Umbilical cord blood cd34(+) stem cells and other mononuclear cell subtypes processed up to 96 h from collection and stored at room temperature maintain a satisfactory functionality for cell therapy

Background and ObjectivesUmbilical cord blood (UCB) is a good stem cell source for cell therapy. We recently demonstrated that cord blood mononuclear cell (MNCs) subtypes were viable and functional until 96h after collection, even stored at room temperature. Now, we analyzed the viability and functionality of the cells before and after cryopreservation. Materials and MethodsTwenty UCB units were analyzed at 24 and 96h after collection, frozen for 6months, thawed and re-evaluated. MNCs were analyzed by flow cytometry, viability by 7-AAD and clonogenic assays (CFU) were performed. ResultsAfter 96h of storage, no substantial loss of MNC was found (median 7320x10(6)x6305x10(6)). Percentage and viability CD34(+) cells, B-cell precursors and mesenchymal stem cells were not affected. However, mature B and T lymphocytes as well as granulocytes had a substantial loss. CFU growth was observed in all samples. Prefreezing storage of 96h was associated with a relative loss of colony formation (median 12%). Postthaw, this loss had a median of 49% (24h samples) to 56% (96h samples). ConclusionThe delay of 96h before UCB processing is possible, without a prohibitive impairment of CD34(+) loss in number and functionality.Umbilical cord blood (UCB) is a good stem cell source for cell therapy. We recently demonstrated that cord blood mononuclear cell (MNCs) subtypes were viable and functional until 96h after collection, even stored at room temperature. Now, we analyzed the10817281sem informaçãosem informaçã

IRS2 silencing increases apoptosis and potentiates the effects of ruxolitinib in jak2v617f-positive myeloproliferative neoplasms

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)The recurrent V617F mutation in JAK2 (JAK2(V617F)) has emerged as the primary contributor to the pathogenesis of myeloproliferative neoplasms (MPN). However, the lack of complete response in most patients treated with the JAK1/2 inhibitor, ruxolitinib, indicates the need for identifying pathways that cooperate with JAK2. Activated JAK2 was found to be associated with the insulin receptor substrate 2 (IRS2) in non-hematological cells. We identified JAK2/IRS2 binding in JAK2(V617F) HEL cells, but not in the JAK2(WT) U937 cell line. In HEL cells, IRS2 silencing decreased STAT5 phosphorylation, reduced cell viability and increased apoptosis; these effects were enhanced when IRS2 silencing was combined with ruxolitinib. In U937 cells, IRS2 silencing neither reduced cell viability nor induced apoptosis. IRS1/2 pharmacological inhibition in primary MPN samples reduced cell viability in JAK2(V617F)-positive but not JAK2(WT) specimens; combination with ruxolitinib had additive effects. IRS2 expression was significantly higher in CD34(+) cells from essential thrombocythemia patients compared to healthy donors, and in JAK2(V617F) MPN patients when compared to JAK2(WT). Our data indicate that IRS2 is a binding partner of JAK2(V617F) in MPN. IRS2 contributes to increased cell viability and reduced apoptosis in JAK2-mutated cells. Combined pharmacological inhibition of IRS2 and JAK2 may have a potential clinical application in MPN.The recurrent V617F mutation in JAK2 (JAK2V617F) has emerged as the primary contributor to the pathogenesis of myeloproliferative neoplasms (MPN). However, the lack of complete response in most patients treated with the JAK1/2 inhibitor, ruxolitinib, indi7669486959sem informaçãoConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)sem informaçã

Molecular Characterization Of Hemoglobin α-d Chains From Geochelone Carbonaria And Geochelone Denticulata Land Turtles

