RESUMEN

AIMS: Narrowing or occlusion of arteries that supply the limbs can evolve to critical limb ischemia. M-CSF promotes proliferation, differentiation and survival of monocytes and macrophages, and polarization of macrophages to M2-subtype, which are essential elements for vessel formation and tissue repair. Based on these properties of M-CSF, we hypothesize that transfection of M-CSF into ischemic limbs may promote vessel formation and repair of ischemic limbs. MAIN METHODS: Hindlimb ischemia was surgically induced in 10-12 weeks old Balb/c and gene therapy was performed with intramuscular application of either uP-MCSF or uP plasmids (100 µg). Macrophage and monocyte subpopulations were assessed by flow cytometry and blood flow was monitored by Laser Doppler Perfusion Imaging (LDPI). Thirty days after transfection, we assessed gastrocnemius mass and muscle force, subsequently collecting the muscle for histology. KEY FINDINGS: We successfully developed the uP-MCSF plasmid, which increases M-CSF expression in the muscle transiently. Thirty days after uP-MCSF gene therapy in ischemic muscles, the treated group presented: improved muscle force, reduced fibrosis and increased arteriogenesis, although LDPI analysis did not show any significant difference in blood flow among groups. Noteworthy, we observed a temporary increase in MHCIIhighCD206high macrophages after uP-MCSF transfection. SIGNIFICANCE: M-CSF gene therapy improved ischemic muscle functionality by promoting arteriogenesis and decreasing fibrosis, likely through increased MHCIIhighCD206high macrophages and not via classically known M2-macrophages.

Asunto(s)

Factor Estimulante de Colonias de Macrófagos , Macrófagos , Animales , Humanos , Macrófagos/metabolismo , Monocitos/metabolismo , Músculo Esquelético/patología , Isquemia/metabolismo , Miembro Posterior/irrigación sanguínea

RESUMEN

Tumor-associated carcinoembryonic antigen (CEA) is a natural target for vaccines against colorectal cancers. Our previous experience with a DNA vaccine with scFv6.C4, a CEA surrogate, showed a CEA-specific immune response with 40% of tumor-free mice after challenge with B16F10-CEA and 47% with MC38-CEA cells. These percentages increased to 63% after using FrC as an adjuvant. To further enhance the vaccine efficacy, we tested GM-CSF and IFNγ as adjuvants. C57BL/6J-CEA2682 mice were immunized 4 times with uP-PS/scFv6.C4, uP-PS/scFv6.C4 + uP-IFNγ, or uP-PS/scFv6.C4 + uP-GMCSF. After one week, the mice were challenged with MC38-CEA, and tumor growth was monitored over 100 days. Immunization with scFv6.C4 and scFv6.C4 + GM-CSF resulted in a gradual increase in the anti-CEA antibody titer, while scFv6.C4 + IFNγ immunization led to a rapid and sustained increase in the titer. The addition of IFNγ also induced higher CD4 + and CD8 + responses. When challenged, almost 80% of the scFv6.C4 + IFNγ-vaccinated mice did not develop tumors, while the others had a significant tumor growth delay. The probability of being tumor-free was 2700% higher using scFv6.C4 + IFNγ than scFv6.C4. The addition of GM-CSF had no additional effect on tumor protection. DNA immunization with scFv6.C4 + IFNγ, but not GM-CSF, increased the antitumor effect via readily sustained specific humoral and cytotoxic responses to CEA.

Asunto(s)

Vacunas contra el Cáncer , Neoplasias , Vacunas de ADN , Ratones , Animales , Antígeno Carcinoembrionario/genética , Ratones Endogámicos C57BL , Interferón gamma , Vacunas contra el Cáncer/genética

