ROS signaling.

Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor-induced mobilization c-Met expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell-derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and c-Met activation.

Efficient transcriptional targeting of human hematopoietic stem cells and blood cell lineages by lentiviral vectors containing the regulatory element of the Wiskott-Aldrich syndrome gene.

The ability to effectively transduce human hematopoietic stem cells (HSCs) and to ensure adequate but "physiological" levels of transgene expression in different hematopoietic lineages represents some primary features of a gene-transfer vector. The ability to carry, integrate, and efficiently sustain transgene expression in HSCs strongly depends on the vector. We have constructed lentiviral vectors (LV) containing fragments of different lengths of the hematopoietic-specific regulatory element of the Wiskott-Aldrich syndrome (WAS) gene-spanning approximately 1,600 and 170 bp-that direct enhanced green fluorescent protein (EGFP) expression.

Blood-forming stem cells are nervous: direct and indirect regulation of immature human CD34+ cells by the nervous system.

The nervous system regulates immunity through hormonal and neuronal routes as part of host defense and repair mechanism. Here, we review the emerging evidence for regulation of human hematopoietic stem and progenitor cells (HSPC) by the nervous system both directly and indirectly via their bone marrow (BM) niche-supporting stromal cells. Functional expression of several neurotransmitter receptors was demonstrated on HSPC, mainly on the more primitive CD34(+)/CD38(-/low) fraction.

Granulocyte-colony stimulating factor upregulates ErbB2 expression on breast cancer cell lines and converts primary resistance to trastuzumab.

The recombinant monoclonal antibody trastuzumab has antiproliferative effect on breast cancer (BC) cells with ErbB2 overexpression. We postulated that a mechanism able to modify ErbB2 expression enhances the antitumor effect of trastuzumab. We analyzed whether granulocyte-colony stimulating factor (G-CSF), widely used in adjuvant cancer therapy to alleviate chemotherapy-induced myelotoxicity, could influence ErbB2 expression in BC cells and patients.

Tumor angiogenesis and progression are enhanced by Sema4D produced by tumor-associated macrophages.

Increased evidence suggests that cancer-associated inflammation supports tumor growth and progression. We have previously shown that semaphorin 4D (Sema4D), a ligand produced by different cell types, is a proangiogenic molecule that acts by binding to its receptor, plexin B1, expressed on endothelial cells (Conrotto, P., D.

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.

Refreezing of cord blood hematopoietic stem cells for allogenic transplantation: in vitro and in vivo validation of a clinical phase I/II protocol in European and Italian Good Manufacturing Practice conditions.

Objective: Several requirements need to be fulfilled for clinical use of expanded hematopoietic stem cells (HSCs). Because most cord blood (CB) samples are frozen in single bags and only an aliquot ( approximately 25%) of the blood can be expanded, the thawing and refreezing of samples must be validated in the current European and Italian Good Manufacturing Practice (eIGMP) conditions. Here, we describe in vitro and in vivo validation of the phase I/II protocol for CD34+ expansion of thawed CB units according to the current Cell Therapy Products (CTPs) Guidelines.

Initiating and cancer-propagating cells in TEL-AML1-associated childhood leukemia.

Understanding cancer pathogenesis requires knowledge of not only the specific contributory genetic mutations but also the cellular framework in which they arise and function. Here we explore the clonal evolution of a form of childhood precursor-B cell acute lymphoblastic leukemia that is characterized by a chromosomal translocation generating a TEL-AML1 fusion gene. We identify a cell compartment in leukemic children that can propagate leukemia when transplanted in mice.

Multilineage engraftment of refrozen cord blood hematopoietic progenitors in NOD/SCID mice.

Seven cord blood (CB) units were tested for their capacity to repopulate irradiated NOD/SCID mice after one or two successive cryopreservation procedures. In primary transplants with frozen or refrozen CB cells we observed equivalent human colonies and percentages of human CD45+ cells, with multilineage engraftment. In secondary transplants flow cytometry and polymerase chain reaction for the a satellite region of chromosome 17 showed equivalent levels of human engraftment.

Serial transplantations in nonobese diabetic/severe combined immunodeficiency mice of transduced human CD34+ cord blood cells: efficient oncoretroviral gene transfer and ex vivo expansion under serum-free conditions.

Stable oncoretroviral gene transfer into hematopoietic stem cells (HSCs) provides permanent genetic disease correction. It is crucial to transplant enough transduced HSCs to compete with and replace the defective host hemopoiesis. To increase the number of transduced cells, the role of ex vivo expansion was investigated.

Proliferative senescence in hematopoietic stem cells during ex-vivo expansion.

The good outcome of hematopoietic stem cell (HSC) transplantation is hampered by low doses of CD34+ cell infusion. Transplanted HSCs undergo a replicative stress that causes accelerated senescence due to rapid telomere shortening. The expansion of human cord blood HSCs is instrumental in obtaining a large number of "good quality" cells, in terms of telomere length and telomerase activity compared to adult HSCs.

Feasibility of cord blood stem cell manipulation with high-energy shock waves: an in vitro and in vivo study.

Objective: Cord blood CD34+ cells are more uncommitted than their adult counterparts as they can be more easily maintained and expanded in vitro and transduced with lentiviral vectors. The aim of this study was to evaluate whether pretreatment with high-energy shock waves (HESW) could further enhance the expansion of cord blood progenitors and the transduction efficiency with lentiviral vectors.

Methods: Human cord blood CD34+ cells underwent HESW treatment with a wide range of energy and number of shots (from 0.

Cycling G1 CD34+/CD38+ cells potentiate the motility and engraftment of quiescent G0 CD34+/CD38-/low severe combined immunodeficiency repopulating cells.

The mechanism of human stem cell expansion ex vivo is not fully understood. Furthermore, little is known about the mechanisms of human stem cell homing/repopulation and the role that differentiating progenitor cells may play in these processes. We report that 2- to 3-day in vitro cytokine stimulation of human cord blood CD34(+)-enriched cells induces the production of short-term repopulating, cycling G1 CD34(+)/CD38(+) cells with increased matrix metalloproteinase (MMP)-9 secretion as well as increased migration capacity to the chemokine stromal cell-derived factor-1 (SDF-1) and homing to the bone marrow of irradiated nonobese diabetic severe/combined immunodeficiency (NOD/SCID) mice.

Involvement of chemokine receptor 4/stromal cell-derived factor 1 system during osteosarcoma tumor progression.

Despite intensive chemotherapy and surgery treatment, lung and bone metastasis develop in about 30% of patients with osteosarcoma. Mechanisms for this preferential metastatic behavior are largely unknown. We investigated the role of the chemokine receptor 4 (CXCR4)/stromal cell-derived factor 1 (SDF-1) system to drive the homing of osteosarcoma cells.