RESUMO
In bone marrow transplantation (BMT), hematopoiesis-reconstituting cells are introduced following myeloablative treatment, which eradicates existing hematopoietic cells and disrupts stroma within the hematopoietic tissue. Both hematopoietic cells and stroma then undergo regeneration. Our study compares the outcomes of a second BMT administered to mice shortly after myeloablative treatment and the first BMT, with those of a second BMT administered to mice experiencing robust hematopoietic regeneration after the initial transplant. We evaluated the efficacy of the second BMT in terms of engraftment efficiency, types of generated blood cells, and longevity of function. Our findings show that regenerating hematopoiesis readily accommodates newly transplanted stem cells, including those endowed with a robust capacity for generating B and T cells. Importantly, our investigation uncovered a window for preferential engraftment of transplanted stem cells coinciding with the resumption of blood cell production. Repeated BMT could intensify hematopoiesis reconstitution and enable therapeutic administration of genetically modified autologous stem cells.
Assuntos
Transplante de Medula Óssea , Hematopoese , Animais , Transplante de Medula Óssea/métodos , Camundongos , Células-Tronco Hematopoéticas/imunologia , Camundongos Endogâmicos C57BL , Reconstituição Imune , RegeneraçãoRESUMO
BACKGROUND: Lymphedema is a chronic condition characterized by progressive edema with complicated treatment. Recently, new treatment strategies inducing lymphangiogenesis were proposed. The aim of our study was to examine the effect of vascular endothelial growth factor C (VEGF-C) and adipose-derived stem cells (ADSCs) on lymphatic regeneration and drainage re-establishment in vascularized lymph node transfer (VLNT) model using a pedicled vascularized lymph node (VLN) groin flap. METHODS: Female Lewis rats with groin VLN flaps were utilized as a lymphedema model. Group A served as the control. Group B received VEGF-C. Group C received both VEGF-C and ADSCs. Group D received ADSCs only. Lymphatic drainage re-establishment was evaluated by ultrasound-photoacoustic imaging (US-PAI) after indocyanine green (ICG) injection. RESULTS: The fastest regeneration of elevated flaps was observed in Groups B and C in all monitored periods. After the first month, ICG positivity was detected in 14.3% of animals in Group A, 71.43% of animals in Group B (odds ratio [OR] = 15; p = 0.048), and 83.33% in Group C (OR = 30; p = 0.027). On the contrary, the difference between control group and Group D (16.67%; p = 0.905) was statistically insignificant. Administration of VEGF-C, ADSC + VEGF-C, and ADSC led to full flap regeneration after 6 months. The control group had the lowest percentage of ICG positivity at all monitored time points. CONCLUSION: We found that the fastest regeneration occurred with the combination of the VLN flap and VEGF-C. The addition of ADSC had an insignificant effect in our study. Furthermore, we proved the feasibility of PAI as an assessment tool of the lymphatic drainage recovery in a VLNT model.
Assuntos
Linfedema , Fator C de Crescimento do Endotélio Vascular , Ratos , Feminino , Animais , Ratos Endogâmicos Lew , Linfonodos/irrigação sanguínea , Linfedema/cirurgia , Linfedema/etiologia , Verde de Indocianina , Regeneração , Células-TroncoRESUMO
Aqueous solutions of some polymers exhibit a lower critical solution temperature (LCST); that is, they form phase-separated aggregates when heated above a threshold temperature. Such polymers found many promising (bio)medical applications, including in situ thermogelling with controlled drug release, polymer-supported radiotherapy (brachytherapy), immunotherapy, and wound dressing, among others. Yet, despite the extensive research on medicinal applications of thermoresponsive polymers, their biodistribution and fate after administration remained unknown. Thus, herein, they studied the pharmacokinetics of four different thermoresponsive polyacrylamides after intramuscular administration in mice. In vivo, these thermoresponsive polymers formed depots that subsequently dissolved with a two-phase kinetics (depot maturation, slow redissolution) with half-lives 2 weeks to 5 months, as depot vitrification prolonged their half-lives. Additionally, the decrease of TCP of a polymer solution increased the density of the intramuscular depot. Moreover, they detected secondary polymer depots in the kidneys and liver; these secondary depots also followed two-phase kinetics (depot maturation and slow dissolution), with half-lives 8 to 38 days (kidneys) and 15 to 22 days (liver). Overall, these findings may be used to tailor the properties of thermoresponsive polymers to meet the demands of their medicinal applications. Their methods may become a benchmark for future studies of polymer biodistribution.
