Augusto Pessina

Prevalidation of a model for predicting acute neutropenia by colony forming unit granulocyte/macrophage (CFU-GM) assay

This report describes an international prevalidation study conducted to optimise the Standard Operating Procedure (SOP) for detecting myelosuppressive agents by CFU-GM assay and to study a model for predicting (by means of this in vitro hematopoietic assay) the acute xenobiotic exposure levels that cause maximum tolerated decreases in absolute neutrophil counts (ANC). In the first phase of the study (Protocol Refinement), two SOPs were assessed, by using two cell culture media (Test A, containing GM-CSF; and Test B, containing G-CSF, GM-CSF, IL-3, IL-6 and SCF), and the two tests were applied to cells from both human (bone marrow and umbilical cord blood) and mouse (bone marrow) CFU-GM. In the second phase (Protocol Transfer), the SOPs were transferred to four laboratories to verify the linearity of the assay response and its interlaboratory reproducibility. After a further phase (Protocol Performance), dedicated to a training set of six anticancer drugs (adriamycin, flavopindol, morpholino-doxorubicin, pyrazoloacridine, taxol and topotecan), a model for predicting neutropenia was verified. Results showed that the assay is linear under SOP conditions, and that the in vitro endpoints used by the clinical prediction model of neutropenia are highly reproducible within and between laboratories. Valid tests represented 95% of all tests attempted. The 90% inhibitory concentration values (IC(90)) from Test A and Test B accurately predicted the human maximum tolerated dose (MTD) for five of six and for four of six myelosuppressive anticancer drugs, respectively, that were selected as prototype xenobiotics. As expected, both tests failed to accurately predict the human MTD of a drug that is a likely protoxicant. It is concluded that Test A offers significant cost advantages compared to Test B, without any loss of performance or predictive accuracy. On the basis of these results, we proposed a formal Phase II validation study using the Test A SOP for 16-18 additional xenobiotics that represent the spectrum of haematotoxic potential.

Mesenchymal Stem/Stromal Cells: A New "Cells as Drugs" Paradigm. Efficacy and Critical Aspects in Cell Therapy

Mesenchymal stem cells (MSCs) were first isolated more than 50 years ago from the bone marrow. Currently MSCs may also be isolated from several alternative sources and they have been used in more than a hundred clinical trials worldwide to treat a wide variety of diseases. The MSCs mechanism of action is undefined and currently under investigation. For in vivo purposes MSCs must be produced in compliance with good manufacturing practices and this has stimulated research on MSCs characterization and safety. The objective of this review is to describe recent developments regarding MSCs properties, physiological effects, delivery, clinical applications and possible side effects.

Mesenchymal Stromal Cells Primed with Paclitaxel Provide a New Approach for Cancer Therapy

Background Mesenchymal stromal cells may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that exposure of mouse bone marrow derived stromal cells to Doxorubicin led them to acquire anti-proliferative potential towards co-cultured haematopoietic stem cells (HSCs). We thus hypothesized whether freshly isolated human bone marrow Mesenchymal stem cells (hMSCs) and mature murine stromal cells (SR4987 line) primed in vitro with anti-cancer drugs and then localized near cancer cells, could inhibit proliferation. Methods and Principal Findings Paclitaxel (PTX) was used to prime culture of hMSCs and SR4987. Incorporation of PTX into hMSCs was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by PTX primed hMSCs (hMSCsPTX) was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of hMSCsPTX and PTX primed SR4987(SR4987PTX), while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting hMSCsPTX and SR4987PTX with cancer cells in mice. Nevertheless, despite a loss of cells due to chemo-induced apoptosis, both hMSCs and SR4987 were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. PTX primed cells acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, hMSCsPTX and SR4987PTX co-injected with human cancer cells (DU145 and U87MG) and mouse melanoma cells (B16) in immunodeficient and in syngenic mice significantly delayed tumor takes and reduced tumor growth. Conclusions These data demonstrate, for the first time, that without any genetic manipulation, mesenchymal stromal cells can uptake and subsequently slowly release PTX. This may lead to potential new tools to increase efficacy of cancer therapy.

