Eduardo Mansilla

Feasibility investigation of allogeneic endometrial regenerative cells.

Endometrial Regenerative Cells (ERC) are a population of mesenchymal-like stem cells having pluripotent differentiation activity and ability to induce neoangiogenesis. In vitro and animal studies suggest ERC are immune privileged and in certain situations actively suppress ongoing immune responses. In this paper we describe the production of clinical grade ERC and initial safety experiences in 4 patients with multiple sclerosis treated intravenously and intrathecally. The case with the longest follow up, of more than one year, revealed no immunological reactions or treatment associated adverse effects. These preliminary data suggest feasibility of clinical ERC administration and support further studies with this novel stem cell type.

New potential therapeutic approach for the treatment of B-Cell malignancies using chlorambucil/hydroxychloroquine-loaded anti-CD20 nanoparticles.

Current B-cell disorder treatments take advantage of dose-intensive chemotherapy regimens and immunotherapy via use of monoclonal antibodies. Unfortunately, they may lead to insufficient tumor distribution of therapeutic agents, and often cause adverse effects on patients. In this contribution, we propose a novel therapeutic approach in which relatively high doses of Hydroxychloroquine and Chlorambucil were loaded into biodegradable nanoparticles coated with an anti-CD20 antibody. We demonstrate their ability to effectively target and internalize in tumor B-cells. Moreover, these nanoparticles were able to kill not only p53 mutated/deleted lymphoma cell lines expressing a low amount of CD20, but also circulating primary cells purified from chronic lymphocitic leukemia patients. Their safety was demonstrated in healthy mice, and their therapeutic effects in a new model of Burkitts lymphoma. The latter serves as a prototype of an aggressive lympho-proliferative disease. In vitro and in vivo data showed the ability of anti-CD20 nanoparticles loaded with Hydroxychloroquine and Chlorambucil to increase tumor cell killing in comparison to free cytotoxic agents or Rituximab. These results shed light on the potential of anti-CD20 nanoparticles carrying Hydroxychloroquine and Chlorambucil for controlling a disseminated model of aggressive lymphoma, and lend credence to the idea of adopting this therapeutic approach for the treatment of B-cell disorders.

The Next Generation of Burns Treatment: Intelligent Films and Matrix, Controlled Enzymatic Debridement, and Adult Stem Cells

We describe a novel technology based on nanoengineered multifunctional acellular biologic scaffolds combined with wound dressings and films of the same kind. This method allows selective delivery and release of shielded biomaterials and bioactive substances to a desired wound or damaged tissue while stimulating the selective anchoring and adhesion of endogenous circulating repairing cells, such as mesenchymal stem cells, to obtain a faster and more physiologic healing process. We also present a new controlled enzymatic debridement process for more effective burned tissue scarolysis. In light of our preliminary in vitro and in vivo data, we are convinced that these approaches can include the use of other kinds of adult stem cells, such as endometrial regenerative cells, to improve the vascularization of the constructs, with great potential in the entire tissue and organ regeneration field but especially for the treatment of severely burned patients, changing the way these lesions may be treated in the future.

Could Metabolic Syndrome, Lipodystrophy, and Aging Be Mesenchymal Stem Cell Exhaustion Syndromes?

One of the most important and complex diseases of modern society is metabolic syndrome. This syndrome has not been completely understood, and therefore an effective treatment is not available yet. We propose a possible stem cell mechanism involved in the development of metabolic syndrome. This way of thinking lets us consider also other significant pathologies that could have similar etiopathogenic pathways, like lipodystrophic syndromes, progeria, and aging. All these clinical situations could be the consequence of a progressive and persistent stem cell exhaustion syndrome (SCES). The main outcome of this SCES would be an irreversible loss of the effective regenerative mesenchymal stem cells (MSCs) pools. In this way, the normal repairing capacities of the organism could become inefficient. Our point of view could open the possibility for a new strategy of treatment in metabolic syndrome, lipodystrophic syndromes, progeria, and even aging: stem cell therapies.