Assuntos
Polímeros , Água , Camundongos , Animais , Distribuição Tecidual , Temperatura , Liberação Controlada de FármacosRESUMO
The immense regenerative power of hematopoietic tissue stems from the activation of the immature stem cells and the progenitor cells. After partial damage, hematopoiesis is reconstituted through a period of intense regeneration when blood cell production originates from erythro-myeloid progenitors in the virtual absence of stem cells. Since the damaged hematopoiesis can also be reconstituted from transplanted hematopoietic cells, we asked whether this also leads to the transient state when activated progenitors initially execute blood cell production. We first showed that the early reconstitution of hematopoiesis from transplanted cells gives rise to extended populations of developmentally advanced but altered progenitor cells, similar to those previously identified in the bone marrow regenerating from endogenous cells. We then identified the cells that give rise to these progenitors after transplantation as LSK CD48- cells. In the submyeloablative irradiated host mice, the transplanted LSK CD48- cells preferably colonized the spleen. Unlike the endogenous hematopoiesis reconstituting cells, the transplanted whole bone marrow cells and sorted LSK CD48- cells had greater potential to differentiate to B-lymphopoiesis. Separate transplantation of the CD150- and CD150+ subsets of LSK CD48- cells suggested that CD150- cells had a greater preference to B-lymphopoiesis than CD150+ cells. In the intensively regenerating hematopoiesis, the CD71/Sca-1 plot of immature murine hematopoietic cells revealed that the expanded populations of altered myeloid progenitors were highly variable in the different places of hematopoietic tissues. This high variability is likely caused by the heterogeneity of the hematopoiesis supporting stroma. Lastly, we demonstrate that during the period when active hematopoiesis resumes from transplanted cells, the hematopoietic tissues still remain highly permissive for further engraftment of transplanted cells, particularly the stem cells. Thus, these results provide a rationale for the transplantation of the hematopoietic stem cells in successive doses that could be used to boost the transplantation outcome.
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The delivery of therapeutics into sites of action by using cargo-delivery platforms potentially minimizes their premature degradation and fast clearance from the bloodstream. Additionally, drug-loaded stimuli-responsive supramolecular assemblies can be produced to respond to the inherent features of tumor microenvironments, such as extracellular acidosis. We report in this framework the use of pH-responsive polymersomes (PSs) manufactured using poly([N-(2-hydroxypropyl)] methacrylamide)35-b-poly[2-(diisopropylamino)ethyl methacrylate]75 as the building unit (PHPMA35-b-PDPA75). The self-assemblies were produced with desired size towards long circulation time and tumor accumulation (hydrodynamic diameter - DH ~ 100 nm), and they could be successfully loaded with 10% w/w DOX (doxorubicin), while maintaining colloidal stability. The DOX loaded amount is presumably mainly burst-released at the acidic microenvironment of tumors thanks to the pH-switchable property of PDPA (pKa ~ 6.8), while reduced drug leakage has been monitored in pH 7.4. Compared to the administration of free DOX, the drug-loaded supramolecular structures greatly enhanced the therapeutic efficacy with effective growth inhibition of EL4 lymphoma tumor model and 100% survival rate in female C57BL/6 black mice over 40 days. The approach also led to reduced cardiotoxic effect. These features highlight the potential application of such nanotechnology-based treatment in a variety of cancer therapies where low local pH is commonly found, and emphasize PHPMA-based nanomedicines as an alternative to PEGylated formulations.