RESUMEN

Ischemia occurs when blood flow is reduced or restricted, leading to a lack of oxygen and nutrient supply and removal of metabolites in a body part. Critical limb ischemia (CLI) is a severe clinical manifestation of peripheral arterial disease. Atherosclerosis serves as the main cause of CLI, which arises from the deposition of lipids in the artery wall, forming atheroma and causing inflammation. Although several therapies exist for the treatment of CLI, pharmacotherapy still has low efficacy, and vascular surgery often cannot be performed due to the pathophysiological heterogeneity of each patient. Gene and cell therapies have emerged as alternative treatments for the treatment of CLI by promoting angiogenesis. However, the delivery of autologous, heterologous or genetically modified cells into the ischemic tissue remains challenging, as these cells can die at the injection site and/or leak into other tissues. The encapsulation of these cells within hydrogels for local delivery is probably one of the promising options today. Hydrogels, three-dimensional (3D) cross-linked polymer networks, enable manipulation of physical and chemical properties to mimic the extracellular matrix. Thus, specific biostructures can be developed by adjusting prepolymer properties and encapsulation process variables, such as viscosity and flow rate of fluids, depending on the final biomedical application. Electrostatic droplet extrusion, micromolding, microfluidics, and 3D printing have been the most commonly used technologies for cell encapsulation due to their versatility in producing different hydrogel-based systems (e.g., microgels, fibers, vascularized architectures and perfusable single vessels) with great potential to treat ischemic diseases. This review discusses the cell encapsulation technologies associated with hydrogels which are currently used for advanced therapies applied to limb ischemia, describing their principles, advantages, disadvantages, potentials, and innovative therapeutic ideas.

RESUMEN

AIMS: The purpose of this study was to investigate the effect of PPRP (pure PRP) and LPRP (PRP with leukocytes) on recovery from limb ischemia and on expression of growth factors involved in angiogenesis, myogenesis and fibrogenesis. MATERIAL AND METHODS: PPRP and LPRP prepared by centrifugation were added to cultures of C2C12 and NIH3T3 cells (1 or 10% PRPs) to evaluate alterations in cell metabolism and expression of growth factors by MTT, ELISA and RT-qPCR, respectively. To evaluate in vivo regenerative effects, PRPs were injected into the ischemic limbs of BALB/c mice and muscle mass/strength and histomorphometry were evaluated after 30 days. KEY FINDINGS: Mice treated with PRPs after limb ischemia showed an increase in the size of myofibers and muscle strength, reduced fibrosis and adipocytes, and decreased capillary density and necrosis scores compared to untreated mice. In cell culture, serum deprivation reduced the viability of C2C12 and NIH3T3 cells to about 50%, but the addition of 1% PRPs completely recovered this loss. Both PRPs, downregulated most of the tested genes; however, angiogenic gene Vegfa in C2C12 and the fibrogenic genes Col1a1 and Col3a1 in NIH3T3 cells were upregulated by LPRP. SIGNIFICANCE: PPRP and LPRP had similar effects in regulation of genes involved in angiogenesis, myogenesis and fibrogenesis. However, the presence of leucocytes did not significantly affect regenerative activities of PRP in the ischemic limb.

Asunto(s)

Miembro Posterior/fisiopatología , Isquemia/fisiopatología , Plasma Rico en Plaquetas/metabolismo , Regeneración/fisiología , Animales , Supervivencia Celular , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/patología , Células 3T3 NIH

RESUMEN

Efficient delivery of nanometric vectors complexed with nanoparticles at a target tissue without spreading to other tissues is one of the main challenges in gene therapy. One means to overcome this problem is to confine such vectors within microgels that can be placed in a target tissue to be released slowly and locally. Herein, a conventional optical microscope coupled to a common smartphone was employed to monitor the microfluidic production of monodisperse alginate microgels containing nanoparticles as a model for the encapsulation of vectors. Alginate microgels (1.2%) exhibited an average diameter of 125 ± 3 µm, which decreased to 106 ± 5 µm after encapsulating 30 nm fluorescent nanoparticles. The encapsulation efficiency was 70.9 ± 18.9%. In a 0.1 M NaCl solution, 55 ± 5% and 92 ± 4.7% of nanoparticles were released in 30 minutes and 48 hours, respectively. Microgel topography assessment by atomic force microscopy revealed that incorporation of nanoparticles into the alginate matrix changes the scaffold's interfacial morphology and induces crystallization with the appearance of oriented domains. The high encapsulation rate of nanoparticles, alongside their continuous release of nanoparticles over time, makes these microgels and the production unit a valuable system for vector encapsulation for gene therapy research.

