Cell-based experimental strategies for myelin repair in multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS), diagnosed at a mean age of 32 years. CNS glia are crucial players in the onset of MS, primarily involving astrocytes and microglia that can cause/allow massive oligodendroglial cells death, without immune cell infiltration. Current therapeutic approaches are aimed at modulating inflammatory reactions during relapsing episodes, but lack the ability to induce very significant repair mechanisms. In this review article, different experimental approaches based mainly on the application of different cell types as therapeutic strategies applied for the induction of myelin repair and/or the amelioration of the disease are discussed. Regarding this issue, different cell sources were applied in various experimental models of MS, with different results, both in significant improvements in remyelination and the reduction of neuroinflammation and glial activation, or in neuroprotection. All cell types tested have advantages and disadvantages, which makes it difficult to choose a better option for therapeutic application in MS. New strategies combining cell-based treatment with other applications would result in further improvements and would be good candidates for MS cell therapy and myelin repair.

Etv4 regulates nociception by controlling peptidergic sensory neuron development and peripheral tissue innervation.

The perception of noxious environmental stimuli by nociceptive sensory neurons is an essential mechanism for the prevention of tissue damage. Etv4 is a transcriptional factor expressed in most nociceptors in dorsal root ganglia (DRG) during the embryonic development. However, its physiological role remains unclear. Here, we show that Etv4 ablation results in defects in the development of the peripheral peptidergic projections in vivo, and in deficits in axonal elongation and growth cone morphology in cultured sensory neurons in response to NGF. From a mechanistic point of view, our findings reveal that NGF regulates Etv4-dependent gene expression of molecules involved in extracellular matrix (ECM) remodeling. Etv4-null mice were less sensitive to noxious heat stimuli and chemical pain, and this behavioral phenotype correlates with a significant reduction in the expression of the pain-transducing ion channel TRPV1 in mutant mice. Together, our data demonstrate that Etv4 is required for the correct innervation and function of peptidergic sensory neurons, regulating a transcriptional program that involves molecules associated with axonal growth and pain transduction.

Nicotine Neurotoxicity Involves Low Wnt1 Signaling in Spinal Locomotor Networks of the Postnatal Rodent Spinal Cord.

The postnatal rodent spinal cord in-vitro is a useful model to investigate early pathophysiological changes after injury. While low dose nicotine (1 µM) induces neuroprotection, how higher doses affect spinal networks is unknown. Using spinal preparations of postnatal wild-type Wistar rat and Wnt1Cre2:Rosa26Tom double-transgenic mouse, we studied the effect of nicotine (0.5-10 µM) on locomotor networks in-vitro. Nicotine 10 µM induced motoneuron depolarization, suppressed monosynaptic reflexes, and decreased fictive locomotion in rat spinal cord. Delayed fall in neuronal numbers (including motoneurons) of central and ventral regions emerged without loss of dorsal neurons. Conversely, nicotine (0.5-1 µM) preserved neurons throughout the spinal cord and strongly activated the Wnt1 signaling pathway. High-dose nicotine enhanced expression of S100 and GFAP in astrocytes indicating a stress response. Excitotoxicity induced by kainate was contrasted by nicotine (10 µM) in the dorsal area and persisted in central and ventral regions with no change in basal Wnt signaling. When combining nicotine with kainate, the activation of Wnt1 was reduced compared to kainate/sham. The present results suggest that high dose nicotine was neurotoxic to central and ventral spinal neurons as the neuroprotective role of Wnt signaling became attenuated. This also corroborates the risk of cigarette smoking for the foetus/newborn since tobacco contains nicotine.

Limitación del esfuerzo terapéutico al final de la vida: una mirada desde la medicina cubana contemporánea / Limitation of the Therapeutic Effort At the End of Life: A View from Contemporary Cuban Medicine

