A Novel Cytomegalovirus-Induced Regulatory-Type T-Cell Subset Increases in Size During Older Life and Links Virus-Specific Immunity to Vascular Pathology

Background. Cytomegalovirus (CMV) infection directly targets vascular endothelium and smooth muscle and at older ages is associated with accelerated vascular pathology and mortality. CMV-specific cellular immunity might directly contribute to this process. Methods. Conventional ex vivo activation–induced T-cell responses to 19 dominant CMV antigens, along with CMV-specific inducible regulatory-type CD4+ T cells (iTregs), were measured in healthy older people, using a novel protocol that included classic Treg markers alongside the activation marker CD134. Measurements were correlated with diastolic, systolic, and mean arterial blood pressure, a surrogate marker for arterial stiffness. Results. CMV-specific iTregs recognized the same antigens as conventional CD4+ T cells and were significantly more frequent at older ages. They suppressed antigen-specific and nonspecific proliferation and in large part expressed Foxp3. Frequencies of CMV-specific iTregs and CD8+ T cells (summated response) were significantly associated with diastolic and mean arterial pressures. Confounders, including age, body mass index, smoking, antihypertensive medication use, or C-reactive protein levels, did not explain these observations. Conclusions. A novel CMV-induced regulatory-type CD4+ T-cell subset is readily detectable in CMV-infected people and, like the aggregate CD8+ T-cell response to the most dominant CMV antigens, is quantitatively associated with arterial stiffness in older life. Whereas CD8+ effector T cells might directly cause vascular injury, iTregs may attenuate this response

Identifizierung und Charakterisierung der CD4+AT2R+ T Zell Subpopulation bei Mensch und Ratte

The study contributes to the understanding of CD4+AT2R+ cell function after myocardial infarction (MI). This potential novel T cell subset, which expresses FoxP3 and various cytokines improves cardiac function and reduces infarct size. Results indicate that the CD4+AT2R+ T cells might be included in therapeutic approaches for the treatment of cardiac diseases, due to its upregulation in the infarcted state. The application of this T cell subset may improve the healing process of compromised heart tissue and contribute to a better quality of life in affected patients

Role of the Immune System and Bioactive Lipids in Trafficking Bone Marrow-Derived Stem Cells in Patients with Ischemic Heart Disease

Acute myocardial infarction (AMI) triggers the mobilization of stem/progenitor cells from bone marrow (BMSPCs) into peripheral blood (PB). The underlying mechanisms orchestrating this mobilization and subsequent homing of BMSPCs to the myocardium are poorly understood. While the role of traditional chemokines in the mobilization and homing of hematopoietic stem cell (HSCs) to BM niches is undisputed, their role in directing BMSPCs to the highly proteolytic environment of the ischemic myocardium is debatable and other redundant mechanism may exist. Based on our observation that bioactive lipids, such as sphingosine-1 phosphate (S1P) and ceramide-1 phosphate (C1P), play an important role in regulating trafficking of HSCs; we explored if they also direct trafficking of BMSPCs in the setting of myocardial ischemia. While BMSPCs expressed S1P receptors regardless of the source, the expression of S1P receptor 1 (S1PR1) and receptor 3 (S1PR3), which are responsible for migration and chemotaxis, was elevated in BMSPCs in naïve BM cells and was reduced following mobilization. This expression correlated to differential response of BMSPCs to S1P in chemotaxis assays. By employing flow cytometry analyses, we observed an increase in circulating PB CD34+, CD133+ and CXCR4+ lineage negative (Lin-)/CD45- cells that are enriched in non-HSCs (P \u3c 0.05 vs. controls). This corroborated our mass spectrometry studies showing a temporal increase in S1P and C1P plasma levels. At the same time, plasma obtained in the early phases following AMI strongly chemoattracted human BM-derived CD34+/Lin- and CXCR4+/Lin- cells in Transwell chemotaxis assays in an S1P dependent fashion. We examined other mechanisms that may contribute to the homing of BMSPCs to the infarcted myocardium due to the reduction of S1PRs upon mobilization. We observed that hypoxia induced higher expression of cathelicidins in cardiac tissues. Indeed, PB cells isolated from patients with AMI migrated more efficiently to low, yet physiological, gradient of SDF-1 in Transwell migration assays compared to SDF-1 alone. Together, these observations suggest that while elevated S1P plasma levels early in the course of AMI may trigger mobilization of non-HSCs into PB, cathelicidins appear to play an important role in their homing to ischemic and damaged myocardium

Functional characterisation of a novel nucleoporin gene NUP98 in zebrafish embryo.

