Eficacia y seguridad de los agentes estimulantes de la eritropoyesis para los síndromes mielodisplásicos: Revisión sistemática de la literatura

Los síndromes mielodisplásicos son un grupo heterogéneo de desórdenes clonales de las células madre hematopoyéticas caracterizadas por displasia y producción inefectiva de células sanguíneas. Los agentes estimulantes de la eritropoyesis (AEE) constituyen una alternativa terapéutica para un grupo de pacientes con síndromes mielodisplásicos. Se realizó una revisión sistemática de la literatura de estudios aleatorizados controlados, que evaluaron la eficacia y seguridad de los AEE en adultos con diagnóstico de síndrome mielodisplásico hasta febrero de 2014. A partir de la búsqueda se encontraron 1071 referencias, se obtuvieron 12 referencias, correspondiente a 9 estudios que cumplieron criterios de selección. Todos evaluaron algún AEE pero el grupo comparador difirió entre ellos; De los estudios seleccionados, ninguno evaluó el desenlace de supervivencia según brazos de tratamiento. 3 de los estudios evaluaron el desenlace de calidad de vida en grupos comparativos diferentes, reportando mejorías no estadísticamente significativas. 7 de los estudios evaluaron la respuesta hematológica reportando resultados divergentes según diversas definiciones de la variable de interés. Los desenlaces de seguridad fueron reportaron en 3 de los estudios, ocurriendo en baja proporción y con incidencia similar entre los grupos comparadores y el tratamiento con AEE. Las evaluaciones de riesgo de sesgo consideraron un estudio con bajo riesgo de sesgo, 4 estudios con riesgo incierto y 4 estudios con riesgo alto de sesgo. Los estudios evaluados fueron considerados en su mayoría con riesgo de sesgo incierto o alto. Se sugiere evaluar dichos desenlaces de interés de manera estandarizada en investigaciones futuras en el tema.

Frecuencia de neoplasia escamosa de la superficie ocular coexistente con pterigio

Introducción: En la literatura, han aparecido reportes de neoplasia escamosa de superficie ocular (NESO) asociado con pterigio en un mismo paciente. Sin embargo, Colombia no cuenta con una estadística para ninguna de estas patologías. Objetivos: Determinar la frecuencia de NESO en pterigios resecados, en la Fundación Oftalmológica Nacional. Identificar factores de riesgo y características clínicas que predispongan a su aparición. Metodología: Estudio descriptivo de corte transversal. Se realizó una clasificación prequirúrgica y estudio histopatológico de los pterigios resecados en 93 pacientes, para confirmar su coexistencia con NESO. Se efectuó un análisis de frecuencias para datos demográficos y factores de riesgo asociados su aparición. Resultados: La frecuencia de NESO asociado a pterigio fue 7,07%. De estos, 28,5% identificados como sospechosos en la evaluación preoperatoria. La mayoría se presentaron en mujeres (71,4%), las ocupaciones con mayor frecuencia: labores domésticas (42,8%) y el comercio (28.5%). La exposición a derivados del petróleo y tabaquismo fue del 14,28%. No se presentaron casos asociados a infección por VIH. No hubo diferencias estadísticamente significativas sobre la presencia de NESO al comparar los casos en edad (p=0,8), procedencia (p=0,6) tabaquismo (p=0,4), leucoplaquia (p=1,0), queratinización (p=0,137), o vasos amputados (p=0,137). Conclusiones: De los pacientes con diagnóstico histopatológico de NESO, un porcentaje mínimo es sospechado clínicamente. Además se encontró este diagnóstico en pacientes más jóvenes que lo reportado en la literatura. Se recomienda realizar estudios con mayor número de pacientes para una mejor identificación de factores de riesgo. Palabras clave:

Differentiation status of limbal epithelial cells cultured on intact and denuded amniotic membrane before and after air-lifting