Assuntos
Doxorrubicina , Neoplasias , Animais , Cardiotoxicidade , Doxorrubicina/uso terapêutico , Portadores de Fármacos/uso terapêutico , Sistemas de Liberação de Medicamentos , Feminino , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológico , Microambiente TumoralRESUMO
Hemochromatosis (iron overload) encompasses a group of diseases that are characterized by a toxic hyperaccumulation of iron in parenchymal organs. Currently, only few treatments for this disease have been approved; however, all these treatments possess severe side effects. In this study, a paradigm for hemochromatosis maintenance/preventive therapy is investigated: polymers with negligible systemic biological availability form stable complexes with iron ions in the gastrointestinal tract, which reduces the biological availability of iron. Macroporous polymer beads are synthesized with three different iron-chelating moieties (benzene-1,2-diol, benzene-1,2,3-triol, and 1,10-phenanthroline). The polymers rapidly chelate iron ions from aqueous solutions in vitro in the course of minutes, and are noncytotoxic and nonprooxidant. Moreover, the in vivo biodistribution and pharmacokinetics show a negligible uptake from the gastrointestinal tract (using 125 I-labeled polymer and single photon emission computed tomography/computed tomography), which generally prevents them from having systemic side effects. The therapeutic efficacy of the prepared polymers is successfully tested in vivo, and exhibits a significant inhibition of iron uptake from the gastrointestinal tract without any noticeable signs of toxicity. Furthermore, an in silico method is developed for the prediction of chelator selectivity. Therefore, this paradigm can be applied to the next-generation maintenance/preventive treatment for hemochromatosis and/or other diseases of similar pathophysiology.
Assuntos
Hemocromatose/tratamento farmacológico , Quelantes de Ferro/farmacologia , Ferro/metabolismo , Modelos Teóricos , Benzeno/química , Benzeno/farmacologia , Trato Gastrointestinal/efeitos dos fármacos , Hemocromatose/diagnóstico por imagem , Hemocromatose/patologia , Humanos , Quelantes de Ferro/química , Fenantrolinas/química , Fenantrolinas/farmacologia , Polímeros/química , Polímeros/farmacologia , Tomografia Computadorizada de EmissãoRESUMO
Regeneration of severely damaged adult tissues is currently only partially understood. Hematopoietic tissue provides a unique opportunity to study tissue regeneration due to its well established steady-state structure and function, easy accessibility, well established research methods, and the well-defined embryonic, fetal, and adult stages of development. Embryonic/fetal liver hematopoiesis and adult hematopoiesis recovering from damage share the need to expand populations of progenitors and stem cells in parallel with increasing production of mature blood cells. In the present study, we analyzed adult hematopoiesis in mice subjected to a submyeloablative dose (6 Gy) of gamma radiation and targeted the period of regeneration characterized by massive production of mature blood cells along with ongoing expansion of immature hematopoietic cells. We uncovered significantly expanded populations of developmentally advanced erythroid and myeloid progenitors with significantly altered immunophenotype. Their population expansion does not require erythropoietin stimulation but requires the SCF/c-Kit receptor signaling. Regenerating hematopoiesis significantly differs from the expanding hematopoiesis in the fetal liver but we find some similarities between the regenerating hematopoiesis and the early embryonic definitive hematopoiesis. These are in (1) the concomitant population expansion of myeloid progenitors and increasing production of myeloid blood cells (2) performing these tasks despite the severely reduced transplantation capacity of the hematopoietic tissues, and (3) the expression of CD16/32 in most progenitors. Our data thus provide a novel insight into tissue regeneration by suggesting that cells other than stem cells and multipotent progenitors can be of fundamental importance for the rapid recovery of tissue function.
