The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells

Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (ADSCs) using the chromatin-modifying agents trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2′-deoxycytidine (5azadC), a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available.

Investigating the role of the isomerase Rrd1/PTPA : from yeast to human

Chez Saccharomyces cerevisiae, les souches mutantes pour Rrd1, une protéine qui possède une activité de peptidyl prolyl cis/trans isomérase, montrent une résistance marquée à la rapamycine et sont sensibles au 4-nitroquinoline 1-oxide, un agent causant des dommages à l’ADN. PTPA, l’homologue de Rrd1 chez les mammifères, est reconnu en tant qu’activateur de protéine phosphatase 2A. Notre laboratoire a précédemment démontré que la surexpression de PTPA mène à l’apoptose de façon indépendante des protéines phosphatase 2A. La fonction moléculaire de Rrd1/PTPA était encore largement inconnue au départ de mon projet de doctorat. Mes recherches ont d’abord montré que Rrd1 est associé à la chromatine ainsi qu’à l’ARN polymérase II. L’analyse in vitro et in vivo par dichroïsme circulaire a révélé que Rrd1 est responsable de changements au niveau de la structure du domaine C-terminal de la grande sous-unité de l’ARN polymérase II, Rpb1, en réponse à la rapamycine et au 4-nitroquinoline 1-oxide. Nous avons également démontré que Rrd1 est requis pour modifier l’occupation de l’ARN polymérase II sur des gènes répondant à un traitement à la rapamycine. Finalement, nous avons montré que suite à un traitement avec la rapamycine, Rrd1 médie la dégradation de l’ARN polymérase II et que ce mécanisme est indépendant de l’ubiquitine. La dernière partie de mon projet était d’acquérir une meilleure connaissance de la fonction de PTPA, l’homologue de Rrd1 chez les mammifères. Nos résultats montrent que le «knockdown» de PTPA n’affecte pas la sensibilité des cellules à différentes drogues telles que la rapamycine, le 4-nitroquinoline 1-oxide ou le peroxyde d’hydrogène (H2O2). Nous avons également tenté d’identifier des partenaires protéiques pour PTPA grâce à la méthode TAP, mais nous ne sommes pas parvenus à identifier de partenaires stables. Nous avons démontré que la surexpression de la protéine PTPA catalytiquement inactive n’induisait pas l’apoptose indiquant que l’activité de PTPA est requise pour produire cet effet. Finalement, nous avons tenté d’étudier PTPA dans un modèle de souris. Dans un premier lieu, nous avons déterminé que PTPA était exprimé surtout au niveau des tissus suivants : la moelle osseuse, le thymus et le cerveau. Nous avons également généré avec succès plusieurs souris chimères dans le but de créer une souris «knockout» pour PTPA, mais l’allèle mutante ne s’est pas transférée au niveau des cellules germinales. Mes résultats ainsi que ceux obtenus par mon laboratoire sur la levure suggèrent un rôle général pour Rrd1 au niveau de la régulation des gènes. La question demeure toujours toutefois à savoir si PTPA peut effectuer un rôle similaire chez les mammifères et une vision différente pour déterminer la fonction de cette protéine sera requise pour adresser adéquatement cette question dans le futur.

In Vivo Comparison of Hard Tissue Regeneration with Human Mesenchymal Stem Cells Processed with Either the FICOLL Method or the BMAC Method

Objective: To compare new bone formation in maxillary sinus augmentation procedures using biomaterial associated with mesenchymal stem cells (MSCs) separated by two different isolation methods. Background: In regenerative medicine open cell concentration systems are only allowed for clinical application under good manufacturing practice conditions. Methods: Mononuclear cells, including MSCs, were concentrated with either the synthetic poylsaccharid (FICOLL) method (classic open system-control group, n = 6 sinus) or the bone marrow aspirate concentrate (BMAC) method (closed system-test group, n = 12 sinus) and transplanted in combination with biomaterial. A sample of the cells was characterized by their ability to differentiate. After 4.1 months (SD +/- 1.0) bone biopsies were obtained and analyzed. Results: The new bone formation in the BMAC group was 19.9% (90% confidence interval [CI], 10.9-29), and in the FICOLL group was 15.5% (90% CI, 8.6-22.4). The 4.4% difference was not significant (90% CI, -4.6-13.5; p = 0.39). MSCs could be differentiated into osteogenic, chondrogenic, and adipogenic lineages. Conclusion: MSCs harvested from bone marrow aspirate in combination with bovine bone matrix particles can form lamellar bone and provide a reliable base for dental implants. The closed BMAC system is suited to substitute the open FICOLL system in bone regeneration procedures.