The key role of adult stem cells: therapeutic perspectives

ABSTRACT Background: The origin, function and physiology of totipotent embryonic cells are configured to construct organs and create cross-talk between cells for the biological and neurophysiologic development of organisms. Adult stem cells are involved in regenerating tissues for renewal and damage repair. Findings: Adult stem cells have been isolated from adult tissue, umbilical cord blood and other non-embryonic sources, and can transform into many tissues and cell types in response to pathophysiological stimuli. Clinical applications of adult stem cells and progenitor cells have potential in the regeneration of blood cells, skin, bone, cartilage and heart muscle, and may have potential in degenerative diseases. Multi-pluripotent adult stem cells can change their phenotype in response to trans-differentiation or fusion and their therapeutic potential could include therapies regulated by pharmacological modulation, for example mobilising endogenous stem cells and directing them within a tissue to stimulate regeneration. Adult stem cells could also provide a vehicle for gene therapy, and genetically-engineered human adult stem cells have shown success in treatment of genetic disease. Conclusion: Deriving embryonic stem cells from early human embryos raises ethical, legal, religious and political questions. The potential uses of stem cells for generating human tissues are the subject of ongoing public debate. Stem cells must be used in standardised and controlled conditions in order to guarantee the best safety conditions for the patients. One critical point will be to verify the risk of tumourigenicity; this issue may be more relevant to embryonic than adult stem cells.

Hematotoxicity Testing by Cell Clonogenic Assay in Drug Development and Preclinical Trials

In vitro clonogenic assays have been developed and widely used since many years to investigate the proliferation and the differentiation both of pluripotent haemopoietic stem cells (PHSC) and of the different progenitors of blood cell lineages: megakaryocytes (Colony Forming Unit-Mk) granulocyte -macrophage (CFU-GM), erythrocytes (BFU-E/CFU-E). As these techniques have been introduced, they appeared to be very useful to investigate the pathogenic mechanisms of drug induced blood disorders and also for screening compound during preclinical safety study. Because the integrity of the hematopoiesis is also essential to guarantee the immunological function, the application to the toxicology of these clonogenic assays, provides an essential tool for better understanding the in vivo observation (experimental and clinical) by also helping in predicting the degree of a possible in vivo hematotoxic in drug treated patients. The review introduces to basic concepts on hematopoiesis and on classical evaluation of a drug hematotoxic phenomenon. It describes the application of each clonogenic test to assess the specific hematotoxicity action. Moreover it report the most recent studies on standardisation, prevalidation and validation of such assays and critically reviews a model for predicting myelotoxicity by discussing the main aspects referred to the evaluation of the in vivo acute neutropenia by using the in vitro CFU-GM assay.

Differential effects of extracellular vesicles secreted by mesenchymal stem cells from different sources on glioblastoma cells

Background: Malignant glial tumors, including glioblastoma multiforme, account for 15 – 20% of pediatric CNS malignancies. They are most resistant to therapy and are associated with a poor prognosis. Objective: Given the ability of mesenchymal stem cells (MSCs) to affect glioma growth, we investigated the effects of extracellular vesicles (EVs) derived from MSCs on U87MG glioblastoma cells line. Methods: EVs were isolated from culture media of MSCs from different sources, including bone marrow (BM), umbilical cord (UC) and adipose tissue (AT) and added to U87MG culture. The internalization and the effects of BM-, UC- and AT-MSC-EVs on proliferation and apoptosis of tumor cells were evaluated. Results: Both confocal microscopy and FACS analysis showed internalization of EVs into tumor cells. BM- and UC-MSC-EVs decreased cell proliferation, while an opposite effect was observed with AT-MSC-EVs. Moreover, both BM- and UC-MSC-EVs induced apoptosis of glioblastoma cells, while AT-MSC-EVs had no effect. Loading UC-MSC-EVs with Vincristine further increased cytotoxicity when compared both to the free drug and to untreated EVs. Conclusions: Different effects of MSC-EVs on cancer cells were observed depending on their tissue of origin. Moreover, MSC-EVs can deliver antiblastic drugs to glioblastoma cells.

Synthesis, Degradation, and Antimicrobial Properties of Targeted Macromolecular Prodrugs of Norfloxacin

ABSTRACT Long-term antibiotic treatment is required to cure tuberculosis. Targeted antibiotics should improve the efficacy of treatment by concentrating the drugs close to the bacteria. The aim of the present study was to synthesize targeted conjugates. For this purpose, we used mannose as a homing device to direct norfloxacin into macrophages. Dextran was used as the polymer bearing both mannose and norfloxacin. Using different peptide spacer arms to link norfloxacin to dextran, we demonstrated that norfloxacin acts as an antibiotic only when it is released in its native form. Also, targeting by using mannose as a homing device is required to achieve antimycobacterial activity in vivo. Thus, norfloxacin, which is inactive against mycobacteria in its native form in vivo, can be transformed into an active drug by targeting.