RESUMO
The lack of cellular and tissue specificities in conventional chemotherapies along with the generation of a complex tumor microenvironment (TME) limits the dosage of active agents that reaches tumor sites, thereby resulting in ineffective responses and side effects. Therefore, the development of selective TME-responsive nanomedicines is of due relevance toward successful chemotherapies, albeit challenging. In this framework, we have synthesized novel, ready-to-use ROS-responsive amphiphilic block copolymers (BCs) with two different spacer chemistry designs to connect a hydrophobic boronic ester-based ROS sensor to the polymer backbone. Hydrodynamic flow focusing nanoprecipitation microfluidics (MF) was used in the preparation of well-defined ROS-responsive PSs; these were further characterized by a combination of techniques [1H NMR, dynamic light scattering (DLS), static light scattering (SLS), transmission electron microscopy (TEM), and cryogenic TEM (cryo-TEM)]. The reaction with hydrogen peroxide releases an amphiphilic phenol or a hydrophilic carboxylic acid, which affects polymersome (PS) stability and cargo release. Therefore, the importance of the spacer chemistry in BC deprotection and PS stability and cargo release is herein highlighted. We have also evaluated the impact of spacer chemistry on the PS-specific release of the chemotherapeutic drug doxorubicin (DOX) into tumors in vitro and in vivo. We demonstrate that by spacer chemistry design one can enhance the efficacy of DOX treatments (decrease in tumor growth and prolonged animal survival) in mice bearing EL4 T cell lymphoma. Side effects (weight loss and cardiotoxicity) were also reduced compared to free DOX administration, highlighting the potential of the well-defined ROS-responsive PSs as TME-selective nanomedicines. The PSs could also find applications in other environments with high ROS levels, such as chronic inflammations, aging, diabetes, cardiovascular diseases, and obesity.
Assuntos
Doxorrubicina , Neoplasias , Animais , Linhagem Celular Tumoral , Portadores de Fármacos , Camundongos , Micelas , Neoplasias/tratamento farmacológico , Espécies Reativas de Oxigênio , Microambiente TumoralRESUMO
The thymidine analogues BrdU (5-bromo-2´-deoxyuridine) and EdU (5-ethynyl-2´-deoxyuridine) are routinely used for determination of the cells synthesizing DNA in the S-phase of the cell cycle. Availability of the anti-BrdU antibody clone MoBu-1 detecting only BrdU allowed to develop a method for the sequential DNA labelling by these two thymidine analogues for determining the cell cycle kinetic parameters.In the current step-by-step protocol, we present` two approaches optimized for in vivo study of the cell cycle and the limitations that such approaches imply: (1) determination of the cell flow rate into the G2-phase by dual EdU/BrdU DNA-labelling method and (2) determination of the outflow of DNA-labelled cells arising from the mitosis.
Assuntos
Ciclo Celular , DNA/biossíntese , Coloração e Rotulagem/métodos , Animais , Células da Medula Óssea/metabolismo , Bromodesoxiuridina/metabolismo , Diferenciação Celular , Análise de Dados , Desoxiuridina/análogos & derivados , Citometria de Fluxo , Imunofenotipagem , Camundongos , Mitose , Reologia , Fase SRESUMO
Magnetite (Fe3O4) nanoparticles with uniform sizes of 10, 20, and 31 nm were prepared by thermal decomposition of Fe(III) oleate or mandelate in a high-boiling point solvent (>320 °C). To render the particles with hydrophilic and antifouling properties, their surface was coated with a PEG-containing bisphosphonate anchoring group. The PEGylated particles were characterized by a range of physicochemical methods, including dynamic light scattering, transmission electron microscopy, thermogravimetric analysis, Fourier transform infrared spectroscopy, and magnetization measurements. As the particle size increased from 10 to 31 nm, the amount of PEG coating decreased from 28.5 to 9 wt.%. The PEG formed a dense brush-like shell on the particle surface, which prevented particles from aggregating in water and PBS (pH 7.4) and maximized the circulation time in vivo. Magnetic resonance relaxometry confirmed that the PEG-modified Fe3O4 nanoparticles had high relaxivity, which increased with increasing particle size. In the in vivo experiments in a mouse model, the particles provided visible contrast enhancement in the magnetic resonance images. Almost 70% of administrated 20-nm magnetic nanoparticles still circulated in the blood stream after four hours; however, their retention in the tumor was rather low, which was likely due to the antifouling properties of PEG.
