Roles of aldehyde dehydrogenase (ALDH) isoforms and retinoic acids in the growth and differentiation potential of human adult cardiac progenitor cells

Aim: We have studied human adult cardiac progenitor cells (CPCs) based on high aldehyde dehydrogenase activity (ALDH-hi), a property shared by many stem cells across tissues and organs. However, the role of ALDH in stem cell function is poorly known. In humans, there are 19 ALDH isoforms with different biological activities. The isoforms responsible for the ALDH-hi phenotype of stem cells are not well known but they may include ALDH1A1 and ALDH1A3 isoforms, which function in all-trans retinoic acid (RA) cell signaling. ALDH activity has been shown to regulate hematopoietic stem cell function via RA. We aimed to analyze ALDH isoform expression and the role of RA in human CPC function. Methods: Human adult CPCs were isolated from atrial appendage samples from patients who underwent heart surgery for coronary artery or valve disease. Atrial samples were either cultured as primary explants or enzymatically digested and sorted for ALDH activity by FACS. ALDH isoforms were determined by qRT-PCR. Cells were cultured in the presence or absence of the specific ALDH inhibitor DEAB, with or without RA. Induction of cardiac-specific genes in cells cultured in differentiation medium was measured by qRT-PCR. Results: While ALDH-hi CPCs grew in culture and could be expanded, ALDH-low cells grew poorly. CPC isolated as primary explant outgrowths expressed high levels of ALDH1A3 but not of other isoforms. CPCs isolated from cardiospheres expressed relatively high levels of all the 11 isoforms tested. In contrast, expanded CPCs and cardiosphere-derived cells expressed low levels of all ALDH isoforms. DEAB inhibited CPC growth in a dose-dependent manner, whereas RA rescued CPC growth in the presence of DEAB. In differentiation medium, ALDH-hi CPCs expressed approximately 300-fold higher levels of cardiac troponin T compared with their ALDH-low counterparts. Conclusions: High ALDH activity identifies human adult cardiac cells with high growth and cardiomyogenic potential. ALDH1A3 and, possibly, ALDH1A1 isoforms account for high ALDH activity and RA-mediated regulation of CPC growth.

Mechanism of action of a nitroimidazole-thiadiazole derivate upon Trypanosoma cruzi tissue culture amastigotes

Megazol (CL 64,855) a very effective drug in experimental infections by Trypanosoma cruzi, and also in in vitro assays with vertebrate forms of the parasite, had its parasite, had its activity upon macromolecule biosynthesis tested using tissue culture-derived amastigote forms. Megazol presented a drastic inhibition of [3H]-uridine incorporation, suggesting a selective activity upon protein synthesis. Comparing the three drugs, megazol was more potent than nifurtimox and benznidazole in inhibiting protein an DNA synthesis. Megazol showed a 91% of inhibition of [3H]-leucine incorporation whereas nifurtimox and benznidazole, 0% and 2%, respectively. These latter two drugs inhibited the incorporation of all the precursors tested at similar levels, but the concentration of benznidazole was always three times higher, suggesting different mechanisms of action or, more probably, a greater efficiency of the 5-nitrofuran derivate in relation to the 2-nitroimidazole. So, wes conclude that the mode of action of megazol is different from the ones of nifurtimox and benznidazole and that its primary effect is associated with an impairment of protein synthesis.

On the transition of genetic differentiation from isolation to panmixia: What we can learn from GST and D.

Population genetic differentiation characterizes the repartition of alleles among populations. It is commonly thought that genetic differentiation measures, such as GST and D, should be near zero when allele frequencies are close to their expected value in panmictic populations, and close to one when they are close to their expected value in isolated populations. To analyse those properties, we first derive analytically a reference function f of known parameters that describes how important features of genetic differentiation (e.g. gene diversity, proportion of private alleles, frequency of the most common allele) are close to their expected panmictic and isolation value. We find that the behaviour of function f differs according to three distinct mutation regimes defined by the scaled mutation rate and the number of populations. Then, we compare GST and D to f, and demonstrate that their signal of differentiation strongly depends on the mutation regime. In particular, we show that D captures well the variations of genetic diversity when mutation is weak, otherwise it overestimates it when panmixia is not met. GST detects population differentiation when mutation is intermediate but has a low sensitivity to the variations of genetic diversity when mutation is weak. When mutation is strong the domain of sensitivity of both measures are altered. Finally, we also point out the importance of the number of populations on genetic differentiation measures, and provide recommendations for the use of GST and D.

CD28/CTLA4-B7 interaction is dispensable for T cell stimulation by mouse mammary tumor virus superantigen but not for B cell differentiation and virus dissemination.