We have investigated differences in bovine limbal epithelial cell differentiation when expanded upon intact (amniotic epithelial cells and basement membrane remaining) and denuded human amniotic membrane, a commonly used substrate in ophthalmic surgery for corneal stem cell transplantation. Ex vivo expansion of the epithelial cells, in supplemented media, continued for 2 weeks followed by 1 week under ‘air-lifting’ conditions. Before and after air-lifting the differentiated (K3/K12 positive) and undifferentiated (K14 positive) cells were quantified by immunohistochemistry, Western blotting and quantitative PCR. Limbal epithelial cells expanded upon amniotic membrane formed 4-6 stratified layers, both on intact and denuded amniotic membrane. On denuded amniotic membrane the proportion of differentiated cells remained unaltered following airlifting. Within cells grown on intact amniotic membrane, however, the number of differentiated cells increased significantly following air-lifting. These results have important implications for both basic and clinical research. Firstly, they show that bovine limbal epithelia can be used as an alternative source of cells for basic research investigating ex vivo limbal stem cells expansion. Secondly, these findings serve as a warning to clinicians that the affect of amniotic membrane on transplantable cells is not fully understood; the use of intact or denuded amniotic membrane can produce different results in terms of the amount of differentiation, once cells are exposed to the air.

Cannabinoids stimulate fibroblastic colony formation by bone marrow cells indirectly via CB2 receptors

Recently, the cannabinoid receptors CB1 and CB2 were shown to modulate bone formation and resorption in vivo, although little is known of the mechanisms underlying this. The effects of cannabinoids on mesenchymal stem cell (MSC) recruitment in whole bone marrow were investigated using either the fibroblastic colony-forming unit (CFU-f) assay or high-density cultures of whole bone marrow. Levels of the CB1 and CB2 receptors were assessed by flow cytometry. Treatment of CFU-f cultures with the endocannabinoid 2-arachidonylglycerol (2-AG) dose-dependently increased fibroblastic and differentiated colony formation along with colony size. The nonspecific agonists CP 55,940 and WIN 55,212 both increased colony numbers, as did the CB2 agonists BML190 and JWH015. The CB1-specific agonist ACEA had no effect, whereas the CB2 antagonist AM630 blocked the effect of the natural cannabinoid tetrahydrocannabivarin, confirming mediation via the CB2 receptor. Treatment of primary bone marrow cultures with 2-AG stimulated proliferation and collagen accumulation, whereas treatment of subcultures of MSC had no effect, suggesting that the target cell is not the MSC but an accessory cell present in bone marrow. Subcultures of MSCs were negative for CB1 and CB2 receptors as shown by flow cytometry, whereas whole bone marrow contained a small population of cells positive for both receptors. These data suggest that cannabinoids may stimulate the recruitment of MSCs from the bone marrow indirectly via an accessory cell and mediated via the CB2 receptor. This recruitment may be one mechanism responsible for the increased bone formation seen after cannabinoid treatment in vivo.

Rat neurosphere cells protect axotomized rat retinal ganglion cells and facilitate their regeneration

We investigated the ability of a population of rat neural stem and precursor cells derived from rat embryonic spinal cord to protect injured neurons in the rat central nervous system (CNS). The neonatal rat optic pathway was used as a model of CNS injury, whereby retinal ganglion cells (RGCs) were axotomized by lesion of the lateral geniculate nucleus one day after birth. Neural stem and precursor cells derived from expanded neurospheres (NS) were transplanted into the lesion site at the time of injury. Application of Fast Blue tracer dye to the lesion site demonstrated that significant numbers of RGCs survived at 4 and 8 weeks in animals that received a transplant, with an average of 28% survival, though in some individual cases survival was greater than 50%. No RGCs survived in animals that received a lesion alone. Furthermore, labeled RGCs were also observed when Fast Blue was applied to the superior colliculus (SC) at 4 weeks, suggesting that neurosphere cells also facilitated RGC to regenerate to their normal target. Transplanted cells did not migrate or express neural markers after transplantation, and secreted several neurotrophic factors in vitro. We conclude that NS cells can protect injured CNS neurons and promote their regeneration. These effects are not attributable to cell replacement, and may be mediated via secretion of neurotrophic factors. Thus, neuroprotection by stem cell populations may be a more viable approach for treatment of CNS disorders than cell replacement therapy.