Oral PresentationINTRODUCTION: The nucleoporin gene nup98 is important for the regulation of cytoplasmic-nuclear trafficking. Frequent disruptions of NUP98 during chromosomal translocation in acute myeloid leukaemia suggest that it may play a role in normal haematopoiesis. nup98-knockout mice has resulted in early embryonic lethality. Therefore, its role in embryonic haematopoiesis remains unclear. In this study, we have cloned a zebrafish nup98 gene and examined its role in embryonic development, with particular reference to haematopoiesis. METHODS: Two expressed sequence tags with translated sequence homologous to human NUP98 were identified. The gene was cloned by PCR from cDNA of zebrafish embryos. Expression of nup98 in zebrafish embryos was investigated spatially by whole-mount in-situ hybridisation and temporally by RT-PCR. The functions of nup98 were examined by morpholino knockdown and the effects on embryonic development evaluated by gene expression studies and confocal microscopy. Cellular functions of zebrafish nup98 were investigated in HeLa cells. RESULTS: Zebrafish nup98 gene shared 65% identity to human NUP98 homolog in protein sequence. The gene was expressed during early embryonic development since 1-cell stage and diffusely in eyes and the developing brain since 18 hpf. About 30% nup98-knockdown embryos developed intracranial haemorrhage at 48 hpf, resulting from disrupted blood vessels. nup98-knockdown upregulated pu.1 and scl as evaluated by quantitative RT-PCR. Moreover, ectopic expression of zebrafish nup98 rescued the defective mRNA export due to NUP98 knockdown in HeLa cells. CONCLUSION: A novel zebrafish nup98 gene was shown to exhibit conserved function in mRNA trafficking. Its role in embryonic development should be further evaluated.published_or_final_versio

Towards Stem Cell Treatment for Duchenne Muscular Dystrophy-Related Cardiomyopathy

Duchenne muscular dystrophy (DMD) is a progressive muscular degenerative disease affecting 1 in 3,500 boys. Cardiomyopathy is observed in 95% of patients aged 18 and older, accounting for 30% of deaths. While innovative treatments have been employed to curb symptoms in skeletal muscle, few show success in the heart, and no cure for DMD exists. Recently, regenerative therapy using cardiac stem cells (CSCs) has shown very promising results, but efficacy in DMD remains undetermined. This dissertation sought to provide preliminary insight into the effects of murine Sca-1+ CSC therapy. Three-dimensional echocardiography was validated and used to gauge cardiomyopathy in the mdx:utrn-/- mouse model. CSCs were isolated from healthy mice and displayed differentiation potential. They were implanted into mdx:utrn-/- mice under ultrasound guidance, and showed preliminary trends towards functional improvement. These results support the notion that stem cells may be an excellent avenue of therapy that should be further investigated

MicroRNAs and GRK2 as modulators of Kiss1/GPR54 system: Physiopathological role in pubertal alterations and obesity induced hypogonadism

The reproductive function is governed by the so-called hypothalamic-pituitary-gonadal (HPG) axis, where an intricated network of central, peripheral and external factors determine hormonal balance and proper functioning of the reproductive system and the gonadal function, guaranteeing the perpetuation of the species 1–3. In recent years, it has been documented that a plethora of central (glutamate, GABA, NKB, NPY) 4–7, peripheral (insulin, leptin, ghrelin) 8,9 and external (nutritional availability, endocrine disruptors, circadian rhythms) 10–13 cues converge (either acting directly or indirectly) onto Kiss1 neurons in the hypothalamus, as major signaling hub of the HPG axis 14, whose products, kisspeptins, act on the GnRH neurons, via its canonical receptor, GPR5415, activating puberty onset and reproductive function. In addition, it is well recognized that reproductive function is altered under conditions of metabolic distress, ranging from subnutrition to obesity, type 2 diabetes and metabolic syndrome, which are bound to numerous perturbations, including disordered puberty, central hypogonadism (mainly in males) and cardiometabolic impairment16,17. MicroRNAs have been recently pointed out as essential players in the control of normal pubertal development18–21, although no study has addressed the specific regulation of Kiss1, at central levels, exerted by miRNAs 21,22. Further, the effects of miRNAs in the pathogenesis of central hypogonadism are completely unexplored. In parallel, the Kiss1/GPR54 system is a key element for the integration of the energetic status and reproductive capacity 23, where GPR54 inactivating mutations were described decades ago as underlying origin of hypogonadotropic hypogonadism 24,25. The G-protein coupled receptor kinase 2 (GRK2) 26, which is largely recognized as pleiotropic regulator of cellular signaling 27–29, has been suggested in vitro as a modulator of GPR5426. Nevertheless, no studies had addressed to date its potential roles in proper pubertal development and maintenance of reproductive capacity. In the above context, this Doctoral Thesis has addressed, as main aims, (i) the putative role of specific miRNAs in the physiological control of puberty via regulation of the Kiss1 system; (ii) the pathophysiological role of miRNAs in obesity-induced hypogonadism (OIH), their interplay with Kiss1 and their potential therapeutic implications; (iii) the role of GRK2 in the control of puberty and the HPG axis through regulation of GPR54 in normal conditions and under nutritional stress; and (iv) the implication of GRK2 in OIH through GPR54 regulation.La función reproductora está determinada por el correcto funcionamiento del eje hipotálamohipofiso-gonadal (HHG), donde una compleja red de factores centrales, periféricos y externos determinan el balance hormonal necesario para la adquisición de la capacidad reproductora y, en consecuencia, para el mantenimiento de las especies 1–3. De este modo, se ha documentado que una multitud de factores centrales (GABA, glutamato, NKB, NPY) 4–7, periféricos (leptina, insulina o ghrelina) 8,9 y externos (disruptores endocrinos, aporte energético, ritmos circadianos) 10–13 convergen (actuando directa o indirectamente) en las neuronas Kiss1 hipotalámicas como principal núcleo del eje HHG 14 y cuyo producto, las kisspeptinas, transmitirán información a la neurona GnRH, por medio de su receptor canónico, GPR5415 , activando la pubertad y la función reproductora. Al mismo tiempo, existen evidencias sobre la afectación de la función reproductora como consecuencia del desequilibrio homeostático presente en situaciones como la subnutrición o la obesidad, diabetes mellitus tipo 2 o síndrome metabólico, que se encuentran vinculadas a numerosos desórdenes, incluyendo alteraciones de la edad de pubertad, el hipogonadismo central (principalmente masculino) y la enfermedad cardiovascular 16,17. Datos recientes señalan que los microRNAs (miRNAs) son elementos implicados en la correcta transición puberal 18–21, aunque existen pocos estudios dirigidos a evaluar al papel de los miRNAs en la regulación específica de la expresión de Kiss1, a nivel central19,22. Además, la desregulación de determinados miRNAs en condiciones de hipogonadismo central, su impacto sobre el sistema Kiss1 y su implicación fisiopatológica en esta condición, permanecen inexplorados. En paralelo, el sistema Kiss1/GPR54 es un elemento clave en la integración del estado energético y la capacidad reproductora23, estando descrito que mutaciones inactivantes en GPR54 son una causa subyacente en determinados casos de hipogonadismo hipogonadotropo 24,25. La quinasa de receptor acoplado a proteína G (GRK2) 26 está reconocida como un regulador pleiotrópico de la señalización celular 27–29 y ha sido demostrada su capacidad para regular GPR54, in vitro 26. En cualquier caso, no existen estudios relacionados con su potencial implicación en la correcta maduración puberal y en el mantenimiento de la capacidad reproductora in vivo. En base a lo anterior, esta Tesis Doctoral ha abordado, como objetivos principales, (i) el estudio del papel específico de microRNAs en la regulación puberal a través de la regulación del sistema Kiss1; (ii) el papel fisiopatológico de los microRNAs en el hipogonadismo central inducido por obesidad (HIO) y sus potenciales implicaciones terapéuticas; (iii) el papel de GRK2 en el control de la puberal y el eje HHG mediante la regulación de GPR54 en condiciones control y de estrés nutricional; y (iv) la implicación de GRK2 en HIO a través de la regulación de GPR54