Outstanding Survival and Regeneration Process by the Use of Intelligent Acellular Dermal Matrices and Mesenchymal Stem Cells in a Burn Pig Model

A pig model with a deep large burn was used to study the regeneration process induced by mesenchymal stem cells (MSCs) and acellular pig dermal matrices, made intelligent by the combination with biodegradable nanofibers loaded with growth factors (granulocyte-macrophage colony-stimulating factor and epidermal growth factor) and coated with the anti-CD44 monoclonal antibody (intelligent acellular dermal matrices, IADMs). These IADMs are specially designed to integrate in the wound bed as new biological scaffolds as well as to specifically recruit and attach circulating and/or externally applied MSCs through the anti-CD44 antibody while delivering precise amounts of growth factors. In this way, the reparative process as well as the aesthetic and functional results were enhanced in our burn model. The animal survived, the wound was completely closed, and total regeneration of the skin was obtained without much scarring. Surprisingly, hair follicles and other skin appendages developed despite the severity and deepness of the burn. Even burned muscles and ribs seemed to have undergone a regenerative process by the end of the study. Based on these findings, we have proposed the use of IADMs and autologous, allogeneic or xenogeneic MSCs, as a new paradigm for the future treatment of large burns and probably other dermatological and cosmetic human conditions.

The lysosomotropic agent, hydroxychloroquine, delivered in a biodegradable nanoparticle system, overcomes drug resistance of B-chronic lymphocytic leukemia cells in vitro.

Nonviral delivery systems are relatively easy to produce in the large scale, are safe, and elicit a negligible immune response. Nanoparticles (NPs) offer promise as nonviral vectors as biocompatible and -degradable carriers of drugs with targeting to specific sites by surface receptors of monoclonal antibodies (mAbs). We investigated the effect of four PEG-PLGA (polyethylene glycol-polylactic-co-glycolic acid) NP systems on drug-resistant B-chronic lymphocytic leukemia (B-CLL) cells in vitro, three of them encapsulating the drug, hydroxylchloroquine (HDQ), two with NP surface coatings of mAbs (NP1) CD20, (NP2) CD19, and CD20, and one (NP3) with no mAb, but tagged with the fluorescent marker, fluorescein isothiocyanate. The fourth NP system (NP4) was coated with anti-CD19/FITC and anti-CD20/Alexa-Fluor((R)) antibodies, but did not contain the active drug, HCQ. Our data indicate that PEG-PLGA nanoparticles with surface mAbs are suitable for selective drug delivery to B-CLL cells and produce a strong apoptotic effect when loaded with the lysosomotropic agent, HDQ.

Apoptosis induced by Magnolia Grandiflora extract in chlorambucil-resistant B-chronic lymphocytic leukemia cells.

BACKGROUND B-cell chronic lymphocytic leukemia (B-CLL) still remains as an uncurable disease. Even the newest antineoplastic agents have demonstrated limitations in their efficacy. For this reason, further research of new compounds must be done. New pharmacological properties can be obtained from a great diversity botanical species. Among these products, Magnolia Grandiflora receives our attention since it mainly contains Honokiol which had demonstrated effect against B-CLL cells activating different cell death pathways. AIM To test the ability of Magnolia Grandiflora extracts to induce apoptosis of B-CLL cells in vitro. MATERIALS AND METHODS Herbs extraction: Twenty grams of powdered material were submitted to three consecutives decoctions with 500 ml of distilled water (96 °C), filtered and followed by ultrafiltration with cellulose membrane, lyophilized and reconstituted in AIM-V medium at a final concentration of 10 mg/ml solution. B-CLL chlorambucil-resistant cells were separated and cultivated in the presence of Magnolias extract. Samples of cells were taken from the cultures at 24, 48 and 72 h for apoptosis analysis by flow cytometry measuring positive annexin V (0.1 μg/ml) cells. STATISTICS Apoptosis values were represented by the mean plus or minus SD (± SD) for five independent experiments. Statistical significance was determined by Students t-test. A P value of 0.05 or less was considered as significant. RESULTS AND CONCLUSION This article discusses the apoptosis properties of Magnolia on B-CLL cells. The evidence suggests a potentially effective repertoire for B-CLL treatment. This herb extract might have promising therapy strategies in treating B-CLL or other hematological disease resistant to alkylating agents in clinical practice.