Asunto(s)

Alginatos/química , Microfluídica/métodos , Microgeles/química , Nanopartículas/química , Unión Competitiva , Ligandos , Microscopía de Fuerza Atómica , Nanopartículas/metabolismo , Tamaño de la Partícula

RESUMEN

BACKGROUND: Peripheral arterial disease (PAD) affects millions of people and compromises quality of life. Critical limb ischemia (CLI), which is the most advanced stage of PAD, can cause nonhealing ulcers and strong chronic pain, and it shortens the patients' life expectancy. Cell-based angiogenic therapies are becoming a real therapeutic approach to treat CLI. Pericytes are cells that surround vascular endothelial cells to reinforce vessel integrity and regulate local blood pressure and metabolism. In the past decade, researchers also found that pericytes may function as stem or progenitor cells in the body, showing the potential to differentiate into several cell types. We investigated the gene expression profiles of pericytes during the early stages of limb ischemia, as well as the alterations in pericyte subpopulations to better understand the behavior of pericytes under ischemic conditions. METHODS: In this study, we used a hindlimb ischemia model to mimic CLI in C57/BL6 mice and explore the role of pericytes in regeneration. To this end, muscle pericytes were isolated at different time points after the induction of ischemia. The phenotypes and transcriptomic profiles of the pericytes isolated at these discrete time points were assessed using flow cytometry and RNA sequencing. RESULTS: Ischemia triggered proliferation and migration and upregulated the expression of myogenesis-related transcripts in pericytes. Furthermore, the transcriptomic analysis also revealed that pericytes induce or upregulate the expression of a number of cytokines with effects on endothelial cells, leukocyte chemoattraction, or the activation of inflammatory cells. CONCLUSIONS: Our findings provide a database that will improve our understanding of skeletal muscle pericyte biology under ischemic conditions, which may be useful for the development of novel pericyte-based cell and gene therapies.

Asunto(s)

Pericitos , Transcriptoma , Animales , Quimiotaxis de Leucocito , Células Endoteliales , Humanos , Isquemia/genética , Ratones , Músculo Esquelético , Calidad de Vida

RESUMEN

Drug delivery for treatment of chronic diseases relies on the effective delivery of payload materials into the target cells in a long-term release. In this context, the present study investigated hybrid microgels as platforms to carry nanoparticles to drug delivery. Hybrid microgels were produced with silk fibroin (SF) and chondroitin sulfate (CS), and alginate (ALG) by droplet microfluidics. ALG/SF, ALG/CS, and ALG/CS/SF microgels, ranging from 70-90 µm, were tested to encapsulate two model nanoparticles, polystyrene latex beads in pristine form (NPs) and NPs coated with bovine serum albumin (NPs-BSA) to simulate hydrophobic and hydrophilic nanocarriers, respectively. IR spectroscopy and fluorescence microscopy analysis confirmed the presence of SF and CS within ALG-based microgels revealing marked differences in their morphology and physicochemical properties. The release profiles of model nanoparticles revealed to be dependent on microgels composition and physicochemical properties. These findings show that SF ternary hybrid microgels facilitated the entrapment of hydrophobic nanocarriers with encapsulation efficiency (EE) from 83 to 98% keeping a better sustainable profile release than nonhybrid ALG microgels. Besides, CS improved the carriage of NPs-BSA (EE = 85%) and their profile release. The results highlight the versatility and tunable properties of these biobased microgels, being a good strategy to be used as an efficient platform in using macro and nanoencapsulated systems for drug delivery.

Asunto(s)

Quitosano , Microgeles , Nanopartículas , Sistemas de Liberación de Medicamentos , Interacciones Hidrofóbicas e Hidrofílicas , Microfluídica