Assuntos
Difosfonatos/química , Nanopartículas de Magnetita/química , Animais , Compostos Férricos , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Polietilenoglicóis/química , Distribuição TecidualRESUMO
Hematopoietic stem and progenitor cells (HSPCs) are crucial for lifelong blood cell production. We analyzed the cell cycle and cell production rate in HSPCs in murine hematopoiesis. The labeling of DNA-synthesizing cells by two thymidine analogues, optimized for in-vivo use, enabled determination of the cell cycle flow rate into G2-phase, the duration of S-phase and the average cell cycle time in Sca-1+ and Sca-1- HSPCs. Determination of cells with 2n DNA content labeled in preceding S-phase was then used to establish the cell flow rates in G1-phase. Our measurements revealed a significant difference in how Sca-1+ and Sca-1- myeloid progenitors self-renew and differentiate. Division of the Sca-1+ progenitors led to loss of the Sca-1 marker in about half of newly produced cells, corresponding to asymmetric cell division. Sca-1- cells arising from cell division entered a new round of the cell cycle, corresponding to symmetric self-renewing cell division. The novel data also enabled the estimation of the cell production rates in Sca-1+ and in three subtypes of Sca-1- HSPCs and revealed Sca-1 negative cells as the major amplification stage in the blood cell development.
Assuntos
Antígenos Ly/metabolismo , Ciclo Celular , Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Membrana/metabolismo , Animais , Contagem de Células , Proliferação de Células , Autorrenovação Celular , DNA/biossíntese , Feminino , Fase G2 , Masculino , Camundongos Endogâmicos C57BL , Reologia , Fase SRESUMO
Transgenic mice expressing green fluorescent protein (GFP) are useful in transplantation experiments. When we used ubiquitin-GFP (UBC-GFP) transgenic mice to study the availability of niches for transplanted hematopoietic stem and progenitor cells, the results were strikingly different from the corresponding experiments that used congenic mice polymorphic in the CD45 antigen. Analysis of these unexpected results revealed that the hematopoiesis of UBC-GFP mice was outcompeted by the hematopoiesis of wild-type (WT) mice. Importantly, UBC-GFP mice engrafted the transplanted bone marrow of WT mice without conditioning. There was a significant bias toward lymphopoiesis in the WT branch of chimeric UBC-GFP/WT hematopoiesis. A fraction of immature Sca-1+ cells in the spleen of UBC-GFP mice expressed GFP at a very high level. The chimeric hematopoiesis was stable in the long term and also after transplantation to secondary recipient mice. The article thus identifies a specific defect in the hematopoiesis of UBC-GFP transgenic mice that compromises the lymphoid-primed hematopoietic stem cells in the bone marrow and spleen. Stem Cells 2018;36:1237-1248.
Assuntos
Proteínas de Fluorescência Verde/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Linfócitos/metabolismo , Ubiquitina/metabolismo , Animais , Medula Óssea/metabolismo , Quimera , Hematopoese , Linfopoese , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Baço/metabolismo , Esplenectomia , Timo/metabolismoRESUMO
A conceptually new bimodal immunoradiotherapy treatment was demonstrated using thermoresponsive polymer ß-glucan-graft-poly(2-isopropyl-2-oxazoline-co-2-butyl-2-oxazoline) bearing complexes of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid with yttrium-90(III) at the graft ends. The behavior of this thermoresponsive polymer in aqueous solutions was studied, and it showed the appropriate cloud point temperature for brachytherapy applications. The polymer was tested in vitro, and it exhibited nontoxicity and active uptake into cancer cells and macrophages with colocalization in the lysosomes and macrophagosomes. Moreover, the observed oxidative burst response of the leukocytes established the immunostimulatory properties of the polymer, which were also studied in vivo after injection into the thigh muscles of healthy mice. The subsequent histological evaluation revealed the extensive immune activation reactions at the site of injection. Furthermore, the production of tumor necrosis factor α induced by the prepared polymer was observed in vitro, denoting the optimistic prognosis of the treatment. The biodistribution study in vivo indicated the formation of the polymer depot, which was gradually degraded and excluded from the body. The radiolabeled polymer was used during in vivo antitumor efficiency experiments on mice with EL4 lymphoma. The immunoradiotherapy group (treated with the radiolabeled polymer) demonstrated the complete inhibition of tumor growth during the beginning of the treatment. Moreover, 7 of the 15 mice were completely cured in this group, while the others exhibited significantly prolonged survival time compared to the control group. The in vivo experiments indicated the considerable synergistic effect of using immunoradiotherapy compared to separately using immunotherapy or radiotherapy.