Limitations of the MRL mouse as a model for cardiac regeneration

Objective Myocardial repair following injury in mammals is restricted such that damaged areas are replaced by scar tissue, impairing cardiac function. MRL mice exhibit exceptional regenerative healing in an ear punch wound model. Some myocardial repair with restoration of heart function has also been reported following cryoinjury. Increased cardiomyocyte proliferation and a foetal liver stem cell population were implicated. We investigated molecular mechanisms facilitating myocardial repair in MRL mice to identify potential therapeutic targets in non-regenerative species. Methods Expressions of specific cell-cycle regulators that might account for regeneration (CDKs 1, 2, 4 and 6; cyclins A, E, D1 and B1; p21, p27 and E2F5) were compared by immunoblotting in MRL and control C57BL/6 ventricles during development. Flow cytometry was used to investigate stem cell populations in livers from foetal mice, and infarct sizes were compared in coronary artery-ligated and sham-treated MRL and C57BL/6 adult mice. Key findings No differences in the expressions of cell cycle regulators were observed between the two strains. Expressions of CD34+Sca1+ckit-, CD34+Sca1+ckit+ and CD34+Sca1-ckit+ increased in livers from C57BL/6 vs MRL mice. No differences were observed in infarct sizes, levels of fibrosis, Ki67 staining or cardiac function between MRL and C57BL/6 mice. Conclusions No intrinsic differences were observed in cell cycle control molecules or stem cell populations between MRL and control C57BL mouse hearts. Pathophysiologically relevant ischaemic injury is not repaired more efficiently in MRL myocardium, questioning the use of the MRL mouse as a reliable model for cardiac regeneration in response to pathophysiologically relevant forms of injury.

Formation of atypical podosomes in extravillous trophoblasts regulates extracellular matrix degradation

Throughout pregnancy the cytotrophoblast, the stem cell of the placenta, gives rise to the differentiated forms of trophoblasts. The two main cell lineages are the syncytiotrophoblast and the invading extravillous trophoblast. A successful pregnancy requires extravillous trophoblasts to migrate and invade through the decidua and then remodel the maternal spiral arteries. Many invasive cells use specialised cellular structures called invadopodia or podosomes in order to degrade extracellular matrix. Despite being highly invasive cells, the presence of invadapodia or podosomes has not previously been investigated in trophoblasts. In this study these structures have been identified and characterised in extravillous trophoblasts. The role of specialised invasive structures in trophoblasts in the degradation of the extracellular matrix was compared with well characterised podosomes and invadopodia in other invasive cells and the trophoblast specific structures were characterised by using a sensitive matrix degradation assay which enabled visualisation of the structures and their dynamics. We show trophoblasts form actin rich protrusive structures which have the ability to degrade the extracellular matrix during invasion. The degradation ability and dynamics of the structures closely resemble podosomes, but have unique characteristics that have not previously been described in other cell types. The composition of these structures does not conform to the classic podosome structure, with no distinct ring of plaque proteins such as paxillin or vinculin. In addition, trophoblast podosomes protrude more deeply into the extracellular matrix than established podosomes, resembling invadopodia in this regard. We also show several significant pathways such as Src kinase, MAPK kinase and PKC along with MMP-2 and 9 as key regulators of extracellular matrix degradation activity in trophoblasts, while podosome activity was regulated by the rigidity of the extracellular matrix.

The mechanical properties of amniotic membrane influence its effect as a biomaterial for ocular surface repair

The human amniotic membrane (AM) is a tissue of fetal origin and has proven to be clinically useful as a biomaterial in the management of various ocular surface disorders including corneal stem cell transplantation. However, its success rate displays a degree of clinical unpredictability. We suggest that the measured variability inAMstiffness offers an explanation for the poor clinical reproducibility when it is used as a substrate for stem cell expansion and transplantation. Corneal epithelial stem cells were expanded upon AM samples possessing different mechanical stiffness. To investigate further the importance of biological substrate stiffness on cell phenotype we replaced AM with type I collagen gels of known stiffness. Substrate stiffness was measured using shear rheometry and surface topography was characterized using scanning electron microscopy and atomic force microscopy. The differentiation status of epithelial cells was examined using RT-PCR, immunohistochemistry and Western blotting. The level of corneal stem cell differentiation was increased in cells expanded upon AM with a high dynamic elastic shear modulus and cell expansion on type I collagen gels confirmed that the level of corneal epithelial stem cell differentiation was related to the substrate’s mechanical properties. In this paper we provide evidence to show that the preparatory method of AM for clinical use can affect its mechanical properties and that these measured differences can influence the level of differentiation within expanded corneal epithelial stem cells.

