Laboratory Of Cellular Therapy G Lanzani

Bergamo, Italy

Laboratory Of Cellular Therapy G Lanzani

Bergamo, Italy

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PubMed | Laboratory Of Cellular Therapy G Lanzani, Irccs Instituto Of Ricerche Farmacologiche Mario Negri and New York Medical College
Type: Journal Article | Journal: Stem cell reports | Year: 2015

The application of cell-based therapies in regenerative medicine is gaining recognition. Here, we show that human bone marrow stromal cells (BMSCs), also known as bone-marrow-derived mesenchymal cells, can be reprogrammed into renal proximal tubular-like epithelial cells using cell-free extracts. Streptolysin-O-permeabilized BMSCs exposed to HK2-cell extracts underwent morphological changes-formation of domes and tubule-like structures-and acquired epithelial functional properties such as transepithelial-resistance, albumin-binding, and uptake and specific markers E-cadherin and aquaporin-1. Transmission electron microscopy revealed the presence of brush border microvilli and tight intercellular contacts. RNA sequencing showed tubular epithelial transcript abundance and revealed the upregulation of components of the EGFR pathway. Reprogrammed BMSCs integrated into self-forming kidney tissue and formed tubular structures. Reprogrammed BMSCs infused in immunodeficient mice with cisplatin-induced acute kidney injury engrafted into proximal tubuli, reduced renal injury and improved function. Thus, reprogrammed BMSCs are a promising cell resource for future cell therapy.


Pievani A.,Laboratory Of Cellular Therapy G Lanzani | Belussi C.,Laboratory Of Cellular Therapy G Lanzani | Klein C.,Hoffmann-La Roche | Rambaldi A.,Laboratory Of Cellular Therapy G Lanzani | And 2 more authors.
Blood | Year: 2011

We have investigated combining adoptive immunotherapy with cytokine-induced killer (CIK) cells and anti-CD20 monoclonal antibodies (mAb) GA101 or rituximab to optimize B-cell non-Hodgkin lymphoma (B-NHL) therapy. CIK cultures alone demonstrated significant cytotoxic activity against B-NHL cell lines or freshly isolated samples in either an autologous or allogeneic combination. This natural cytotoxicity (NC) was mainly due to the predominating CD3+CD56 + CIK population (40%-75%) present in the cultures. The addition of anti-CD20 mAb GA101 or rituximab further increased cytotoxicity by 35% and 15%, respectively. This enhancement was mainly due to antibody-dependent cytotoxicity (ADCC) mediated by the 1%-10% NK cells contaminating CIK cultures. The addition of human serum (HS) inhibited NK-cell activation induced by rituximab, but not activation induced by GA101.Overall lysis in presence of serum, even of a resistant B-NHL cell line, was significantly increased by 100 μg/mL of rituximab, but even more so by GA101, with respect to CIK cultures alone. This was due to the combined action of complement-mediated cytotoxicity (CDC), ADCC, and CIK-mediated NC. These data suggest that rituximab, and even more so GA101, could be used in vivo to enhance CIK therapeutic activity in B-NHL.© 2011 by The American Society of Hematology.


Alzani R.,Nerviano Medical science Srl | Pedrini O.,Laboratory Of Cellular Therapy G Lanzani | Albanese C.,Nerviano Medical science Srl | Ceruti R.,Nerviano Medical science Srl | And 8 more authors.
Experimental Hematology | Year: 2010