Los dilemas éticos respecto al cuidado al final de la vida son los más delicados e importantes en la práctica médica contemporánea; rebasan el campo de la bioética por sus implicaciones legales, sociales, culturales y religiosas. Independientemente de la universalidad de los postulados bioéticos, estos deben ser contextualizados atendiendo las características propias de cada nación. El panorama demográfico y epidemiológico de Cuba hace que la problemática tenga un comportamiento similar al mundo desarrollado, lo cual nos motivó a realizar un acercamiento teórico a la práctica de la limitación del esfuerzo terapéutico. Para ello partimos de los principios éticos de la nacionalidad cubana, el escenario social-demográfico, el marco legal vigente y la política de salud en el contexto de las transformaciones económicas que vive el país. Esa estrategia terapéutica no guarda relación alguna con la eutanasia y permite retirar del escenario clínico la distanasia. A la vez, constituye un punto de partida hacia los cuidados paliativos con impacto en lo económico y en el ordenamiento eficiente de los servicios de salud potencialmente beneficioso. Existe en la literatura nacional un movimiento favorable respecto a esa práctica médica; pero la principal debilidad en el país es la falta de un marco legal que condene la distanasia y reconozca la limitación del esfuerzo terapéutico como la medida que la evita(AU)

Ethical dilemmas regarding end-of-life care are the most delicate and important in contemporary medical practice; this care goes beyond the field of bioethics due to legal, social, cultural and religious implications. Regardless of the universality of bioethical postulates, they must be contextualized taking into account the characteristics of each nation. The demographic and epidemiological panorama of Cuba makes the problem behave similarly to the developed world, which motivated us to make a theoretical approach to the practice of limiting the therapeutic effort. For this purpose, we start from the ethical principles of Cuban nationality, the social-demographic scenario, the current legal framework and the health policy in the context of the economic transformations that the country is experiencing. This therapeutic strategy is not related to euthanasia and it allows dysthanasia to be removed from the clinical setting. At the same time, it constitutes a starting point towards palliative care with an economic impact and in the efficient organization of potentially beneficial health services. There is a favorable movement in the national literature regarding this medical practice; but the main weakness in the country is the lack of a legal framework that condemns dysthanasia and it recognizes the limitation of therapeutic effort as the measure that avoids it(AU)

Diverse cellular origins of adult blood vascular endothelial cells.

During embryonic stages, vascular endothelial cells (ECs) originate from the mesoderm, at specific extraembryonic and embryonic regions, through a process called vasculogenesis. In the adult, EC renewal/replacement mostly depend on local resident ECs or endothelial progenitor cells (EPCs). Nevertheless, contribution from circulating ECs/EPCs was also reported. In addition, cells lacking from EC/EPC markers with in vitro extended plasticity were shown to originate endothelial-like cells (ELCs). Most of these cells consist of mesenchymal stromal progenitors, which would eventually get mobilized from the bone marrow after injury. Based on that, current knowledge on different mouse and human bone marrow stromal cell (BM-SC) subpopulations, able to contribute with mesenchymal stromal/stem cells (MSCs), is herein reviewed. Such analyses underline an unexpected heterogeneity among sinusoidal LepR+ stromal/CAR cells. For instance, in a recent report a subgroup of LepR+ stromal/CAR progenitors, which express GLAST and is traced in Wnt1Cre;R26RTom mice, was found to contribute with ELCs in vivo. These GLAST â€‹+ â€‹Wnt1+ BM-SCs were shown to get mobilized to the peripheral blood and to contribute with liver regeneration. Other sources of ELCs, such as adipose, neural and dental pulp tissues, were also published. Finally, mechanisms likely involved in the enhanced cellular plasticity properties of bone marrow/adipose tissue stromal cells, able to originate ELCs, are assessed. In the future, strategies to analyze the in vivo expression profile of stromal cells, with MSC properties, in combination with screening of active genomic regions at the single cell-level, during early postnatal development and/or after injury, will likely help understanding properties of these ELC sources.

GABAergic Mechanisms Can Redress the Tilted Balance between Excitation and Inhibition in Damaged Spinal Networks.