A novel function of the proneural factor Ascl1 in progenitor proliferation identified by genome-wide characterization of its targets

Proneural genes such as Ascl1 are known to promote cell cycle exit and neuronal differentiation when expressed in neural progenitor cells. The mechanisms by which proneural genes activate neurogenesis--and, in particular, the genes that they regulate--however, are mostly unknown. We performed a genome-wide characterization of the transcriptional targets of Ascl1 in the embryonic brain and in neural stem cell cultures by location analysis and expression profiling of embryos overexpressing or mutant for Ascl1. The wide range of molecular and cellular functions represented among these targets suggests that Ascl1 directly controls the specification of neural progenitors as well as the later steps of neuronal differentiation and neurite outgrowth. Surprisingly, Ascl1 also regulates the expression of a large number of genes involved in cell cycle progression, including canonical cell cycle regulators and oncogenic transcription factors. Mutational analysis in the embryonic brain and manipulation of Ascl1 activity in neural stem cell cultures revealed that Ascl1 is indeed required for normal proliferation of neural progenitors. This study identified a novel and unexpected activity of the proneural gene Ascl1, and revealed a direct molecular link between the phase of expansion of neural progenitors and the subsequent phases of cell cycle exit and neuronal differentiation.

Clonal expansion of early to mid-life mitochondrial DNA point mutations drives mitochondrial dysfunction during human ageing

Age-related decline in the integrity of mitochondria is an important contributor to the human ageing process. In a number of ageing stem cell populations, this decline in mitochondrial function is due to clonal expansion of individual mitochondrial DNA (mtDNA) point mutations within single cells. However the dynamics of this process and when these mtDNA mutations occur initially are poorly understood. Using human colorectal epithelium as an exemplar tissue with a well-defined stem cell population, we analysed samples from 207 healthy participants aged 17-78 years using a combination of techniques (Random Mutation Capture, Next Generation Sequencing and mitochondrial enzyme histochemistry), and show that: 1) non-pathogenic mtDNA mutations are present from early embryogenesis or may be transmitted through the germline, whereas pathogenic mtDNA mutations are detected in the somatic cells, providing evidence for purifying selection in humans, 2) pathogenic mtDNA mutations are present from early adulthood (<20 years of age), at both low levels and as clonal expansions, 3) low level mtDNA mutation frequency does not change significantly with age, suggesting that mtDNA mutation rate does not increase significantly with age, and 4) clonally expanded mtDNA mutations increase dramatically with age. These data confirm that clonal expansion of mtDNA mutations, some of which are generated very early in life, is the major driving force behind the mitochondrial dysfunction associated with ageing of the human colorectal epithelium.

Nuclear Factor-kappaB controls the reaggregation of 3D neurosphere cultures in vitro