Objective: The aim of the work was to determine and characterize, in vitro and in vivo, the therapeutic activity of PHA-793887, a new potent pan-cdk inhibitor, in the context of hematopoietic neoplasms. Materials and Methods: Thirteen leukemic cell lines bearing different cytogenetic abnormalities and normal hematopoietic cells were used in cytotoxicity and colony assays. The drug activity at the molecular level was analyzed by Western blotting. PHA-793887 was also tested in vivo in several leukemia xenograft models. Results: PHA-793887 was cytotoxic for leukemic cell lines in vitro, with IC50 ranging from 0.3 to 7 μM (mean: 2.9 μM), regardless of any specific chromosomal aberration. At these doses, the drug was not cytotoxic for normal unstimulated peripheral blood mononuclear cells or CD34+ hematopoietic stem cells. Interestingly, in colony assays PHA-793887 showed very high activity against leukemia cell lines, with an IC50 <0.1 μM (mean: 0.08 μM), indicating that it has efficient and prolonged antiproliferative activity. PHA-793887 induced cell-cycle arrest, inhibited Rb and nucleophosmin phosphorylation, and modulated cyclin E and cdc6 expression at low doses (0.2-1 μM) and induced apoptosis at the highest dose (5 μM). It was also effective in vivo in both subcutaneous xenograft and primary leukemic disseminated models that better mimic naturally occurring human disease. Interestingly, in one disseminated model derived from a relapsed Philadelphia-positive acute lymphoid leukemia patient, PHA-793887 showed strong therapeutic activity also when treatment was started after establishment of high disease burden. Conclusions: We conclude that PHA-793887 has promising therapeutic activity against acute leukemias in vitro and in vivo. © 2010 ISEH - Society for Hematology and Stem Cells.


Verdelli D.,University of Milan | Nobili L.,University of Milan | Todoerti K.,Laboratory of Pre Clinical and Translational Research | Mosca L.,University of Milan | And 11 more authors.
Genes Chromosomes and Cancer | Year: 2014

We explored the molecular mechanisms involved in the establishement of CMA-03/06, an IL-6-independent variant of the multiple myeloma cell line CMA-03 previously generated in our Institution. CMA-03/06 cells grow in the absence of IL-6 with a doubling time comparable with that of CMA-03 cells; neither the addition of IL6 (IL-6) to the culture medium nor co-culture with multipotent mesenchymal stromal cells increases the proliferation rate, although they maintain the responsiveness to IL-6 stimulation as demonstrated by STAT1, STAT3, and STAT5 induction. IL-6 independence of CMA-03/06 cells is not apparently due to the development of an autocrine IL-6 loop, nor to the observed moderate constitutive activation of STAT5 and STAT3, since STAT3 silencing does not affect cell viability or proliferation. When compared to the parental cell line, CMA-03/06 cells showed an activated pattern of the NF-κB pathway. This finding is supported by gene expression profiling (GEP) analysis identifying an appreciable fraction of modulated genes (28/308) in the CMA-03/06 subclone reported to be involved in this pathway. Furthermore, although more resistant to apoptotic stimuli compared to the parental cell line, CMA-03/06 cells display a higher sensibility to NF-κB inhibition induced by bortezomib. Finally, GEP analysis suggests an involvement of a number of cytokines, which might contribute to IL-6 independence of CMA-03/06 by stimulating growth and antiapoptotic processes. In conclusion, the parental cell-line CMA-03 and its variant CMA-03/06 represent a suitable model to further investigate molecular mechanisms involved in the IL-6-independent growth of myeloma cells. © 2013 Wiley Periodicals, Inc.


Pievani A.,Laboratory Of Cellular Therapy G Lanzani | Borleri G.,Laboratory Of Cellular Therapy G Lanzani | Pende D.,Instituto Nazionale Per la Ricerca Sul Cancro | Moretta L.,Instituto Giannina Gaslini | And 3 more authors.
Blood | Year: 2011