Correct operation of neuronal networks depends on the interplay between synaptic excitation and inhibition processes leading to a dynamic state termed balanced network. In the spinal cord, balanced network activity is fundamental for the expression of locomotor patterns necessary for rhythmic activation of limb extensor and flexor muscles. After spinal cord lesion, paralysis ensues often followed by spasticity. These conditions imply that, below the damaged site, the state of balanced networks has been disrupted and that restoration might be attempted by modulating the excitability of sublesional spinal neurons. Because of the widespread expression of inhibitory GABAergic neurons in the spinal cord, their role in the early and late phases of spinal cord injury deserves full attention. Thus, an early surge in extracellular GABA might be involved in the onset of spinal shock while a relative deficit of GABAergic mechanisms may be a contributor to spasticity. We discuss the role of GABA A receptors at synaptic and extrasynaptic level to modulate network excitability and to offer a pharmacological target for symptom control. In particular, it is proposed that activation of GABA A receptors with synthetic GABA agonists may downregulate motoneuron hyperexcitability (due to enhanced persistent ionic currents) and, therefore, diminish spasticity. This approach might constitute a complementary strategy to regulate network excitability after injury so that reconstruction of damaged spinal networks with new materials or cell transplants might proceed more successfully.

Contribution of neural crest and GLAST+ Wnt1+ bone marrow pericytes with liver fibrogenesis and/or regeneration.

BACKGROUND AND AIMS: Liver fibrosis results from cycles of liver damage and scar formation. We herein aimed at analysing neural crest cells and/or bone marrow stromal cells contribution to the liver. METHODS: Two liver fibrosis and one hepatectomy model were applied on double-transgenic loxP-Cre mouse lines. RESULTS: Increased numbers of glia with more complex processes were found in fibrotic livers. During embryonic development, only few cells were traced in the liver and bone marrow, in a minor fraction of mice of different neural crest reporter strains analysed: therefore, a neural crest origin of such cells is doubtful. In the fibrotic liver, a significantly higher incidence of endothelial cells and hepatocyte-like cells expressing the reporter gene Tomato were found in Wnt1-Cre-Tom and GLAST-CreERT2-Tom mice. Consistently, during early fibrogenesis stromal Wnt1-traced cells, with progenitor (CFU-F) properties, get likely mobilized to peripheral blood. Circulating adult Wnt1-traced cells are stromal cells and lack from the expression of other bone marrow and endothelial progenitor cells markers. Furthermore, in a 70% hepatectomy model GLAST+ Wnt1-traced pericytes were found to be mobilized from the bone marrow and the incidence of GLAST-traced hepatocyte-like cells was increased. Finally, GLAST-traced hepatocyte like-cells were found to maintain the expression of stromal markers. CONCLUSIONS: Our data suggest a gliosis process during liver fibrogenesis. While neural crest cells probably do not contribute with other liver cell types than glia, GLAST+ Wnt1-traced bone marrow pericytes are likely a source of endothelial and hepatocyte-like cells after liver injury and do not contribute to scarring.

Medicina holística y complementaria. El problema no está en el qué, sino en el cómo / Holistic and complementary medicine. The problem is not in the what, but in the how

En su revista se publicó una comunicación titulada: Contra la pseudociencia y las medicinas con apellidos, en la que el profesor Dr. Rojas Ochoa comparte con los lectores fragmentos de una crítica a la medicina alternativa que divulgó el diario español El País. El artículo, noticiado en ese rotativo, sustenta su cuestionamiento en los resultados del estudio: Use of Alternative Medicine for Cancer and Its Impact on Survival, del Dr. Skyler Johnson y colaboradores.1) Consideramos la investigación del Dr. Johnson, que se publicó originalmente en Journal of the National Cancer Institute, de excelente diseño y resultados irrefutables. Lo cuestionable es la valoración que hace el Dr. Esteve Fernández de los mismos y el fin que persigue con ello, que parece compartir el profesor Dr. Rojas Ochoa.2 Concluir que las terapias alternativas son inefectivas para el tratamiento de las neoplasias de mama, colon, pulmón y próstata es acertado. Calificarlas como inútiles para todo propósito porque no sean efectivas en pacientes oncológicos, es generalista y absolutista. El debate contra la medicina complementaria también es espacio...(AU)

Cistoadenoma mucinoso del apéndice: un caso con presentación inusual / Appendiceal mucinous cystoadenom: inusual presentation case