RESUMEN

BACKGROUND: After traumatic skeletal muscle injury, muscle healing is often incomplete and produces extensive fibrosis. The sequence of M1 and M2 macrophage accumulation and the duration of each subtype in the injured area may help to direct the relative extent of fibrogenesis and myogenesis during healing. We hypothesized that increasing the number of M1 macrophages early after traumatic muscle injury would produce more cellular and molecular substrates for myogenesis and fewer substrates for fibrosis, leading to better muscle healing. METHODS: To test this hypothesis, we transfected skeletal muscle with a plasmid vector to transiently express GM-CSF shortly after injury to drive the polarization of macrophages towards the M1 subset. C57BL/6 mouse tibialis anterior (TA) muscles were injured by contusion and electroporated with uP-mGM, which is a plasmid vector that transiently expresses GM-CSF. Myogenesis, angiogenesis, and fibrosis were evaluated by histology, immunohistochemistry, and RT-qPCR; subpopulations of macrophages by flow cytometry; and muscle functioning by the maximum running speed on the treadmill and the recovery of muscle mass. RESULTS: Muscle injury increased the number of local M1-like macrophages and decreased the number of M2-like macrophages on day 4, and uP-mGM treatment enhanced this variation. uP-mGM treatment decreased TGF-ß1 protein expression on day 4, and the Sirius Red-positive area decreased from 35.93 ± 15.45% (no treatment) to 2.9% ± 6.5% (p < 0.01) on day 30. uP-mGM electroporation also increased Hgf, Hif1α, and Mtor gene expression; arteriole density; and muscle fiber number during regeneration. The improvement in the quality of the muscle tissue after treatment with uP-mGM affected the increase in the TA muscle mass and the maximum running speed on a treadmill. CONCLUSION: Collectively, our data show that increasing the number of M1-like macrophages immediately after traumatic muscle injury promotes muscle recovery with less fibrosis, and this can be achieved by the transient expression of GM-CSF.

Asunto(s)

Factor Estimulante de Colonias de Granulocitos y Macrófagos , Macrófagos , Animales , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético , Cicatrización de Heridas

RESUMEN

The concept of angiogenic therapy emerged in the early 1990s. The method employs genes that encode growth factors to promote formation of new vessels and remodeling of collateral vessels. Since the procedure involved in this therapy usually only consists of local injections of vectors, the process is minimally invasive, quick, and simple to perform. However, since the first clinical evidence of the effects of gene therapy with vascular endothelial growth factor (VEGF) was observed in patients with peripheral artery disease, to date only two angiogenic therapy drugs have been approved, one in Russia and another in Japan, which seem a very small number, in view of the large volume of investment made in pre-clinical and clinical studies. After all, can we conclude that angiogenic therapy is a reality?

RESUMEN

One of the most remarkable examples of cell-penetrating peptides (CPPs) is Penetratin, a 16-mer fragment derived from the Drosophila Antennapedia homeobox. Understanding the structure of Penetratin/DNA complexes is a key factor for the successful design of new vectors for gene delivery and may assist in optimizing molecular carriers based on CPPs. Herein, we present a comprehensive study on the nanoscale structure of noncovalent complexes formed between Penetratin and DNA. The strong cationic nature of the peptide makes it a very efficient agent for condensing DNA strands via electrostatic attraction, and we show for the first time that DNA condensation is accompanied by random-to-ß-sheet transitions of Penetratin secondary structure, demonstrating that nucleic acids behave as a structuring agent upon complexation. For the first time, nanoscale-resolved spectroscopy is used to provide single-particle infrared data from DNA carriers based on CPPs, and they show that the structures are stabilized by Penetratin ß-sheet cores, whereas larger DNA fractions are preferentially located in the periphery of aggregates. In-solution infrared assays indicate that phosphate diester groups are strongly affected upon DNA condensation, presumably as a consequence of charge delocalization induced by the proximity of cationic amide groups in Penetratin. The morphology is characterized by nanoassemblies with surface fractal features, and short-range order is found in the inner structure of the scaffolds. Interestingly, the formation of beads-on-a-string arrays is found, producing nanoscale architectures that resemble structures observed in early steps of chromatin condensation. A complexation pathway where DNA condensation and peptide pairing into ß-sheets are key steps for organization is proposed.

Asunto(s)

Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/síntesis química , ADN/química , Nanoestructuras , Dicroismo Circular , Espectroscopía Infrarroja por Transformada de Fourier