CD3 +CD56 + cytokine-induced killer (CIK) cells display a potent cytolytic activity. The adhesion molecule lymphocyte function-associated antigen-1 plays a crucial role in binding as well as in cytolytic activity of CIK cells against tumor target cells expressing the corresponding ligands. CIK cells express activating natural killer (NK) receptors, including NKG2D, DNAX accessory molecule-1 (DNAM-1), and low levels of NKp30. Cell signaling not only through TCR/CD3 but also through NKG2D, DNAM-1, and NKp30 leads to CIK cell activation resulting in granule exocytosis, cytokine secretion, and cytotoxicity. Antibody blocking experiments showed that DNAM-1, NKG2D, and NKp30 are involved in the TCR-independent tumor cell recognition and killing. Anti-CMV-specific CIK cells could be expanded in standard CIK cultures and mediate both specific, MHC-restricted recognition and TCR-independent NK-like cytolytic activity against leukemic cell lines or fresh leukemic blasts. Antibody blocking of lymphocyte function-associated antigen-1 and DNAM-1 led to significant reduction of both CTL and NK-cell functions, whereas blocking of NKG2D and NKp30 only inhibited NK-like cytotoxicity. Their dual-effector function suggests that CIK cells, when used in a clinical setting, may control both neoplastic relapses and viral infections, 2 frequently associated complications in patients who received a transplant. © 2011 by The American Society of Hematology.


Tomasoni S.,Mario Negri Institute for Pharmacological Research | Longaretti L.,Mario Negri Institute for Pharmacological Research | Rota C.,Mario Negri Institute for Pharmacological Research | Morigi M.,Mario Negri Institute for Pharmacological Research | And 6 more authors.
Stem Cells and Development | Year: 2013

Bone marrow-mesenchymal stem cells (BM-MSC) ameliorate renal dysfunction and repair tubular damage of acute kidney injury by locally releasing growth factors, including the insulin-like growth factor-1 (IGF-1). The restricted homing of BM-MSC at the site of injury led us to investigate a possible gene-based communication mechanism between BM-MSC and tubular cells. Human BM-MSC (hBM-MSC) released microparticles and exosomes (Exo) enriched in mRNAs. A selected pattern of transcripts was detected in Exo versus parental cells. Exo expressed the IGF-1 receptor (IGF-1R), but not IGF-1 mRNA, while hBM-MSC contained both mRNAs. R- cells lacking IGF-1R exposed to hBM-MSC-derived Exo acquired the human IGF-1R transcript that was translated in the corresponding protein. Transfer of IGF-1R mRNA from Exo to cisplatin-damaged proximal tubular cells (proximal tubular epithelial cell [PTEC]) increased PTEC proliferation. Coincubation of damaged PTEC with Exo and soluble IGF-1 further enhanced cell proliferation. These findings suggest that horizontal transfer of the mRNA for IGF-1R to tubular cells through Exo potentiates tubular cell sensitivity to locally produced IGF-1 providing a new mechanism underlying the powerful renoprotection of few BM-MSC observed in vivo. © 2013, Mary Ann Liebert, Inc.


Golay J.,Laboratory Of Cellular Therapy G Lanzani | Introna M.,Laboratory Of Cellular Therapy G Lanzani
Archives of Biochemistry and Biophysics | Year: 2012

Therapeutic monoclonal antibodies (mAbs) are mostly used in cancer, as anti-infectious agents and as immunomodulatory drugs, and are amongst the most active area of research and development in the pharmaceutical industry. This class of drugs comprises unconjugated antibodies or antibody fragments, antibody-drug conjugates, radio-immunoconjugates and bispecific/trispecific molecules. A better understanding of the mechanism of action of successful mAbs is fundamental for the selection of more active and less toxic mAbs of new generation. Furthermore reliable screening of new compounds at an early stage of preclinical development, for both efficacy and toxicity, should allow the selection of the best molecules at an early stage, and improve the rate of success of this class of drugs. Here we review the major methods that are employed for testing the activity of therapeutic mAbs in vitro and in vivo in small animal models and point out to some of the pitfalls in these assays. © 2011 Elsevier Inc. All rights reserved.