RESUMEN Fundamento: los tumores mucinosos representan alrededor del 8 % de las neoplasias apendiculares y originan dilatación quística del apéndice debido a la acumulación de material gelatinoso. El cistoadenoma mucinoso del apéndice es una rara enfermedad, que cursa de manera asintomática y se diagnostica de manera incidental mediante estudios imagenológicos o intra operatorio y representa de un 0,2 % a 0,6 % de las apendicectomías. Objetivo: presentar el caso inusual de una joven operada por apendicitis aguda que tenía además un cistoadenoma mucinoso del apéndice. Caso clínico: paciente nuligesta, de 19 años de edad con antecedentes de pérdida de peso en un período de seis meses y cuadros recurrentes de dolor en epigastrio que aliviaban con analgésicos, acudió por dolor en el cuadrante inferior derecho del abdomen de 48 horas de evolución, acompañado de náuseas y pérdida del apetito. Al examen físico se constató taquicardia y dolor abdominal a la palpación en la fosa iliaca derecha, con maniobra de Blumberg positiva, el tacto vaginal y rectal resultaron dolorosos. En el acto quirúrgico se constató apéndice cecal engrosado en el tercio proximal, turgente, con una tumoración dura en tercio distal y se realizó apendicectomía sin complicaciones. La evolución fue favorable y fue dada de alta. Conclusiones: a pesar de lo raro de esta afección, el cirujano debe conocer que en un cuadro apendicular puede estar presente este tipo de tumor aún en pacientes jóvenes a fin de tomar la conducta quirúrgica adecuada y evitar complicaciones al enfermo.

ABSTRACT Background: mucinous tumors represent about 8% of appendix neoplasms and cause cystic dilatation of the appendix due to the accumulation of gelatinous material. Mucinous cystadenoma of the appendix is a rare disease, which occurs asymptomatically and is diagnosed incidentally through imaging studies or intra-operatively and accounts for 0.2% to 0.6% of appendectomies. Objective: to present the unusual case of a 19-year-old girl operated on for acute appendicitis who also had a mucinous cystadenoma of the appendix. Clinical case: patient without previous pregnancy, with a history of weight loss in a period of 6 months and recurrent pain in the epigastrium that relieved with analgesics, came for pain in the lower right quadrant of the abdomen of 48 hours of evolution, accompanied by nausea and loss of appetite. Physical examination revealed tachycardia and abdominal pain on palpation in the right iliac fossa, with a positive Blumberg maneuver; vaginal and rectal examinations were painful. In the surgical act, a cecal appendix was found thickened in the proximal third, turgid, with a hard mass in the distal third and an appendectomy was performed without complications. The evolution was favorable and the patient was discharged. Conclusions: despite the rareness of this condition, the surgeon must know that this type of tumor may be present in an appendicular frame even in young patients in order to take the appropriate surgical behavior and avoid complications to the patient.

Cistoadenoma mucinoso del apéndice: un caso con presentación inusual / Appendiceal mucinous cystoadenom: inusual presentation case

Fundamento: los tumores mucinosos representan alrededor del 8 porciento de las neoplasias apendiculares y originan dilatación quística del apéndice debido a la acumulación de material gelatinoso. El cistoadenoma mucinoso del apéndice es una rara enfermedad, que cursa de manera asintomática y se diagnostica de manera incidental mediante estudios imagenológicos o intra operatorio y representa de un 0,2 porciento a 0,6 porciento de las apendicectomías. Objetivo: presentar el caso inusual de una joven operada por apendicitis aguda que tenía además un cistoadenoma mucinoso del apéndice. Caso clínico: paciente nuligesta, de 19 años de edad con antecedentes de pérdida de peso en un período de seis meses y cuadros recurrentes de dolor en epigastrio que aliviaban con analgésicos, acudió por dolor en el cuadrante inferior derecho del abdomen de 48 horas de evolución, acompañado de náuseas y pérdida del apetito. Al examen físico se constató taquicardia y dolor abdominal a la palpación en la fosa iliaca derecha, con maniobra de Blumberg positiva, el tacto vaginal y rectal resultaron dolorosos. En el acto quirúrgico se constató apéndice cecal engrosado en el tercio proximal, turgente, con una tumoración dura en tercio distal y se realizó apendicectomía sin complicaciones. La evolución fue favorable y fue dada de alta. Conclusiones: a pesar de lo raro de esta afección, el cirujano debe conocer que en un cuadro apendicular puede estar presente este tipo de tumor aún en pacientes jóvenes a fin de tomar la conducta quirúrgica adecuada y evitar complicaciones al enfermo(AU)