RESUMEN

Chitosan has received a lot of attention as a carrier for small interfering RNA (siRNA), due to its capacity for complexation and intracellular release of these molecules. However, one of its limitations is its insolubility at neutral pH and the tendency towards aggregation of its nanoparticles in isotonic ionic strength. In this study, a series of amphipathic chitosans were synthesized by varying the degree of acetylation (DA) from ˜2 to ˜30 mol% and the degree of substitution (DS) from 5 to 25%. by tertiary amino groups (DEAE) The results showed that the adjustment of these parameters decreases the interparticle interactions mediated by hydrogen bonding to obtain nanoparticles with improved colloidal stability. siRNA-containing nanoparticles of 100 to 150 nm with low polydispersities (0.15-0.2) and slightly positive zeta potentials (˜+ 5 mV) were resistant to aggregation at pH 7.4 and ionic strength of 150 mM. This resistance to aggregation is provided by changes on the nanoparticle surface and highlights the importance of more organized self-assembly in providing colloidal stability at physiological conditions. Additionally, the PEGylation of the most promising vectors conferred favorable physicochemical properties to nanoparticles. The chitosans and their nanoparticles exhibited low toxicity and an efficient cell uptake, as probed by confocal microscopy of rhodamine labeled vectors. The results provide a new approach to overcome the limited stability of chitosan nanoparticles at physiological conditions and show the potential of these amphipathic chitosans as siRNA carriers.

Asunto(s)

Quitosano/análogos & derivados , Portadores de Fármacos/química , Nanopartículas/química , ARN Interferente Pequeño/administración & dosificación , Tensoactivos/química , Anhídridos Acéticos/química , Acetilación , Animales , Quitosano/síntesis química , Quitosano/metabolismo , Quitosano/toxicidad , Dietilaminas/química , Portadores de Fármacos/síntesis química , Portadores de Fármacos/metabolismo , Portadores de Fármacos/toxicidad , Fluorescencia , Colorantes Fluorescentes/química , Concentración de Iones de Hidrógeno , Ratones , Nanopartículas/metabolismo , Nanopartículas/toxicidad , Tamaño de la Partícula , Polietilenglicoles/síntesis química , Polietilenglicoles/química , Polietilenglicoles/metabolismo , Polietilenglicoles/toxicidad , Células RAW 264.7 , ARN Interferente Pequeño/química , Rodaminas/química , Tensoactivos/síntesis química , Tensoactivos/metabolismo , Tensoactivos/toxicidad

RESUMEN

The carcinoembryonic antigen (CEA) is the main tumor-associated antigen of colorectal cancers. Previously, we developed a DNA vaccine using scFv6.C4, a CEA surrogate, against CEA-expressing tumors; 40% of the vaccinated mice were tumor-free after tumor challenge. In order to enhance vaccine efficacy, fragment C of Tetanus Toxin (FrC) was tested as adjuvant. C57BL/6J-CEA2682 mice were electroporated intramuscularly 4 times with uP-PS/scFv6.C4-FrC or uP-PS/scFv6.C4, challenged by s.c. injection of 1 × 105 MC38-CEA cells, and tumor growth was monitored over 100 days. The humoral and cellular immune responses were assessed by ELISA, immunocytochemistry, in-vitro lymphocyte proliferation, and CTL cytotoxicity assays. Immunization with uP-PS/scFv6.C4-FrC or uP-PS/scFv6.C4 induced similar anti-CEA antibody titers. However, immunocytochemistry analysis showed stronger staining with uP-PS/scFv6.C4-FrC-immunized mice sera. When challenged with MC38-CEA cells, 63% of the FrC-vaccinated mice did not develop tumors, half of the rest had a significant tumor growth delay, and the probability of being free of tumors was on average 40% higher than that of scFv6.C4-immunized mice. Addition of the adjuvant led to higher CD4+ and CD8+ proliferative responses and strong CD8+ CTL response against MC38-CEA cells. DNA immunization with scFv6.C4 and FrC increased antitumor effect via induction of high and specific humoral and cellular immune responses to CEA.

Asunto(s)

Vacunas contra el Cáncer/inmunología , Antígeno Carcinoembrionario/inmunología , Anticuerpos de Cadena Única/inmunología , Toxina Tetánica/inmunología , Animales , Vacunas contra el Cáncer/genética , Antígeno Carcinoembrionario/genética , Línea Celular Tumoral , Humanos , Inmunogenicidad Vacunal , Ratones , Ratones Endogámicos C57BL , Anticuerpos de Cadena Única/genética , Toxina Tetánica/genética , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología

RESUMEN

Dystrophin deficiency caused by mutations of the related gene leads to muscle wasting in Duchenne muscular dystrophy (DMD). Some patients with DMD also present with intellectual disability and various degrees of neurological disorders, which have been related to a decreased number of postsynaptic gamma-aminobutyric acid type A receptors (GABAARs) in the hippocampus (HPC) and cerebellum (CBL). The aim of this study was to examine the relevance of dystrophin in the presynaptic GABAergic function in brain regions in which this protein is normally abundant. [3H]-GABA release, induced by nicotinic receptor (nAChR) activation or K+ depolarization, and [3H]-GABA uptake were determined using synaptosomes extracted from the cortex (CTX), HPC, and CBL of littermate control and mdx mice. Superfusion of the synaptosomes with nicotine or high K+ solutions led to a concentration-dependent and Ca2+-dependent [3H]-GABA release in control and mdx synaptosomes. [3H]-GABA release induced by 10 µM nicotine in mdx CBL synaptosomes was 47% less than that in control mice. K+-induced [3H]-GABA release did not differ between control and mdx synaptosomes. α7-containing and ß2-containing nAChRs were involved in nicotine-induced [3H]-GABA release in control and mdx synaptosomes. Kinetic analysis of [3H]-GABA uptake in mdx CBL synaptosomes showed a reduced (50%) half-maximal uptake time (t1/2) and increased (44%) rate of [3H]-GABA uptake (Vmax) compared to controls. The apparent transporter affinity (Km) for GABA was not altered. Our findings show that dystrophin deficiency in mdx mice is associated with significant changes in the release and uptake of GABA in the CBL. These presynaptic alterations may be related to the reported decrease in postsynaptic GABAAR in the same brain region. The results indicate possible dysfunction of GABAergic synapses associated with dystrophin deficiency in the CBL, which may contribute to the cognitive and neurobehavioral disorders in mdx mice and patients with DMD.

Asunto(s)

Cerebelo/metabolismo , Distrofina/deficiencia , Distrofia Muscular de Duchenne/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Cerebelo/ultraestructura , Distrofina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/genética

RESUMEN

α-Synuclein is the major component of neuronal cytoplasmic aggregates called Lewy bodies, the main pathological hallmark of Parkinson disease. Although neurons are the predominant cells expressing α-synuclein in the brain, recent studies have demonstrated that primary astrocytes in culture also express α-synuclein and regulate α-synuclein trafficking. Astrocytes have a neuroprotective role in several detrimental brain conditions; we therefore analyzed the effects of the overexpression of wild-type α-synuclein and its A30P and A53T mutants on autophagy and apoptosis. We observed that in immortalized astrocyte cell lines, overexpression of α-synuclein proteins promotes the decrease of LC3-II and the increase of p62 protein levels, suggesting the inhibition of autophagy. When these cells were treated with rotenone, there was a loss of mitochondrial membrane potential, especially in cells expressing mutant α-synuclein. The level of this decrease was related to the toxicity of the mutants because they show a more intense and sustained effect. The decrease in autophagy and the mitochondrial changes in conjunction with parkin expression levels may sensitize astrocytes to apoptosis.

Asunto(s)

Apoptosis/fisiología , Astrocitos/metabolismo , Autofagia/fisiología , alfa-Sinucleína/biosíntesis , Animales , Astrocitos/patología , Línea Celular Transformada , Células Cultivadas , Femenino , Expresión Génica , Masculino , Ratas , Ratas Wistar , alfa-Sinucleína/genética

RESUMEN

The subventricular zone (SVZ) of the adult mammalian brain hosts full potential neural stem cells (NSCs). NSCs are able to respond to extracellular signals in the brain, amplifying the pool of progenitor cells and giving rise to neuroblasts that show ability to migrate towards an injury site. These signals can come from vascular system, cerebrospinal fluid, glial cells, or projections of neurons in adjoining regions. CXCL12, a chemokine secreted after brain injury, reaches the SVZ in a gradient manner and drives neuroblasts towards the lesion area. Among many other molecules, matrix metalloproteinase 2 and 9 (MMP-2/9) are also released during brain injury. MMP-2/9 can cleave CXCL12 generating a new molecule, CXCL12(5-67), and its effects on NSCs viability is not well described. Here we produced recombinant CXCL12 and CXCL12(5-67) and evaluated their effect in murine adult NSCs migration and survival in vitro. We showed CXCL12(5-67) does not promote NSCs migration, but does induce cell death. The NSC death induced by CXCL12(5-67) involves caspases 9 and 3/7 activation, implying the intrinsic apoptotic pathway in this phenomenon. Our evidences in vitro make CXCL12(5-67) and its receptor potential candidates for brain injuries and neurodegeneration studies.