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Antineoplásicos/síntese química , Compostos Aza/química , Complexos de Coordenação/química , Compostos Heterocíclicos com 1 Anel/química , Oxazóis/química , Polímeros/química , Radioimunoterapia/métodos , beta-Glucanas/química , Animais , Antibacterianos/síntese química , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Braquiterapia/métodos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Sistema Imunitário/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Camundongos Endogâmicos C57BL , Oxirredução , Staphylococcus aureus/efeitos dos fármacos , Temperatura , Radioisótopos de Ítrio/químicaRESUMO
This study continues our earlier findings on the hematopoiesis-modulating effects of adenosine A1 and A3 receptor agonists that were performed on committed hematopoietic progenitor and precursor cell populations. In the earlier experiments, N (6)-cyclopentyladenosine (CPA), an adenosine A1 receptor agonist, was found to inhibit proliferation in the above-mentioned hematopoietic cell systems, whereas N (6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), an adenosine A3 receptor agonist, was found to stimulate it. The topic of this study was to evaluate the possibility that the above-mentioned adenosine receptor agonists modulate the behavior of early hematopoietic progenitor cells and hematopoietic stem cells. Flow cytometric analysis of hematopoietic stem cells in mice was employed, as well as a functional test of hematopoietic stem and progenitor cells (HSPCs). These techniques enabled us to study the effect of the agonists on both short-term repopulating ability and long-term repopulating ability, representing multipotent progenitors and hematopoietic stem cells, respectively. In a series of studies, we did not find any significant effect of adenosine agonists on HSPCs in terms of their numbers, proliferation, or functional activity. Thus, it can be concluded that CPA and IB-MECA do not significantly influence the primitive hematopoietic stem and progenitor cell pool and that the hematopoiesis-modulating action of these adenosine receptor agonists is restricted to more mature compartments of hematopoietic progenitor and precursor cells.
Assuntos
Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Células-Tronco Multipotentes/fisiologia , Receptor A1 de Adenosina/metabolismo , Receptor A3 de Adenosina/metabolismo , Animais , Citometria de Fluxo , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Multipotentes/efeitos dos fármacos , Agonistas do Receptor Purinérgico P1/farmacologiaRESUMO
Two mouse HPV16-transformed cell lines, viz. MK16 cells, which induce metastasizing tumors, and TC-1 cells, which induce non-metastasizing tumors were transduced with the gene for mouse endostatin. Two clones constitutively expressing endostatin were isolated from each of them. They were denoted ME3 and ME9, and TE2 and TE5, respectively. When inoculated into mice, ME3 cells were non-oncogenic. Nearly all mice inoculated with ME9 cells developed tumors, but considerably later than did the parental MK16 cells and metastasis formation was strongly reduced in these animals. On the other hand, TE2 and TE5 cells displayed oncogenic potential similar to that of the parental cells. To provide more information on these different effects of endostatin production, cell lysates of all six lines studied were tested for the content of 25 factors known to be involved in angiogenesis. The parental MK16 cells differed from the parental TC-1 cells and also from all endostatin producing sublines by a markedly higher production of interleukin 1alpha (IL-1alpha) and, to a lesser extent, by a higher production of several other factors tested. Additional experiments indicated that the suppression of the production of IL-1alpha by the parental MK16 caused by endostatin was due to an autocrine mechanism.