Background: mucinous tumors represent about 8 percent of appendix neoplasms and cause cystic dilatation of the appendix due to the accumulation of gelatinous material. Mucinous cystadenoma of the appendix is a rare disease, which occurs asymptomatically and is diagnosed incidentally through imaging studies or intra-operatively and accounts for 0.2percent to 0.6percent of appendectomies. Objective: to present the unusual case of a 19-year-old girl operated on for acute appendicitis who also had a mucinous cystadenoma of the appendix.Clinical case: patient without previous pregnancy, with a history of weight loss in a period of 6 months and recurrent pain in the epigastrium that relieved with analgesics, came for pain in the lower right quadrant of the abdomen of 48 hours of evolution, accompanied by nausea and loss of appetite. Physical examination revealed tachycardia and abdominal pain on palpation in the right iliac fossa, with a positive Blumberg maneuver; vaginal and rectal examinations were painful. In the surgical act, a cecal appendix was found thickened in the proximal third, turgid, with a hard mass in the distal third and an appendectomy was performed without complications. The evolution was favorable and the patient was discharged. Conclusions: despite the rareness of this condition, the surgeon must know that this type of tumor may be present in an appendicular frame even in young patients in order to take the appropriate surgical behavior and avoid complications to the patient(AU)

Schwann cell precursors in health and disease.

Schwann cell precursors (SCPs) are frequently regarded as neural crest-derived cells (NCDCs) found in contact with axons during nerve formation. Nevertheless, cells with SCPs properties can be found up to the adulthood. They are well characterized with regard to both gene expression profile and cellular behavior -for instance, proliferation, migratory capabilities and survival requirements-. They differ in origin regarding their anatomic location: even though most of them are derived from migratory NCCs, there is also contribution of the boundary cap neural crest cells (bNCCs) to the skin and other tissues. Many functions are known for SCPs in normal development, including nerve fasciculation and target innervation, arterial branching patterning and differentiation, and other morphogenetic processes. In addition, SCPs are now known to be a source of many neural (glia, endoneural fibroblasts, melanocytes, visceral neurons, and chromaffin cells) and non-neural-like (mesenchymal stromal cells, able e.g., to generate dentine-producing odontoblasts) cell types. Until now no reports of endoderm-like derivatives were reported so far. Interestingly, in the Schwann cell lineage only early SCPs are likely able to differentiate into melanocytes and bone marrow mesenchymal stromal cells. We have also herein discussed the literature regarding their role in repair as well as in disease mechanisms, such as in diverse cancers. Moreover, many caveats in our knowledge of SCPs biology are highlighted all through this article. Future research should expand more into the relevance of SCPs in pathologies and in other regenerative mechanisms which might bring new unexpected clinically-relevant knowledge.

[Mesenchymal stem cells and regenerative medicine in liver cirrhosis]. / Células madre mesenquimales y medicina regenerativa en la cirrosis hepática.

Mesenchymal stem cells (MSCs) are multipotent cells with self-renewal capacity which are present in diverse tissues. Recently, significant progresses have been made in the field of MSCs because of its therapeutic potential in regenerative medicine. MSCs selectively migrate toward sites of damage and remodeling, and have the ability to evade the immune system and to promote tissue repair through the production of a number of growth factors and cytokines. Many pre-clinical and clinical studies have been carried out to study its therapeutic effect in liver cirrhosis with promising results. In addition, experimental studies showed that this therapeutic effect can be improved by engineering MSCs to produce therapeutic genes. In this work, the role of MSCs in regenerative medicine and its clinical and pre-clinical applications are reviewed, with an emphasis on its potential as vehicles for therapeutic genes.

Células madre mesenquimales y medicina regenerativa en la cirrosis hepática / Mesenchymal stem cells and regenerative medicine in liver cirrhosis

Las células madre mesenquimales (MSCs) son células multipotentes con capacidad de auto-renovación, presentes en diferentes tejidos del organismo. En los últimos años se ha avanzado significativamente en su estudio debido a su potencial terapéutico en medicina regenerativa. Las MSCs se caracterizan por migrar selectivamente a sitios de injuria y remodelación y por su capacidad para evadir al sistema inmunitario y colaborar en la reparación tisular mediante la secreción de factores tróficos. Numerosos estudios pre-clínicos y clínicos analizan su potencial efecto terapéutico en la cirrosis hepática con resultados alentadores. Diversas evidencias experimentales sugieren que este efecto podría ser superior si se utilizaran MSCs como vehículo de genes terapéuticos. En este trabajo se revisa el rol de las MSCs en medicina regenerativa y su empleo en estudios clínicos y pre-clínicos, con énfasis en su potencial como vehículo de genes terapéuticos.