PubMed | Laboratory Of Cellular Therapy G Lanzani
Type: Journal Article | Journal: Archives of biochemistry and biophysics | Year: 2012

Therapeutic monoclonal antibodies (mAbs) are mostly used in cancer, as anti-infectious agents and as immunomodulatory drugs, and are amongst the most active area of research and development in the pharmaceutical industry. This class of drugs comprises unconjugated antibodies or antibody fragments, antibody-drug conjugates, radio-immunoconjugates and bispecific/trispecific molecules. A better understanding of the mechanism of action of successful mAbs is fundamental for the selection of more active and less toxic mAbs of new generation. Furthermore reliable screening of new compounds at an early stage of preclinical development, for both efficacy and toxicity, should allow the selection of the best molecules at an early stage, and improve the rate of success of this class of drugs. Here we review the major methods that are employed for testing the activity of therapeutic mAbs in vitro and in vivo in small animal models and point out to some of the pitfalls in these assays.


PubMed | Laboratory Of Cellular Therapy G Lanzani
Type: Comparative Study | Journal: Journal of immunology (Baltimore, Md. : 1950) | Year: 2011

We analyzed in B-chronic lymphocytic leukemia (B-CLL) whole blood assays the activity of therapeutic mAbs alemtuzumab, rituximab, and type II glycoengineered anti-CD20 mAb GA101. Whole blood samples were treated with Abs, and death of CD19(+) B-CLL was measured by flow cytometry. Alemtuzumab efficiently lysed B-CLL targets with maximal lysis at 1-4 h (62%). In contrast, rituximab induced a more limited cell death (21%) that was maximal only at 24 h. GA101 killed B-CLL targets to a similar extent but more rapidly than rituximab, with 19.2 and 23.5% cell death at 4 and 24 h, respectively, compared with 7.9 and 21.4% for rituximab. Lysis by both rituximab and GA101 correlated directly with CD20 expression levels (r(2) = 0.88 and 0.85, respectively). Interestingly, lysis by all three Abs at high concentrations was mostly complement dependent, because it was blocked by the anti-C5 Ab eculizumab by 90% in the case of alemtuzumab and rituximab and by 64% in the case of GA101. Although GA101 caused homotypic adhesion, it induced only limited (3%) direct cell death of purified B-CLL cells. Both rituximab and GA101 showed the same efficiency in phagocytosis assays, but phagocytosis was not significant in whole blood due to excess Igs. Finally, GA101 at 1-100 g/ml induced 2- to 3-fold more efficient NK cell degranulation than rituximab in isolated B-CLL or normal PBMCs. GA101, but not rituximab, also mediated significant NK cell degranulation in whole blood samples. Thus, complement and Ab-dependent cellular cytotoxicity are believed to be the major effector mechanisms of GA101 in whole blood assays.


PubMed | Laboratory Of Cellular Therapy G Lanzani
Type: Journal Article | Journal: Blood | Year: 2011

CD3(+)CD56(+) cytokine-induced killer (CIK) cells display a potent cytolytic activity. The adhesion molecule lymphocyte function-associated antigen-1 plays a crucial role in binding as well as in cytolytic activity of CIK cells against tumor target cells expressing the corresponding ligands. CIK cells express activating natural killer (NK) receptors, including NKG2D, DNAX accessory molecule-1 (DNAM-1), and low levels of NKp30. Cell signaling not only through TCR/CD3 but also through NKG2D, DNAM-1, and NKp30 leads to CIK cell activation resulting in granule exocytosis, cytokine secretion, and cytotoxicity. Antibody blocking experiments showed that DNAM-1, NKG2D, and NKp30 are involved in the TCR-independent tumor cell recognition and killing. Anti-CMV-specific CIK cells could be expanded in standard CIK cultures and mediate both specific, MHC-restricted recognition and TCR-independent NK-like cytolytic activity against leukemic cell lines or fresh leukemic blasts. Antibody blocking of lymphocyte function-associated antigen-1 and DNAM-1 led to significant reduction of both CTL and NK-cell functions, whereas blocking of NKG2D and NKp30 only inhibited NK-like cytotoxicity. Their dual-effector function suggests that CIK cells, when used in a clinical setting, may control both neoplastic relapses and viral infections, 2 frequently associated complications in patients who received a transplant.

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