Clinical correlations of human cytomegalovirus strains and viral load in kidney transplant recipients

Little is known about clinical differences associated with cytomegalovirus (CMV) infection by distinct strains in renal transplant patients. Different clinical pictures may be associated with specific viral genotypes. viral load, as well as host factors. The objective of this study was to identify CMV strains to determine viral load (antigenemia), and their correlation with clinical data in renal transplant recipients. Seventy-one patients were enrolled, comprising 91 samples. After selection, polymorphonuclear cells were used to amplify and sequence the gB region of CMV DNA. The sequences were analyzed to ascertain the frequency of different genotypes. Additionally, the results of this Study showed that the gB coding gene presents a great variability, revealing a variety of patterns: classical gB (1.4%), gB1V (46.4%), classical gB2 (35.2%), gB2V (2.8%), gB3 (1.4%), classical gB4 (4.9%) and gB4V (4.9%). The mean viral load in kidney transplant patient was 75.1 positive cells (1-1000). A higher viral load was observed in patients with genotype 4 infection. Statistically significant differences were detected between gB1 and gB4 (p=0.010), and between gB2 and gB4 (p=0.021). The average numbers of positive cells in relation to clinical presentation were: 34.5 in asymptomatic, 49.5 in CMV associated syndrome and 120.7 in patients with invasive disease (p=0.048). As a group, gB1 was the most frequent strain and revealed a potential risk for developing invasive disease. Viral load also seemed to be important as a marker associated with clinical presentation of the disease. (C) 2008 Elsevier B.V. All rights reserved.

Molecular characterization of human erythrovirus B19 strains obtained from patients with several clinical presentations in the Amazon region of Brazil

Background: Human erythrovirus B 19, endemic in the Amazon region since 1990, is associated with a wide spectrum of clinical presentations. Objectives: To assess the prevalence of erythrovirus B 19 infection and the relative frequency of erythrovirus B 19 genotypes in patients in the Amazon region with various clinical presentations. Study design: A total of 487 clinical samples obtained from patients with symptoms suggestive of erythrovirus infection were tested using specific IgM and IgG antibody assays (ELISA) and PCR for viral DNA detection. Partial VP1 and VP2 regions were sequenced and genotyped by phylogenetic reconstruction. Results: B 19 DNA was detected in 117 (24%) of 487 samples. Of these, 106 (91%) isolates were genotype I and II (9%) were genotype 3. No genotype 2 was found. Genotype I had three clusters (A1, A2 and 13) and all genotype 3 sequences were subtype 3b. All patients with hernatological disorders within cluster B of genotype I were infected by the sarne B 19 lineage, suggesting that this lineage of B 19 may have been transmitted via transfusion of blood products. Conclusion: We reported two genotypes, I and 3b, with three genotype I clusters co-circulating in the Amazon region during the past 10 years. (C) 2008 Elsevier B.V. All rights reserved.

Structure, Dynamics, and RNA Interaction Analysis of the Human SBDS Protein

Shwachman-Bodian-Diamond syndrome is an autosomal recessive genetic syndrome with pleiotropic phenotypes, including pancreatic deficiencies, bone marrow dysfunctions with increased risk of myelodysplasia or leukemia, and skeletal abnormalities. This syndrome has been associated with mutations in the SBDS gene, which encodes a conserved protein showing orthologs in Archaea and eukaryotes. The Shwachman-Bodian-Diamond syndrome pleiotropic phenotypes may be an indication of different cell type requirements for a fully functional SBDS protein. RNA-binding activity has been predicted for archaeal and yeast SBDS orthologs, with the latter also being implicated in ribosome biogenesis. However, full-length SBDS orthologs function in a species-specific manner, indicating that the knowledge obtained from model systems may be of limited use in understanding major unresolved issues regarding SBDS function, namely, the effect of mutations in human SBDS on its biochemical function and the specificity of RNA interaction. We determined the solution structure and backbone dynamics of the human SBDS protein and describe its RNA binding site using NMR spectroscopy. Similarly to the crystal structures of Archaea, the overall structure of human SBDS comprises three well-folded domains. However, significant conformational exchange was observed in NMR dynamics experiments for the flexible linker between the N-terminal domain and the central domain, and these experiments also reflect the relative motions of the domains. RNA titrations monitored by heteronuclear correlation experiments and chemical shift mapping analysis identified a classic RNA binding site at the N-terminal FYSH (fungal, Yhr087wp, Shwachman) domain that concentrates most of the mutations described for the human SBDS. (C) 2010 Elsevier Ltd. All rights reserved.