Mesenchymal stem cells (MSCs) are multipotent cells with self-renewal capacity which are present in diverse tissues. Recently, significant progresses have been made in the field of MSCs because of its therapeutic potential in regenerative medicine. MSCs selectively migrate toward sites of damage and remodeling, and have the ability to evade the immune system and to promote tissue repair through the production of a number of growth factors and cytokines. Many pre-clinical and clinical studies have been carried out to study its therapeutic effect in liver cirrhosis with promising results. In addition, experimental studies showed that this therapeutic effect can be improved by engineering MSCs to produce therapeutic genes. In this work, the role of MSCs in regenerative medicine and its clinical and pre-clinical applications are reviewed, with an emphasis on its potential as vehicles for therapeutic genes.

Uncovering the In Vivo Source of Adult Neural Crest Stem Cells.

Some late embryonic and adult postmigratory neural crest-derived cells (NCDCs) from diverse tissues were shown to grow as multipotent neurospheres. Neural crest stem cells (NCSCs) contained in these spheres were found to give rise not only to neuroectodermal derivatives but also to some of the progeny of the other embryonic germ layers. In this review, evidences regarding the in vivo properties of NCDCs contributing to NCSCs are discussed. Even though in many cases the final proof for the phenotype identity of in vivo cells generating NCSCs is lacking, some evidences suggest that such postmigratory NCDCs would differ from neural crest cells. The streamline of this review follows a historical perspective that helps understanding the advancements in knowledge of this field of research and highlighting its importance, in an appropriate context. Finally, the potential for regenerative medicine purpose of NCDCs and more specifically of tissues that can be a source of peripheral glia progenitors in the adult is underlined.

Involvement of hepatic macrophages in the antifibrotic effect of IGF-I-overexpressing mesenchymal stromal cells.

BACKGROUND: Cirrhosis is a major health problem worldwide and new therapies are needed. Hepatic macrophages (hMø) have a pivotal role in liver fibrosis, being able to act in both its promotion and its resolution. It is well-known that mesenchymal stromal cells (MSCs) can modulate the immune/inflammatory cells. However, the effects of MSCs over hMø in the context of liver fibrosis remain unclear. We previously described evidence of the antifibrotic effects of in vivo applying MSCs, which were enhanced by forced overexpression of insulin-like growth factor 1 (AdIGF-I-MSCs). The aim of this work was to analyze the effect of MSCs on hMø behavior in the context of liver fibrosis resolution. METHODS: Fibrosis was induced in BALB/c mice by chronic administration of thioacetamide (8 weeks). In vivo gene expression analyses, in vitro experiments using hMø isolated from the nonparenchymal liver cells fraction, and in vivo experiments with depletion of Mø were performed. RESULTS: One day after treatment, hMø from fibrotic livers of MSCs-treated animals showed reduced pro-inflammatory and pro-fibrogenic gene expression profiles. These shifts were more pronounced in AdIGF-I-MSCs condition. This group showed a significant upregulation in the expression of arginase-1 and a higher downregulation of iNOS expression thus suggesting decreased levels of oxidative stress. An upregulation in IGF-I and HGF expression was observed in hMø from AdIGF-I-MSCs-treated mice suggesting a restorative phenotype in these cells. Factors secreted by hMø, preconditioned with MSCs supernatant, caused a reduction in the expression levels of hepatic stellate cells pro-fibrogenic and activation markers. Interestingly, hMø depletion abrogated the therapeutic effect achieved with AdIGF-I-MSCs therapy. Expression profile analyses for cell cycle markers were performed on fibrotic livers after treatment with AdIGF-I-MSCs and showed a significant regulation in genes related to DNA synthesis and repair quality control, cell cycle progression, and DNA damage/cellular stress compatible with early induction of pro-regenerative and hepatoprotective mechanisms. Moreover, depletion of hMø abrogated such effects on the expression of the most highly regulated genes. CONCLUSIONS: Our results indicate that AdIGF-I-MSCs are able to induce a pro-fibrotic to resolutive phenotype shift on hepatic macrophages, which is a key early event driving liver fibrosis amelioration.

