Defective hematopoietic differentiation of immune aplastic anemia patient-derived iPSCs

In acquired immune aplastic anemia (AA), pathogenic cytotoxic Th1 cells are activated and expanded, driving an immune response against the hematopoietic stem and progenitor cells (HSPCs) that provokes cell depletion and causes bone marrow failure. However, additional HSPC defects may contribute to hematopoietic failure, reflecting on disease outcomes and response to immunosuppression. Here we derived induced pluripotent stem cells (iPSCs) from peripheral blood (PB) erythroblasts obtained from patients diagnosed with immune AA using non-integrating plasmids to model the disease. Erythroblasts were harvested after hematologic response to immunosuppression was achieved. Patients were screened for germline pathogenic variants in bone marrow failure-related genes and no variant was identified. Reprogramming was equally successful for erythroblasts collected from the three immune AA patients and the three healthy subjects. However, the hematopoietic differentiation potential of AA-iPSCs was significantly reduced both quantitatively and qualitatively as compared to healthy-iPSCs, reliably recapitulating disease: differentiation appeared to be more severely affected in cells from the two patients with partial response as compared to the one patient with complete response. Telomere elongation and the telomerase machinery were preserved during reprogramming and differentiation in all AA-iPSCs. Our results indicate that iPSCs are a reliable platform to model immune AA and recapitulate clinical phenotypes. We propose that the immune attack may cause specific epigenetic changes in the HSPCs that limit adequate proliferation and differentiation.Subject terms: Anaemia, Induced pluripotent stem cells     

Effect of the Alzheimer amyloid fragment Abeta(25-35) on Akt/PKB kinase and survival of PC12 cells

The phosphatidylinositol 3 kinase (PI3K)-Akt/PKB pathway protects neurons from apoptosis caused by diverse stress stimuli. However, its protective role against the amyloid beta peptide (Abeta), a major constituent of Alzheimer's disease plaques, has not been studied. We investigated the effect of the Abeta-derived Abeta(25-35) peptide on apoptosis and on the Akt survival pathway in PC12 cells. Cells submitted to micromolar concentrations of Abeta(25-35) exhibited increased production of reactive oxygen species (ROS) and morphological alterations consistent with apoptosis. Akt1 was activated shortly after incubation with Abeta(25-35) and Abeta(1-40) with a kinetics different to that of nerve-derived growth factor. Akt1 activation was blocked by the PI3K inhibitor wortmannin. We tested the hypothesis that Akt1 might modify the vulnerability of neural cells to apoptosis induced by Abeta(25-35). Overexpression of an active version of Akt1 attenuated the apoptotic effect of Abeta(25-35) as determined by flow cytometry. Moreover, PC12 cells overexpressing a membrane-targeted N-myristylated fusion protein of enhanced green fluorescence protein (EGFP) and mouse Akt1 exhibited lower levels of ROS than control EGFP-transfected cells. The present findings demonstrate that Akt1 is activated in response to Abeta(25-35) in a PI3K-dependent manner and that active Akt1 protects PC12 cells against the pro-apoptotic action of this peptide.     

Control of muscle fibre and motoneuron diversification

Recent studies have elucidated both the mechanism of early formation of diverse muscle fibre types and the matching of diverse populations of motoneurons to their appropriate muscle targets. Highlights include the demonstration that distinct signals are necessary for the formation of several distinct myoblast populations in the vertebrate somite, the identification of motoneuron subtypes, studies of how motoneurons target appropriate muscles, and rapid progress on the Drosophila neuromuscular system. We propose a model in which four classes of decision control the patterning of both motoneurons and muscles.     

Genotype and allele frequencies of polymorphic cytochromes P450 CYP1A2 and CYP2E1 in Mexicans

CYP1A2 and CYP2E1 are two of the main cytochrome P450 isoforms involved in the metabolism of commonly used drugs and xenobiotic compounds considered to be responsible for or possible participants in the development of several human diseases. Individual susceptibility to developing these pathologies relies, among other factors, on genetic polymorphism which depends on ethnic differences, as the frequency of mutant genotypes varies in different human populations. Thus the aim of this study was to investigate the frequency of CYP1A2 5'-flanking region and CYP2E1 Rsa I/Pst I polymorphisms in Mexicans by PCR-RFLP methods. The DNA of 159 subjects was analysed and mutant allele frequencies of 30% for CYP2E1 Rsa I/Pst I sites and 43% for CYP1A2 5'-flanking region were found. These frequencies are higher than those previously reported for other human populations.     

Inhibition of vesicular stomatitis virus replication by prostaglandin A1 in Aedes albopictus cells

Cyclopentenone prostaglandins (PGs) exhibit antiviral activity against RNA and DNA viruses in mammalian cell lines, and this effect has been associated with the induction of a heat shock protein (hsp70). We investigated the effect of prostaglandin A1 (PGA1) on the replication of vesicular stomatitis virus (VSV) in Aedes albopictus (mosquito) cells. PGA1 was found to inhibit VSV replication dose dependently. Virus yield was reduced to 50% (3 microg PGA1/ml) and to 95% with 8 microg PGA1/ml. Even with the dramatic reduction of virus production observed in cells treated with PGA1, VSV-specific protein synthesis was unaltered. Treatment of cells with PGA1 (5 microg/ml) stimulated the synthesis of a polypeptide identified as a heat-shock protein (hsp) by immunoblot analysis. PGA1 induced hsp70 synthesis in uninfected cells. However, in VSV-infected cells the induction of hsp70 by PGA1 was reduced. This is the first report of antiviral effects of PGs affecting the replication of VSV in a mosquito cell line.     

Coumarin content and physicochemical profile of Mikania laevigata extracts

The 'guaco' lianous herb Mikania laevigata, which is widespread in Southern Brazil, is traditionally used to treat bronchitis, asthma and cough. This work investigates the influence of the extraction method, solvent:drug ratio, ethanol proportion, harvest season (summer or winter) and solvent heating on the physicochemical profile of the extracts (dry weight, density, pH) and the coumarin (1,2-benzopyrone) content determined by LC. Among the results obtained, it is observed that higher ethanol content increases the amount of coumarin in the extract. Leaves harvested in summer also produce an extract with a high coumarin yield. The most efficient method of extraction is percolation, independent of the solvent used.     

Effect of angiotensin II and losartan on the phagocytic activity of peritoneal macrophages from Balb/C mice

Angiotensin II (AII), a product of rennin-angiotensin system, exerts an important role on the function of immune system cells. In this study, the effect of AII on the phagocytic activity of mouse peritoneal macrophages was assessed. Mice peritoneal macrophages were cultured for 48 h and the influence of different concentrations of AII (10(-14) to 10(-7) M) and/or losartan, 10(-16) to 10(-6) M), an AT1 angiotensin receptor antagonist, on phagocytic activity and superoxide anion production was determined. Dimethylthiazoldiphenyltetrazolium bromide reduction and the nucleic acid content were used to assess the cvtotoxicity of losartan. A stimulatory effect on phagocytic activity (P < 0.05) was observed with 10(-13) M and 10(-12 M) AII concentrations. The addition of losartan (up to10(-14) M) to the cell cultures blocked (P < 0.001) the phagocytosis indicating the involvement of AT1 receptors. In contrast, superoxide anion production was not affected by AII or losartan. The existence of AT1 and AT2 receptors in peritoneal macrophages was demonstrated by immunofluorescence microscopy. These results support the hypothesis that AII receptors can modulate murine macrophage activity and phagocytosis, and suggest that AII may have a therapeutic role as an immunomodulatory agent in modifying the host resistance to infection.