B cells are the primary targets of infection for mouse mammary tumor virus (MMTV). However, for productive retroviral infection, T cell stimulation through the virally-encoded superantigen (SAG) is necessary. It activates B cells and leads to cell division and differentiation. To characterize the role of B cell differentiation for the MMTV life cycle, we studied the course of infection in transgenic mice deficient for CD28/CTLA4-B7 interactions (mCTLA4-H gamma 1 transgenic mice). B cell infection occurred in CTLA4-H gamma 1 transgenic mice as integrated proviral DNA could be detected in draining lymph node cells early after infection by polymerase chain reaction analysis. In mice expressing I-E, B cells were able to present the viral SAG efficiently to V beta 6+ T cells. These cells expanded specifically and were triggered to express the activation marker CD69. Further stages of progression of infection appeared to be defective. Kinetics experiments indicated that T and B cell stimulation stopped more rapidly than in control mice. B cells acquired an activated CD69+ phenotype, were induced to produce IgM but only partially switched to IgG secretion. Finally, the dissemination of infected cells to other lymph nodes and spleen was reduced and the peripheral deletion of V beta 6+ T cells was minimal. In contrast, in mice lacking I-E, T cell stimulation was also impaired and B cell activation undetectable. These data implicate B7-dependent cellular interactions for superantigenic T cell stimulation by low-affinity TCR ligands and suggest a role of B cell differentiation in viral dissemination and peripheral T cell deletion.

Determinants of renal tissue oxygenation as measured with BOLD-MRI in chronic kidney disease and hypertension in humans.

Experimentally renal tissue hypoxia appears to play an important role in the pathogenesis of chronic kidney disease (CKD) and arterial hypertension (AHT). In this study we measured renal tissue oxygenation and its determinants in humans using blood oxygenation level-dependent magnetic resonance imaging (BOLD-MRI) under standardized hydration conditions. Four coronal slices were selected, and a multi gradient echo sequence was used to acquire T2* weighted images. The mean cortical and medullary R2* values ( = 1/T2*) were calculated before and after administration of IV furosemide, a low R2* indicating a high tissue oxygenation. We studied 195 subjects (95 CKD, 58 treated AHT, and 42 healthy controls). Mean cortical R2 and medullary R2* were not significantly different between the groups at baseline. In stimulated conditions (furosemide injection), the decrease in R2* was significantly blunted in patients with CKD and AHT. In multivariate linear regression analyses, neither cortical nor medullary R2* were associated with eGFR or blood pressure, but cortical R2* correlated positively with male gender, blood glucose and uric acid levels. In conclusion, our data show that kidney oxygenation is tightly regulated in CKD and hypertensive patients at rest. However, the metabolic response to acute changes in sodium transport is altered in CKD and in AHT, despite preserved renal function in the latter group. This suggests the presence of early renal metabolic alterations in hypertension. The correlations between cortical R2* values, male gender, glycemia and uric acid levels suggest that these factors interfere with the regulation of renal tissue oxygenation.

Atteintes cardiaques dans les connectivites: l'exemple du lupus érythémateux systémique [Cardiac involvement in connective tissue disease: the example of systemic lupus erythematosus]

When we think of cardiac affection in the context of systemic lupus erythematosus (SLE), we usually refer to pericarditis first. As frequent as this affection is, it is actually not the only cardio-vascular problem that occurs with this systemic inflammatory disease. Are the cardiac events--ranging from multiple heart valve involvements to increased cardiovascular risks--clinically significant? And are they involving a specific follow-up, treatment or support? We are therefore trying to evaluate these questions in order to give some recommendations to any practitioners following up a lupus patient, or a patient suffering from any other inflammatory systemic disease.

The distribution of agglutinins and lytic activity against Trypanosoma rangeli and erythrocytes in Rhodnius prolixus and Triatoma infestans tissue extracts and haemolymph

Haemolymph, heads, salivary glands, crops, midguts, hindguts, and Malpighian tubules from Rhodnius prolixus and Triatoma infestans were extracted in phosphate or Tris buffer saline with calcium, and tested for agglutination and lytic activities by microtitration against both vertebrateerythrocytes and cultured epimatigote forms of Trypanosoma rangeli. Haemagglutination activity against rabbit erythrocytes was found in the crop, midgut and hindgut extracts of T. infestans but only in the haemolymph of R. prolixus. Higher titres of parasite agglutinins were found in R. prolixus haemolymph than T. infestans, whilst the converse occurred for the tissue extracts. In addition, the extracts of T. infestans salivary glands, but not those of R. prolixus, showed a trypanolytic activity that was heat-inactivated and was not abolished by pre-incubation with any of the sugars or glycoproteins tested. T. infestans, which is refractory to infection by T. rangeli, thus appears to contain a much wider distribution of agglutinating and trypanolytic factors in its tissues than the more susceptible species, R. prolixus

Hyperplasia of elastic tissue in hepatic schistosomal fibrosis

Elastic tissue hyperplasia, revealed by means of histological, immunocytochemical and ultrastructural methods, appeared as a prominent change in surgical liver biopsies taken from 61 patients with schistosomal periportal and septal fibrosis. Such hyperplasia was absent in ecperimental murine schistosomiasis, including mice with "pipe-stem" fibrosis. Displaced connective tissue cells in periportal areas, such as smooth muscle cells, more frequently observed in human material, could be the site of excessive elastin synthesis, and could explain the differences observed in human and experimental materials. Elastic tissue, sometimes represented by its microfibrillar components, also appeared to be more condensed in areas of matrix (collagen) degradation, suggesting a participation of this tissue in the remodelling of the extracellular matrix. By its rectratile properties elastic tissue hyperplasia in hepatic schistosomiasis can cause vascular narrowing and thus play a role in the pathogenesis of portal hypeertension.