Comparison of in vitro drug-sensitivity of human granulocyte-macrophage progenitors from two different origins: umbilical cord blood and bone marrow.

Predictive in vitro hematotoxicity assays using human cells will provide estimation of tolerable level and aid considerably the development of agents with greater therapeutic activity and less toxicity. Human hematopoietic cells can be derived from three sources: human bone marrow by sternal or femoral aspiration, mobilized peripheral blood, or umbilical cord blood samples collected from placentas after deliveries. Because of the difficulties to have a continuous supply of bone marrow cells from normal human donors and the related ethical problems, we performed a study to compare the sensitivity of human bone marrow cells (h-BMC) and human cord blood cells (h-CBC) to chemicals in order to confirm if h-CBC can readily replace bone marrow cells in checking the sensitivity of GM-CFU progenitors to drugs as preliminarily reported in literature. Our results showed that the prediction of IC50 values in human model is quite similar by using h-BMC or h-CBC. On the contrary, the type of medium influenced in a significant way the ICs determination of some drugs.

Mesenchymal stromal cells primed with Paclitaxel attract and kill leukaemia cells, inhibit angiogenesis and improve survival of leukaemia‐bearing mice

Current leukaemia therapy focuses on increasing chemotherapy efficacy. Mesenchymal stromal cells (MSCs) have been proposed for carrying and delivery drugs to improve killing of cancer cells. We have shown that MSCs loaded with Paclitaxel (PTX) acquire a potent anti‐tumour activity. We investigated the effect of human MSCs (hMSCs) and mouse SR4987 loaded with PTX (hMSCsPTX and SR4987PTX) on MOLT‐4 and L1210, two leukaemia cell (LCs) lines of human and mouse origin, respectively. SR4987PTX and hMSCsPTX showed strong anti‐LC activity. hMSCsPTX, co‐injected with MOLT‐4 cells or intra‐tumour injected into established subcutaneous MOLT‐4 nodules, strongly inhibited growth and angiogenesis. In BDF1‐mice‐bearing L1210, the intraperitoneal administration of SR4987PTX doubled mouse survival time. In vitro, both hMSCs and hMSCsPTX released chemotactic factors, bound and formed rosettes with LCs. In ultrastructural analysis of rosettes, hMSCsPTX showed no morphological alterations while the attached LCs were apoptotic and necrotic. hMSCs and hMSCsPTX released molecules that reduced LC adhesion to microvascular endothelium (hMECs) and down‐modulated ICAM1 and VCAM1 on hMECs. Priming hMSCs with PTX is a simple procedure that does not require any genetic cell manipulation. Once the effectiveness of hMSCsPTX on established cancers in mice is proven, this procedure could be proposed for leukaemia therapy in humans.

Establishment and characterization of a new murine cell line (SR-4987) derived from marrow stromal cells

A new murine cell line designated as SR-4987 was established by treating a long-term bone marrow culture with the supernatant from Y-1 cells which actively produce viral C-particles (MuLV). The line showed a fibrolbast-like morphology and its mesodermal origin was confirmed by immunocytochemical staining. Flow cytometric analysis of DNA index evidenced a tetraploid number of chromosomes whereas cell cycle analysis showed 34.8% of cells in S phase and 60.7% in G1.In vitro growth studies demonstrated a population doubling time of 14.7h, a good plating efficiency (52.3%) and a very poor agar clonogenic capacity (0.6%). SR-4987 was tumorigenic only in syngeneic mice in which sarcomas were induced. The line produced M-CSF in the culture supermatant whereas G-CSF, IL-3 and GM-CSF were not detected. Studies are in progress to assess the production of other cytokines and to verify if same autocrine growth factor is involved in the control of SR-4987 proliferation. Our line provides a further model of stromal cells for studying the interaction between hemopoietic progenitors and their micro-environment, as well as to study factors produced by stromal cells acting as modulators of proliferation and differentiation of related cell populations.