Bcl-2 expression and apoptosis induction in human HL60 leukaemic cells treated with a novel organotellurium(IV) compound RT-04

Organotellurium(]V) compounds have been reported to have multiple biological activities including cysteine protease-inhibitory activity, mainly cathepsin B. As cathepsin B is a highly predictive indicator for prognosis and diagnosis of cancer, a possible antitumor potential for these new compounds is expected. In this work, it was investigated the effectiveness of organotellurium(IV) RT-04 to produce lethal effects in the human promyelocytic leukaemia cell line HL60. Using the MTT tetrazolium reduction test, and trypan blue exclusion assay, the IC50 for the compound after 24 h incubation was 6.8 and 0.35 mu M, respectively. Moreover, the compound was found to trigger apoptosis in HL60 cells, inducing DNA fragmentation and caspase-3, -6, and -9 activations. The apoptsosis-induced by RT-04 is probably related to the diminished Bcl-2 expression, observed by RT-PCR, in HL60-treated cells. In vivo studies demonstrated that the RT-04 treatment (2.76 mg/kg given for three consecutive days) produces no significant toxic effects for bone marrow and spleen CFU-GM. However, higher doses (5.0 and 10 mg/kg) produced a dose-dependent reduction in the number of CFU-GM of RT-04-treated mice. These results suggest that RT-04 is able to induce apoptosis in HL60 cells by Bcl-2 expression down-modulation. Further studies are necessary to better clarify the effects of this compound on bone marrow normal cells. (C) 2008 Elsevier Ltd. All rights reserved.

HISTORICAL PERSPECTIVE and HUMAN CONSEQUENCES of AFRICANIZED BEE STINGS IN THE AMERICAS

In 1956, Africanized bees began to spread in the American continent from southern Brazil, where original African bees mated with European bees. A few years later, in 1990, these Africanized bees reached the United States and were found in Texas. Currently, these hybrid bees are found in several North American states and will probably reach the Canadian border in the future. Although the presence of Africanized bees had produced positive effects on Brazilian economy, including improvement in crop pollination and in honey production, turning Brazil into a major exporter, the negative impacts-such as swarming, aggressive behavior, and the ability to mass attack-resulted in serious and fatal envenomation with humans and animals. Victims of bee attacks usually develop a severe envenomation syndrome characterized by the release of a large amount of cytokines [interleukins (IL) IL-1, IL-6, IL-8], and tumor necrosis factor (TNF). Subsequently, such cytokines produce an acute inflammatory response that triggers adverse effects on skeletal muscles; bone marrow; hepatic and renal functions; and cardiovascular, central nervous, and immune systems. Finally, the aim of the present review is to study historical characteristics and current status of Africanized bees' spread, the composition of their venom, the impact of the bees on the Brazilian economy and ecology, and clinical aspects of their stings including immune response, and to suggest a protocol for bee sting management since there is no safe and effective antivenom available.

Cysteine proteinase inhibitors regulate human and mouse osteoclastogenesis by interfering with RANK signaling

The cysteine proteinase inhibitor cystatin C inhibited RANKL-stimulated osteoclast formation in mouse bone marrow macrophage cultures, an effect associated with decreased mRNA expression of Acp5, Calcr, Ctsk, Mmp9, Itgb3, and Atp6i, without effect on proliferation or apoptosis. The effects were concentration dependent with half-maximal inhibition at 0.3 μM. Cystatin C also inhibited osteoclast formation when RANKL-stimulated osteoclasts were cultured on bone, leading to decreased formation of resorption pits. RANKL-stimulated cells retained characteristics of phagocytotic macrophages when cotreated with cystatin C. Three other cysteine proteinase inhibitors, cystatin D, Z-RLVG-CHN2 (IC50 0.1 μM), and E-64 (IC 50 3 μM), also inhibited osteoclast formation in RANKL-stimulated macrophages. In addition, cystatin C, Z-RLVG-CHN2, and E-64 inhibited osteoclastic differentiation of RANKL-stimulated CD14+ human monocytes. The effect by cystatin C on differentiation of bone marrow macrophages was exerted at an early stage after RANKL stimulation and was associated with early (4 h) inhibition of c-Fos expression and decreased protein and nuclear translocation of c-Fos. Subsequently, p52, p65, IκBα, and Nfatc1 mRNA were decreased. Cystatin C was internalized in osteoclast progenitors, a process requiring RANKL stimulation. These data show that cystatin C inhibits osteoclast differentiation and formation by interfering intracellularly with signaling pathways downstream RANK. © FASEB.