Plant tissue culture techniques

Plant cell and tissue culture in a simple fashion refers to techniques which utilize either single plant cells, groups of unorganized cells (callus) or organized tissues or organs put in culture, under controlled sterile conditions.

Differentiation of rat striatal embryonic stem cells in vitro: monolayer culture vs. three-dimensional coculture with differentiated brain cells.

Several groups have demonstrated the existence of self-renewing stem cells in embryonic and adult mouse brain. In vitro, these cells proliferate in response to epidermal growth factor, forming clusters of nestin-positive cells that may be dissociated and subcultured repetitively. Here we show that, in stem cell clusters derived from rat embryonic striatum, cell proliferation decreased with increasing number of passages and in response to elevated concentrations of potassium (30 mM KCl). In monolayer culture, the appearance of microtubule-associated protein type-5-immunoreactive (MAP-5(+)) cells (presumptive neurons) in response to basic fibroblast growth factor (bFGF) was reduced at low cell density and with increasing number of passages. In the presence of bFGF, elevated potassium caused a more differentiated neuronal phenotype, characterized by an increased proportion of MAP-5(+) cells, extensive neuritic branching, and higher specific activity of glutamic acid decarboxylase. Dissociated stem cells were able to invade cultured brain cell aggregates containing different proportions of neurons and glial cells, whereas they required the presence of a considerable proportion of glial cells in the host cultures to become neurofilament H-positive. The latter observation supports the view that astrocyte-derived factors influence early differentiation of the neuronal cell lineage.

Tyrosine phosphorylation is an early and specific event involved in primary keratinocyte differentiation.

Very little is known about early molecular events triggering epithelial cell differentiation. We have examined the possible role of tyrosine phosphorylation in this process, as observed in cultures of primary mouse keratinocytes after exposure to calcium or 12-O-tetradecanoylphorbol-13-acetate (TPA). Immunoblotting with phosphotyrosine-specific antibodies as well as direct phosphoamino acid analysis revealed that induction of tyrosine phosphorylation occurs as a very early and specific event in keratinocyte differentiation. Very little or no induction of tyrosine phosphorylation was observed in a keratinocyte cell line resistant to the differentiating effects of calcium. Treatment of cells with tyrosine kinase inhibitors prevented induction of tyrosine phosphorylation by calcium and TPA and interfered with the differentiative effects of these agents. These results suggest that specific activation of tyrosine kinase(s) may play an important regulatory role in keratinocyte differentiation.

The murine model of infection with Leishmania major and its importance for the deciphering of mechanisms underlying differences in Th cell differentiation in mice from different genetic backgrounds.

Mice from the majority of inbred strains are resistant to infection by Leishmania major, an obligate intracellular protozoan parasite of macrophages in the mammalian host. In contrast, mice from BALB strains are unable to control infection and develop progressive disease. In this model of infection, genetically determined resistance and susceptibility have been clearly shown to result from the appearance of parasite-specific CD4+ T helper 1 or T helper 2 cells, respectively. This murine model of infection is considered as one of the best experimental systems for the study of the mechanisms operating in vivo at the initiation of polarised T helper 1 and T helper 2 cell maturation. Among the several factors influencing Th cell development, cytokines themselves critically regulate this process. The results accumulated during the last years have clarified some aspects of the role played by cytokines in Th cell differentiation. They are providing critical information that may ultimately lead to the rational devise of means by which to tailor immune responses to the effector functions that are most efficient in preventing and/or controlling infections with pathogens.

Cell-based computational modeling of vascular morphogenesis using Tissue Simulation Toolkit.

Computational modeling has become a widely used tool for unraveling the mechanisms of higher level cooperative cell behavior during vascular morphogenesis. However, experimenting with published simulation models or adding new assumptions to those models can be daunting for novice and even for experienced computational scientists. Here, we present a step-by-step, practical tutorial for building cell-based simulations of vascular morphogenesis using the Tissue Simulation Toolkit (TST). The TST is a freely available, open-source C++ library for developing simulations with the two-dimensional cellular Potts model, a stochastic, agent-based framework to simulate collective cell behavior. We will show the basic use of the TST to simulate and experiment with published simulations of vascular network formation. Then, we will present step-by-step instructions and explanations for building a recent simulation model of tumor angiogenesis. Demonstrated mechanisms include cell-cell adhesion, chemotaxis, cell elongation, haptotaxis, and haptokinesis.