Cell-based therapies for ischemic cardiomyopathy : investigations of intramyocardial retention and safety of high dose intracoronary delivery of c-kit positive cardiac progenitor cells, and therapeutic utility of a novel population of cardiac mesenchymal stem cells expressing stage-specific embryonic antigen-3 (SSEA-3).

Over the last decade attempts at reducing morbidity and mortality of patients with chronic heart failure have been made via the development and implementation of novel cell based therapies. Substantial advances in cell based therapies with indications of efficacy have been shown along with a robust safety profile. Despite these advances, there is a substantial unmet need for novel therapies, specifically addressing repair and regeneration of the damaged or lost myocardium and its vasculature. Accordingly, cardiac cell-based therapies have gained attention. Various cell-types have been utilized, including bone marrow-derived mononuclear cells, bone marrow-derived mesenchymal stem cells, mobilized CD34+ cells, and more recently, cardiosphere-derived cells and cardiac-derived c-kit positive progenitor cells. Early studies have suggested a potential of cell-based therapies to reduce cardiac scar size and to improve cardiac function in patients with ischemic cardiomyopathy. However, variability of results has been observed necessitating improvement of current methodologies related to optimizing the cell type(s), infusion techniques, timing, dosage, acuity related to ischemic injury, and perhaps repeat dosing over time among others, all the while ensuring complete and total patient safety. Accordingly, present efforts and goals of my research are aimed at i.) Optimizing methodologies utilized within the recent phase I clinical trial (SCIPIO) that showed intracoronary infusion of 1 million c-kit positive cardiac progenitor cells was safe with indications of efficacy in cardiac repair, as well as, ii.) Development of a novel cell based approach with a newly discovered cardiac cell type. Within the present dissertation, I explored the impact of coronary stop-flow on cardiac retention of intracoronarily infused c-kit positive cardiac progenitor cells given that balloon inflation in a non-stented coronary artery is inherently dangerous, especially in already damaged hearts. I demonstrate that intracardiac retention with or without stop-flow is equivalent and balloon inflation confers an undue risk to patients. Furthermore, I investigated the safety of intracoronary infusion of 20 million c-kit positive cardiac progenitor cells in pigs, an equivalent dose 40 times larger than was used in the SCIPIO trial. High dose of cells delivered intracoronarily is safe and does not result in myocardial injury or functional deficit. Therefore, larger doses may reasonably be utilized in future clinical trials. Finally, I describe a novel adult cardiac cell type that maintains expression of an embryonic stem cell associated marker, stage-specific embryonic antigen (SSEA)-3, resides within the native adult heart, and can be isolated and utilized for cardiac repair as a cell based therapy