The approach of reaggregation involves the regeneration and self-renewal of histotypical 3D spheres from isolated tissue kept in suspension culture. Reaggregated spheres can be used as tumour, genetic, biohybrid and neurosphere models. In addition the functional superiority of 3D aggregates over conventional 2D cultures developed the use of neurospheres for brain engineering of CNS diseases. Thus 3D aggregate cultures created enormous interest in mechanisms that regulate the formation of multicellular aggregates in vitro. Here we analyzed mechanisms guiding the development of 3D neurosphere cultures. Adult neural stem cells can be cultured as self-adherent clusters, called neurospheres. Neurospheres are characterised as heterogeneous clusters containing unequal stem cell sub-types. Tumour necrosis factor-alpha (TNF-alpha is one of the crucial inflammatory cytokines with multiple actions on several cell types. TNF-alpha strongly activates the canonical Nuclear Factor Kappa-B (NF- kappaB) pathway. In order to investigate further functions of TNF in neural stem cells (NSCs) we tested the hypothesis that TNF is able to modulate the motility and/or migratory behaviour of SVZ derived adult neural stem cells. We observed a significantly faster sphere formation in TNF treated cultures than in untreated controls. The very fast aggregation of isolated NSCs (<2h) is a commonly observed phenomenon, though the mechanisms of 3D neurosphere formation remain largely unclear. Here we demonstrate for the first time, increased aggregation and enhanced motility of isolated NSCs in response to the TNF-stimulus. Moreover, this phenomenon is largely dependent on activated transcription factor NF-kappaB. Both, the pharmacological blockade of NF-kappaB pathway by pyrrolidine dithiocarbamate (PDTC) or Bay11-7082 and genetic blockade by expression of a transdominant-negative super-repressor IkappaB-AA1 led to decreased aggregation.

Schwann cells can be reprogrammed to multipotency by culture

Adult neural crest related-stem cells persist in adulthood, making them an ideal and easily accessible source of multipotent cells for potential clinical use. Recently, we reported the presence of neural crest-related stem cells within adult palatal ridges, thus raising the question of their localization in their endogenous niche. Using immunocytochemistry, reverse transcription-polymerase chain reaction, and correlative fluorescence and transmission electron microscopy, we identified myelinating Schwann cells within palatal ridges as a putative neural crest stem cell source. Palatal Schwann cells expressed nestin, p75(NTR), and S100. Correlative fluorescence and transmission electron microscopy revealed the exclusive nestin expression within myelinating Schwann cells. Palatal neural crest stem cells and nestin-positive Schwann cells isolated from adult sciatic nerves were able to grow under serum-free conditions as neurospheres in presence of FGF-2 and EGF. Spheres of palatal and sciatic origin showed overlapping expression pattern of neural crest stem cell and Schwann cell markers. Expression of the pluripotency factors Sox2, Klf4, c-Myc, Oct4, the NF-κB subunits p65, p50, and the NF-κB-inhibitor IκB-β were up-regulated in conventionally cultivated sciatic nerve Schwann cells and in neurosphere cultures. Finally, neurospheres of palatal and sciatic origin were able to differentiate into ectodermal, mesodermal, and endodermal cell types emphasizing their multipotency. Taken together, we show that nestin-positive myelinating Schwann cells can be reprogrammed into multipotent adult neural crest stem cells under appropriate culture conditions.

An upgrade on the rabbit model of anthracycline-induced cardiomyopathy: shorter protocol, reduced mortality, and higher incidence of overt dilated cardiomyopathy

Current protocols of anthracycline-induced cardiomyopathy in rabbits present with high premature mortality and nephrotoxicity, thus rendering them unsuitable for studies requiring long-term functional evaluation of myocardial function (e.g., stem cell therapy). We compared two previously described protocols to an in-house developed protocol in three groups: Group DOX2 received doxorubicin 2 mg/kg/week (8 weeks); Group DAU3 received daunorubicin 3 mg/kg/week (10 weeks); and Group DAU4 received daunorubicin 4 mg/kg/week (6 weeks). A cohort of rabbits received saline (control). Results of blood tests, cardiac troponin I, echocardiography, and histopathology were analysed. Whilst DOX2 and DAU3 rabbits showed high premature mortality (50% and 33%, resp.), DAU4 rabbits showed 7.6% premature mortality. None of DOX2 rabbits developed overt dilated cardiomyopathy; 66% of DAU3 rabbits developed overt dilated cardiomyopathy and quickly progressed to severe congestive heart failure. Interestingly, 92% of DAU4 rabbits showed overt dilated cardiomyopathy and 67% developed congestive heart failure exhibiting stable disease. DOX2 and DAU3 rabbits showed alterations of renal function, with DAU3 also exhibiting hepatic function compromise. Thus, a shortened protocol of anthracycline-induced cardiomyopathy as in DAU4 group results in high incidence of overt dilated cardiomyopathy, which insidiously progressed to congestive heart failure, associated to reduced systemic compromise and very low premature mortality.