Asunto(s)

Quimiocina CXCL12/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis/fisiología , Secuencia de Bases , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Quimiocina CXCL12/farmacología , Quimiotaxis/efectos de los fármacos , Femenino , Células HEK293 , Humanos , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Receptores CXCR4/metabolismo , Proteínas Recombinantes/farmacología

RESUMEN

BACKGROUND: It is well known that platelet-rich plasma (PRP) preparations are not the same and that not all preparations include white blood cells, but the part that leukocytes play on the healing role of PRP is still unknown. PURPOSE: The primary aim of this study was to evaluate the influence of leukocytes in different PRP preparations with a special emphasis on growth factor concentrations. The secondary aim was to evaluate the influence of PRP on muscle healing. STUDY DESIGN: Controlled laboratory study. METHODS: Two PRP preparation procedures were evaluated. Blood fractions were stained with Rapid Panoptic, and growth factors (transforming growth factor beta 1 [TGF-ß1], vascular endothelial growth factor [VEGF], insulin-like growth factor [IGF], epidermal growth factor [EGF], hepatocyte growth factor [HGF], and platelet-derived growth factor [PDGF]) were quantified by enzyme-linked immunosorbent assay. Western blotting analysis was performed for Fms-related tyrosine kinase 1 (Flt-1). A muscle contusion injury was created and treated with PRP at different time points. RESULTS: Leukocytes were the main source of VEGF, and all other growth factors measured had a higher concentration in the preparations that included the buffy coat and consequently had a higher concentration of white blood cells. Flt-1 was also found in platelet-poor plasma (PPP). There were higher concentrations of PDGF and HGF in the preparations that encompassed the buffy coat. A PRP injection 7 days after the injury provided significantly increased exercise performance and decreased the fibrotic area when compared with other PRP-treated groups. CONCLUSION: VEGF is only present in PRP's buffy coat, while Flt-1 is present in PPP. A PRP injection 7 days after an injury resulted in improved exercise performance. CLINICAL RELEVANCE: The presence of Flt-1 in PRP provides yet another explanation for results described in the literature after a PRP injection. This information is relevant for selecting the best PRP for each type of injury.

Asunto(s)

Contusiones/tratamiento farmacológico , Leucocitos/metabolismo , Músculos/lesiones , Plasma Rico en Plaquetas/química , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Contusiones/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Músculos/metabolismo , Músculos/fisiopatología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de Heridas/efectos de los fármacos

RESUMEN

Trypanosoma cruzi extracellular amastigotes (EAs) display unique mechanisms for cell invasion that are highly dependent on host actin filaments. Protein kinase D1 (PKD1) phosphorylates and modulates the activity of cortactin, a key regulator of actin dynamics. We evaluated the role of host cortactin and PKD1 in actin filament dynamics during HeLa cell invasion by EAs. Host cortactin, PKD1 and actin are recruited by EAs based on experiments in fixed and live cells by time lapse confocal microscopy. EAs trigger PKD1 and extracellular signal-regulated kinase 1/2 activation, but not Src family kinases, and selectively phosphorylate cortactin. Heat-killed EAs and non-infective epimastigotes both triggered distinct host responses and did not recruit the molecules studied herein. EA invasion was influenced by depletion or overexpression of host cortactin and PKD1, respectively, suggesting the involvement of both proteins in this event. Collectively, these results show new host cell mechanisms subverted during EA internalization into non-phagocytic cells.