Epidemiology of human immunodeficiency virus-visceral leishmaniasis-co-infection

In Brazil, the rates of mother-to-child-transmission (MTCT) of human immunodeficiency virus (HIV) decreased from 20% to 1-2% in some regions. However, the country contains 90% of individuals infected with visceral leishmaniasis (VL) in Latin America, and the west region of São Paulo state faces an alarming expansion of the disease. We describe the epidemiological aspects of the expanding infection of VL and a case report of an HIV-VL-co-infected child from the west region of São Paulo state. The patient was an AIDS-C3 with low levels of CD4, high viral load, severe diarrhea, oral and perineal candidiasis, severe thrombocytopenia, and protein-caloric malnourishment. She evolved with sepsis, renal and cardiac failure. An rK rapid diagnosis test, indirect fluorescent antibody test (IFAT), and bone marrow aspirate were performed for VL. Her symptoms improved significantly after liposomal amphotericin B administration. From the 45 municipalities that compose the Regional Health Department of Presidente Prudente, Lutzomyia longipalpis vectors were found in 58% of them. VL infected dogs were found in 33% of those municipalities, infected dogs and humans were found in 29%, 20% are starting and 33% of the municipalities are preparing VL investigation. It is likely, in this patient, that VL advanced the clinical progression of the HIV disease and the development of AIDS severity. Supported by favorable conditions, the region becomes a new frontier of VL in Brazil. © 2013.

Human immature dental pulp stem cells share key characteristic features with limbal stem cells

Objectives: Limbal stem cells (LSC) are self-renewing, highly proliferative cells in vitro, which express a set of specific markers and in vivo have the capacity to reconstruct the entire corneal epithelium in cases of ocular surface injury. Currently, LSC transplantation is a commonly used procedure in patients with either uni- or bilateral total limbal stem cells deficiency (TLSCD). Although LSC transplantation holds great promise for patients, several problems need to be overcome. In order to find an alternative source of cells that can partially substitute LSC in cornea epithelium reconstruction, we aimed at investigating whether human immature dental pulp stem cells (hIDPSC) would present similar key characteristics as LSC and whether they could be used for corneal surface reconstruction in a rabbit TLSCD model. Materials: We used hIDPSC, which co-express mesenchymal and embryonic stem cell markers and present the capacity to differentiate into derivative cells of the three germinal layers. TLSCD was induced by chemical burn in one eye of rabbits. After 30 days, the opaque tissue formed was removed by superficial keratectomy. Experimental group received undifferentiated hIDPSC, while control group only received amniotic membrane (AM). Both groups were sacrificed after 3 months. Results and conclusions: We have demonstrated, using immunohistochemistry and reverse transcription-polymerase chain reaction, that hIDPSCs express markers in common with LSC, such as ABCG2, integrin beta 1, vimentin, p63, connexin 43 and cytokeratins 3/12. They were also capable of reconstructing the eye surface after induction of unilateral TLSCD in rabbits, as shown by morphological and immunohistochemical analysis using human-specific antibodies against limbal and corneal epithelium. Our data suggest that hIDPSCs share similar characteristics with LSC and might be used as a potential alternative source of cells for corneal reconstruction.

Human telomere disease due to disruption of the CCAAT box of the TERC promoter

Mutations in the coding region of telomerase complex genes can result in accelerated telomere attrition and human disease. Manifestations of telomere disease include the bone marrow failure syndromes dyskeratosis congenita and aplastic anemia, acute myeloid leukemia, liver cirrhosis, and pulmonary fibrosis. Here, we describe a mutation in the CCAAT box (GCAAT) of the TERC gene promoter in a family in which multiple members had typical features of telomeropathy. The genetic alteration in this critical regulatory sequence resulted in reduced reporter gene activity and absent binding of transcription factor NF-Y, likely responsible for reduced TERC levels, decreased telomerase activity, and short telomeres. This is the first description of a pathogenic mutation in the highly con-served CCAAT box and the first instance of a mutation in the promoter region of TERC producing a telomeropathy. We propose that current mutation-screening strategies should include gene promoter regions for the diagnosis of telomere diseases. This clinical trial was registered at www.clinicaltrials.gov as #NCT00071045. (Blood. 2012;119(13):3060-3063)