Tumor Microenvironment Remodeling by 4-Methylumbelliferone Boosts the Antitumor Effect of Combined Immunotherapy in Murine Colorectal Carcinoma.

We have previously demonstrated that a low dose of cyclophosphamide (Cy) combined with gene therapy of interleukin-12 (AdIL-12) has a synergistic, although limited, antitumoral effect in mice with colorectal carcinoma. The main mechanism involved in the efficacy of Cy+AdIL-12 was the induction of a specific immune response mediated by cytotoxic T lymphocytes. Our current aims were to evaluate the effects of 4-methylumbelliferone (4Mu), a selective inhibitor of hyaluronan (HA) synthesis, on tumor microenvironment (TME) and to investigate how 4Mu affects the therapeutic efficacy of Cy+AdIL-12. The results showed that 4Mu significantly reduced the amount of tumoral HA leading to a significant decrease in tumor interstitial pressure (TIP). As a consequence, tumor perfusion was improved allowing an increased adenoviral transgene expression. In addition, treatment with 4Mu boosted the number of cytotoxic T lymphocytes that reach the tumor after adoptive transfer resulting in a potent inhibition of tumor growth. Importantly, we observed complete tumor regression in 75% of mice when 4Mu was administrated in combination with Cy+AdIL-12. The triple combination 4Mu+Cy+AdIL-12 also induced a shift toward antiangiogenic factors production in tumor milieu. Our results showed that TME remodeling is an interesting strategy to increase the efficacy of anticancer immunotherapies based on gene and/or cell therapy.

Mesenchymal Stem/Stromal Cells in Liver Fibrosis: Recent Findings, Old/New Caveats and Future Perspectives.

Mesenchymal stem/stromal cells (MSCs) are progenitors which share plastic-adherence capacity and cell surface markers but have different properties according to their cell and tissue sources and to culture conditions applied. Many recent publications suggest that MSCs can differentiate into hepatic-like cells, which can be a consequence of either a positive selection of rare in vivo pluripotent cells or of the original plasticity of some cells contributing to MSC cultures. A possible role of MSCs in hereditary transmission of obesity and/or diabetes as well as properties of MSCs regarding immunomodulation, cell fusion and exosome release capacities are discussed according to recent literature. Limitations in methods used to track MSCs in vivo especially in the context of liver cirrhosis are addressed as well as strategies explored to enhance their migratory, survival and proliferation properties, which are known to be relevant for their future clinical use. Current knowledge regarding mechanisms involved in liver cirrhosis amelioration mediated by naïve and genetically modified MSCs as well as the effects of applying preconditioning and combined strategies to improve their therapeutic effects are evaluated. Finally, first reports of GMP guidelines and biosafety issues in MSCs applications are discussed.

Mesenchymal stromal cells engineered to produce IGF-I by recombinant adenovirus ameliorate liver fibrosis in mice.

Liver cirrhosis involves chronic wound healing and fibrotic processes. Mesenchymal stromal cells (MSCs) are multipotent adult progenitor cells that are used as vehicles of therapeutic genes. Insulin growth factor like-I (IGF-I) was shown to counteract liver fibrosis. We aimed at analyzing the effect of applying IGF-I overexpressing mouse bone marrow-derived MSCs on hepatic fibrosis. Fibrosis was induced by chronic thioacetamide application or bile duct ligation. MSCs engineered to produce green fluorescent protein (GFP) (AdGFP-MSCs) or IGF-I (AdIGF-I-MSCs) were applied systemically, and changes in collagen deposition and in the expression of key pro-fibrogenic and pro-regenerative genes/proteins were assessed. In addition, immunogenicity of transduced cells was analyzed. Liver fibrosis was further ameliorated after a single-dose application of AdIGF-I-MSCs when compared with AdGFP-MSCs and/or recombinant IGF-I treatments. Interestingly, an early and transitory upregulation in IGF-I and hepatocyte growth factor (HGF) mRNA expression was found in the liver of MSC-treated animals, which was more pronounced in AdIGF-I-MSCs condition. A reduction in hepatic stellate cell activation status was found after incubation with MSCs conditioned media. In addition, the AdIGF-I-MSCs cell-free supernatant induced the expression of IGF-I and HGF in primary cultured hepatocytes. From day 1 after transplantation, the proliferation marker proliferating cell nuclear antigen was upregulated in the liver of AdIGF-I-MSCs group, mainly in hepatocytes. MSCs were in vivo traced till day 14 after injection. In addition, multiple doses of Ad-IGF-I-MSCs likely suppressed antiviral immune response and it further reduced collagen deposition. Our results uncover early events that are likely involved in the anti-fibrogenic effect of genetically modified MSCs and overall would support the use of AdIGF-I-MSCs in treatment of liver fibrosis.