Asunto(s)

Actinas/metabolismo , Cortactina/metabolismo , Endocitosis , Interacciones Huésped-Patógeno , Proteína Quinasa C/metabolismo , Transducción de Señal , Trypanosoma cruzi/fisiología , Células Epiteliales/parasitología , Células Epiteliales/fisiología , Células HeLa , Humanos , Microscopía Confocal , Análisis de Secuencia de ADN , Imagen de Lapso de Tiempo

RESUMEN

Glioblastoma (GBM) is an infiltrative tumor that is difficult to eradicate. Treating GBM with mesenchymal stem cells (MSCs) that have been modified with the HSV-Tk suicide gene has brought significant advances mainly because MSCs are chemoattracted to GBM and kill tumor cells via a bystander effect. To use this strategy, abundantly present adipose-tissue-derived mesenchymal stem cells (AT-MSCs) were evaluated for the treatment of GBM in mice. AT-MSCs were prepared using a mechanical protocol to avoid contamination with animal protein and transduced with HSV-Tk via a lentiviral vector. The U-87 glioblastoma cells cultured with AT-MSC-HSV-Tk died in the presence of 25 or 50 µM ganciclovir (GCV). U-87 glioblastoma cells injected into the brains of nude mice generated tumors larger than 3.5 mm2 after 4 weeks, but the injection of AT-MSC-HSV-Tk cells one week after the U-87 injection, combined with GCV treatment, drastically reduced tumors to smaller than 0.5 mm2. Immunohistochemical analysis of the tumors showed the presence of AT-MSC-HSV-Tk cells only within the tumor and its vicinity, but not in other areas of the brain, showing chemoattraction between them. The abundance of AT-MSCs and the easier to obtain them mechanically are strong advantages when compared to using MSCs from other tissues.

Asunto(s)

Tejido Adiposo/metabolismo , Glioblastoma/metabolismo , Células Madre Mesenquimatosas/enzimología , Simplexvirus/genética , Timidina Quinasa/biosíntesis , Transducción Genética , Proteínas Virales/biosíntesis , Tejido Adiposo/patología , Animales , Efecto Espectador/efectos de los fármacos , Línea Celular Tumoral , Femenino , Ganciclovir/farmacología , Glioblastoma/patología , Glioblastoma/terapia , Humanos , Células Madre Mesenquimatosas/patología , Ratones , Ratones Desnudos , Simplexvirus/enzimología , Timidina Quinasa/genética , Proteínas Virales/genética

RESUMEN

BACKGROUND: Mucopolysaccharidose type I (MPSI) is a lysosomal monogenic disease caused by mutations in the gene for α- L-iduronidase (IDUA). MPSI patients need a constant supply of IDUA to alleviate progression of the disease. IDUA gene transfer using integrative vectors might provide a definitive solution and support advancement to clinical trials, although studies have not yet been satisfactory. To achieve a stable IDUA gene expression in vivo, phiC31 was tested in the present study. METHODS: Several plasmid vectors were constructed and IDUA-/- mice were treated with cyclophosphamide and transfected with these vectors hydrodynamically via tail veins. IDUA expression was monitored over time. Treated and nontreated mice underwent an open-field test at age 8 months, and IDUA activity and glycosaminoglycan (GAG) content of tissues were evaluated. RESULTS: High levels of IDUA activity were detected initially (>1000 U/ml), although these levels decayed over time. The reinjection of vectors produced a similar profile of IDUA decay. Three out of six treated mice had IDUA activity in the livers, and also showed lower GAG content, reduced lysosomes and better locomotion. To investigate unsustained IDUA production, wild-type mice were submitted to the same gene therapy procedure, which generated a similar profile of IDUA decay. Anti-IDUA antibody was detected in the sera of these animals. In addition, we also found three methylated sites in the cytomegalovirus promoter region. CONCLUSIONS: phiC31-mediated gene therapy resulted in an important improvement in IDUA-/- mice, including locomotion, although the obstacles that need to be overcome to enable long-term gene therapy for MPSI are also noted.

Asunto(s)

Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos/genética , Iduronidasa/genética , Mucopolisacaridosis I/genética , Animales , Conducta Animal , Línea Celular , Metilación de ADN , Modelos Animales de Enfermedad , Activación Enzimática , Femenino , Expresión Génica , Orden Génico , Genes Reporteros , Vectores Genéticos/administración & dosificación , Células HEK293 , Recombinación Homóloga , Humanos , Iduronidasa/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Noqueados , Actividad Motora , Mucopolisacaridosis I/metabolismo , Mucopolisacaridosis I/terapia , Motivos de Nucleótidos , Regiones Promotoras Genéticas , Transfección