Non-classical HLA-E gene variability in Brazilians: a nearly invariable locus surrounded by the most variable genes in the human genome

The non-classical human leukocyte antigen (HLA) class I genes present a very low rate of variation. So far, only 10 HLA-E alleles encoding three proteins have been described, but only two are frequently found in worldwide populations. Because of its historical background, Brazilians are very suitable for population genetic studies. Therefore, 104 bone marrow donors from Brazil were evaluated for HLA-E exons 14. Seven variation sites were found, including two known single nucleotide polymorphisms (SNPs) at positions +424 and +756 and five new SNPs at positions +170 (intron 1), +1294 (intron 3), +1625, +1645 and +1857 (exon 4). Haplotyping analysis did show eight haplotypes, three of them known as E*01:01:01, E*01:03:01 and E*01:03:02:01 and five HLA-E new alleles that carry the new variation sites. The HLA-E*01:01:01 allele was the predominant haplotype (62.50%), followed by E*01:03:02:01 (24.52%). Selective neutrality tests have disclosed an interesting pattern of selective pressures in which balancing selection is probably shaping allele frequency distributions at an SNP at exon 3 (codon 107), sequence diversity at exon 4 and the non-coding regions is facing significant purifying pressure. Even in an admixed population such as the Brazilian one, the HLA-E locus is very conserved, presenting few polymorphic SNPs in the coding region.

Hedgehog signaling and osteoblast gene expression are regulated by purmorphamine in human mesenchymal stem cells

Several biological events are controlled by Hedgehog (Hh) signaling, including osteoblast phenotype development. This study aimed at evaluating the gene expression profile of human mesenchymal stem cells (hMSCs) treated with the Hh agonist, purmorphamine, focusing on Hh signaling and osteoblast differentiation. hMSCs from bone marrow were cultured in non-osteogenic medium with or without purmorphamine (2 mu M) for periods of up to 14 days. Purmorphamine up-regulated gene expression of the mediators of Hh pathway, SMO, PTCH1, GLI1, and GLI2. The activation of Hh pathway by purmorphamine increased the expression of several genes (e.g., RUNX2 and BMPs) related to osteogenesis. Our results indicated that purmorphamine triggers Hh signaling pathway in hMSCs, inducing an increase in the expression of a set of genes involved in the osteoblast differentiation program. Thus, we conclude that Hh is a crucial pathway in the commitment of undifferentiated cells to the osteoblast lineage. J. Cell. Biochem. 113: 204208, 2012. (C) 2011 Wiley Periodicals, Inc.

Multiple Intravenous Administrations of Human Umbilical Cord Blood Cells Benefit in a Mouse Model of ALS

Background: A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) is the use of cell-based therapies that can protect motor neurons and thereby retard disease progression. We recently showed that a single large dose (25x10(6) cells) of mononuclear cells from human umbilical cord blood (MNC hUCB) administered intravenously to pre-symptomatic G93A SOD1 mice is optimal in delaying disease progression and increasing lifespan. However, this single high cell dose is impractical for clinical use. The aim of the present pre-clinical translation study was therefore to evaluate the effects of multiple low dose systemic injections of MNC hUCB cell into G93A SOD1 mice at different disease stages. Methodology/Principal Findings: Mice received weekly intravenous injections of MNC hUCB or media. Symptomatic mice received 10(6) or 2.5x10(6) cells from 13 weeks of age. A third, pre-symptomatic, group received 10(6) cells from 9 weeks of age. Control groups were media-injected G93A and mice carrying the normal hSOD1 gene. Motor function tests and various assays determined cell effects. Administered cell distribution, motor neuron counts, and glial cell densities were analyzed in mouse spinal cords. Results showed that mice receiving 10(6) cells pre-symptomatically or 2.5x10(6) cells symptomatically significantly delayed functional deterioration, increased lifespan and had higher motor neuron counts than media mice. Astrocytes and microglia were significantly reduced in all cell-treated groups. Conclusions/Significance: These results demonstrate that multiple injections of MNC hUCB cells, even beginning at the symptomatic disease stage, could benefit disease outcomes by protecting motor neurons from inflammatory effectors. This multiple cell infusion approach may promote future clinical studies.