Combined therapy for gastrointestinal carcinomas: exploiting synergies between gene therapy and classical chemo-radiotherapy.

Surgical resection is the only curative option for patients with gastrointestinal carcinomas. Unfortunately, the majority of patients are diagnosed in advanced stages when surgery is not possible. Moreover, the incidence and mortality for certain type of tumors such as hepatocellular carcinoma or pancreatic cancer are steadily increasing worldwide. In spite of the advances in the development of molecular targeted therapies for cancer, the impact on patient survival has been rather limited. It is unlikely that individual agents would be ultimately successful as monotherapy. There is a growing area of research focused on the combination of classical chemotherapy (e.g. cyclophosphamide, gemcitabine, paclitaxel and doxorubicin) with radiotherapy and/or gene therapy strategies. Combined approaches seem to be required due to multiple resistance mechanisms that tumors utilize to limit the activity of chemotherapeutic agents (e.g. the occurrence of multidrug resistance or epigenetic alterations), evade immune responses (e.g. induction of regulatory T cells or myeloid-derived suppressor cells) and to generate resistance against anti-angiogenesis or to radiotherapy by, for example, the induction of hypoxia-inducible factor 1. In addition, new studies suggest that combination of low dose of conventional chemotherapy and gene therapy could allow the development of synergic mechanisms able to achieve significant therapeutic effects against diverse tumors. Although cancer gene therapy is not yet available in clinical practice, advances being recently made look promising, especially when it was applied in combination with standard chemo- or radiotherapy protocols.

The pan-caspase inhibitor Emricasan (IDN-6556) decreases liver injury and fibrosis in a murine model of non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Hepatocyte apoptosis, the hallmark of non-alcoholic steatohepatitis (NASH) contributes to liver injury and fibrosis. Although, both the intrinsic and extrinsic apoptotic pathways are involved in the pathogenesis of NASH, the final common step of apoptosis is executed by a family of cysteine-proteases termed caspases. Thus, our aim was to ascertain if administration of Emricasan, a pan-caspase inhibitor, ameliorates liver injury and fibrosis in a murine model of NASH. METHODS: C57/BL6J-mice were fed regular chow or high fat diet (HFD) for 20 weeks. All mice were treated with vehicle or Emricasan. RESULTS: Mice fed a HFD diet demonstrate a five-fold increase in hepatocyte apoptosis by the TUNEL assay and a 1.5-fold and 1.3-fold increase in caspase-3 and-8 activities respectively; this increase in apoptosis was substantially attenuated in mice fed a HFD treated with Emricasan (HFD-Em). Likewise, liver injury and inflammation were reduced in mice fed HFD-Em as compare to HFD by measuring serum aspartate aminotransferase and alanine aminotransferase levels, NAS histological score and IL 1-ß, TNF-α, monocyte chemoattractant protein (MCP-1) and C-X-C chemokine ligand-2 (CXCL2) quantitative reverse-transcription polymerase chain reaction (qPCR). These differences could not be attributed to differences in hepatic steatosis as liver triglycerides content were similar in both HFD groups. Hepatic fibrosis was reduced by Emricasan in HFD animals by decreasing αSMA (a marker for hepatic stellate cell activation), fibrosis score, Sirius red staining, hydroxyproline liver content and profibrogenic cytokines by qPCR. CONCLUSION: In conclusion, these data demonstrate that in a murine model of NASH, liver injury and fibrosis are suppressed by inhibiting hepatocytes apoptosis and suggests that Emricasan may be an attractive antifibrotic therapy in NASH.