Human Multipotent Mesenchymal Stromal Cells from Distinct Sources Show Different In Vivo Potential to Differentiate into Muscle Cells When Injected in Dystrophic Mice

Limb-girdle muscular dystrophies are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence or deficiency of muscle proteins. The murine model of Limb-Girdle Muscular Dystrophy 2B, the SJL mice, carries a deletion in the dysferlin gene. Functionally, this mouse model shows discrete muscle weakness, starting at the age of 4-6 weeks. The possibility to restore the expression of the defective protein and improve muscular performance by cell therapy is a promising approach for the future treatment of progressive muscular dystrophies (PMD). We and others have recently shown that human adipose multipotent mesenchymal stromal cells (hASCs) can differentiate into skeletal muscle when in contact with dystrophic muscle cells in vitro and in vivo. Umbilical cord tissue and adipose tissue are known rich sources of multipotent mesenchymal stromal cells (MSCs), widely used for cell-based therapy studies. The main objective of the present study is to evaluate if MSCs from these two different sources have the same potential to reach and differentiate in muscle cells in vivo or if this capability is influenced by the niche from where they were obtained. In order to address this question we injected human derived umbilical cord tissue MSCs (hUCT MSCs) into the caudal vein of SJL mice with the same protocol previously used for hASCs; we evaluated the ability of these cells to engraft into recipient dystrophic muscle after systemic delivery, to express human muscle proteins in the dystrophic host and their effect in functional performance. These results are of great interest for future therapeutic application.

Animal models for genetic neuromuscular diseases

The neuromuscular disorders are a heterogeneous group of genetic diseases, caused by mutations in genes coding sarcolemmal, sarcomeric, and citosolic muscle proteins. Deficiencies or loss of function of these proteins leads to variable degree of progressive loss of motor ability. Several animal models, manifesting phenotypes observed in neuromuscular diseases, have been identified in nature or generated in laboratory. These models generally present physiological alterations observed in human patients and can be used as important tools for genetic, clinic, and histopathological studies. The mdx mouse is the most widely used animal model for Duchenne muscular dystrophy (DMD). Although it is a good genetic and biochemical model, presenting total deficiency of the protein dystrophin in the muscle, this mouse is not useful for clinical trials because of its very mild phenotype. The canine golden retriever MD model represents a more clinically similar model of DMD due to its larger size and significant muscle weakness. Autosomal recessive limb-girdle MD forms models include the SJL/J mice, which develop a spontaneous myopathy resulting from a mutation in the Dysferlin gene, being a model for LGMD2B. For the human sarcoglycanopahties (SG), the BIO14.6 hamster is the spontaneous animal model for delta-SG deficiency, whereas some canine models with deficiency of SG proteins have also been identified. More recently, using the homologous recombination technique in embryonic stem cell, several mouse models have been developed with null mutations in each one of the four SG genes. All sarcoglycan-null animals display a progressive muscular dystrophy of variable severity and share the property of a significant secondary reduction in the expression of the other members of the sarcoglycan subcomplex and other components of the Dystrophin-glycoprotein complex. Mouse models for congenital MD include the dy/dy (dystrophia-muscularis) mouse and the allelic mutant dy(2J)/dy(2J) mouse, both presenting significant reduction of alpha 2-laminin in the muscle and a severe phenotype. The myodystrophy mouse (Large(myd)) harbors a mutation in the glycosyltransferase Large, which leads to altered glycosylation of alpha-DG, and also a severe phenotype. Other informative models for muscle proteins include the knockout mouse for myostatin, which demonstrated that this protein is a negative regulator of muscle growth. Additionally, the stress syndrome in pigs, caused by mutations in the porcine RYR1 gene, helped to localize the gene causing malignant hypertermia and Central Core myopathy in humans. The study of animal models for genetic diseases, in spite of the existence of differences in some phenotypes, can provide important clues to the understanding of the pathogenesis of these disorders and are also very valuable for testing strategies for therapeutic approaches.