Exploratory Study on the Effects of Biodegradable Nanoparticles with Drugs on Malignant B Cells and on a Human/Mouse Model of Burkitt Lymphoma

The aim of this study was to determine if Rituximab coated Biodegradable Nanoparticles (BNPs) loaded with Chlorambucil and Hydroxychloroquine could induce apoptosis of B-Chronic Lymphocytic Leukemia (B-CLL), MEC-1 and BJAB cells in vitro and evaluate their toxic and therapeutic effects on a Human/Mouse Model of Burkitt Lymphoma at an exploratory, proof of concept scale. We found that Rituximab-Chlorambucil-Hydroxychloroquine BNPs induce a decrease in cell viability of malignant B cells in a dose-dependent manner. The mediated cytotoxicity resulted from apoptosis, and was confirmed by monitoring the B-CLL cells after Annexin V/propidium iodide staining. Additional data revealed that these BNPs were non toxic for healthy animals, and had prolonged survival in this mice model of human lymphoma.

Enhanced Singlet Oxygen Production by Photodynamic Therapy and a Novel Method for Its Intracellular Measurement

The generation of singlet oxygen (SO) in the presence of specific photosensitizers (PSs) or semiconductor quantum dots (QDs) and its application in photodynamic therapy (PDT) is of great interest to develop cancer therapies with no need of surgery, chemotherapy, and/or radiotherapy. This work was focused on the identification of the main factors leading to the enhancement of SO production using Rose Bengal (RB), and Methylene Blue (MB) as PS species in organic and aqueous mediums. Subsequently, the capacity of zinc oxide (ZnO), zinc sulfide (ZnS), and ZnO/ZnS core-shell QDs as well as manganese (Mn(+2)) doped ZnO and ZnS nanoparticles (NPs) as potential PS was also investigated. Many variable parameters such as type of quencher, PSs, NPs, as well as its different concentrations, light source, excitation wavelength, reaction time, distance from light source, and nature of solvent were used. The degradation kinetics of the quenchers generated by SO species and the corresponding quantum yields were determined by monitoring the photo-oxidation of the chemical quencher and measuring its disappearance by fluorometry and spectrophotometry in the presence of NPs. Small intracellular changes of SO induced by these metal Zn (zinc) NPs and PDT could execute and accelerate deadly programs in these leukemic cells, providing in this way an innovative modality of treatment. In order to perform further more specific in vitro cytotoxic studies on B-chronic lymphocytic leukemia cells exposed to Zn NPs and PDT, we needed first to measure and ascertain those possible intracellular SO variations generated by this type of treatment; for this purpose, we have also developed and tested a novel method first described by us.

A new approach for the treatment of CLL using chlorambucil/hydroxychloroquine-loaded anti-CD20 nanoparticles

Current approaches for the treatment of chronic lymphocytic leukemia (CLL) have greatly improved the prognosis for survival, but some patients remain refractive to these therapeutic regimens. Hence, in addition to reducing the long-term sideeffects of therapeutics for all leukemia patients, there is an urgent need for novel therapeutic strategies for difficult-to-treat leukemia cases. Due to the cytotoxicity of drugs, the major challenge currently is to deliver the therapeutic agents to neoplastic cells while preserving the viability of non-malignant cells. In this study, we propose a therapeutic approach in which high doses of hydroxychloroquine and chlorambucil were loaded into biodegradable polymeric nanoparticles coated with an anti-CD20 antibody.We first demonstrated the ability of the nanoparticles to target and internalize in tumor B-cells. Moreover, these nanoparticles could kill not only p53-mutated/deleted leukemia cells expressing a low amount of CD20, but also circulating primary cells isolated from chronic lymphocytic leukemia patients. The safety of these nanoparticles was also demonstrated in healthy mice, and their therapeutic effects were shown in a new model of aggressive leukemia. These results showed that anti-CD20 nanoparticles containing hydroxychloroquine and chlorambucil can be effective in controlling aggressive leukemia and provided a rationale for adopting this approach for the treatment of other B-cell disorders.