In order to help elucidate the evolution of α-globins, the complete cDNA and amino acid sequences of Geochelone carbonaria and Geochelone denticulata land turtles α-D chains have been described. In G. carbonaria, the cDNA is 539 bp with ATG start codon located at position 46, TGA stop codon at position 469 and AATAAA polyadenylation signal at position 520. In G. denticulata, the cDNA is 536 bp with ATG start codon located at position 46, TGA stop codon at position 469 and AATAAA polyadenylation signal at position 517. Both cDNAs codify 141 amino acid residues, differing from each other in only four amino acid residues. When comparing with human Hb α-chain, alterations in important regions can be noted: α110 Ala-Gly, α114 Pro-Gly, α117 Phe-Tyr and α122 His-Gln. There is a high homology between the amino acids of these turtles when compared with chicken α-D chains, progressively decreasing when compared with human, crocodile, snake, frog and fish α-chains. Phylogenetic analysis of α-D chains shows that those of turtles are closer to those of birds than to snakes and lizards. © 2002 Elsevier Science Inc. All rights reserved.1342389395Baldwin, J.M., The structure of human carbonmonoxy haemoglobin at 2.7 A resolution (1980) J. Mol. Biol., 136, pp. 103-128Bordin, S., Meza, N.A., Saad, S.T.O., Ogo, S.H., Costa, F.F., CDNA-derived amino-acid sequence of a land turtle (Geochelone carbonaria) β-chain hemoglobin (1997) Biochem. Mol. Biol. Int., 42, pp. 255-260Bunn, H.F., Forget, B.G., Hemoglobin structure (1986) Hemoglobin: Molecular, Genetic and Clinical Aspects, pp. 13-35. , Philadelphia, PA: W.B. Saunders CompanyFushitani, K., Higashiyama, K., Moriyama, E.M., Imai, K., Hosokawa, K., The amino acid sequences of two α chains of hemoglobins from Komodo dragon Varanus komodoensis and phylogenetic relationships of amniotes (1996) Mol. Biol. Evol., 13, pp. 1039-1043Gorr, T.A., Mable, B.K., Kleinschmidt, T., Phylogenetic analysis of reptilian hemoglobins: Trees, rates, and divergences (1998) J. Mol. Evol., 47, pp. 471-485Hashimoto, M., Ishimori, K., Imai, K., Site-directed mutagenesis in hemoglobin: Functional and structural study of the intersubunit hydrogen bond of threonine-38(C3)alpha at the alpha 1-beta 2 interface in human hemoglobin (1993) Biochemistry, 32, pp. 13688-13695Komiyama, N.H., Miyazaki, G., Tame, J., Nagai, K., Transplanting a unique allosteric effect from crocodile into human haemoglobin (1995) Nature, 37, pp. 244-246Mylvaganam, S.E., Bonaventura, C., Bonaventura, J., Getzoff, E.D., Structural basis for the root effect in haemoglobin (1996) Nature Struct. Biol., 3, pp. 275-283Petruzzelli, R., Aureli, G., Lania, A., Galtieri, A., Desideri, A., Giardina, B., Diving behaviour and haemoglobin function: The primary structure of the α- and β-chains of the sea turtle (Caretta caretta) and its functional implications (1996) Biochem. J., 316, pp. 959-965Rawn, J.D., Amino acids and the primary structure of proteins (1989) Biochemistry, p. 54. , L.P. Daisy, K.C. Hodgin, T.L. O'Quin, S. Olsen, & J.A. Swan. Burlington, NC: Neil Patterson PublishersRücknagel, K.P., Reischl, E., Braunitzer, G., Hemoglobins of reptiles. Expression of alpha-D-genes in the turtles, Chrysemys picta bellii and Phrynops hilarii (Testudines) (1984) Hoppe-Seyler's Z. Physiol. Chem., 365, pp. 1163-1171Rücknagel, K.P., Braunitzer, G., The primary structure of the major and minor hemoglobin component of adult western painted turtle (Chrysemys picta bellii) (1988) Biol. Chem. Hoppe-Seyler, 369, pp. 123-131Saitou, N., Nei, M., The neighbor-joining method: A new method for reconstructing phylogenetic trees (1987) Mol. Biol. Evol., 4, pp. 406-425Shishikura, F., Takami, K., The amino acid sequences of the alpha- and beta-globin chains of hemoglobin from the aldabra giant tortoises, Geochelone gigantea (2001) Zool. Sci., 18, pp. 515-526Shishikura, F., The primary structure of hemoglobin D from the Aldabra giant tortoise, Geochelone gigantea (2002) Zool. Sci., 19, pp. 197-206Thompson, J.D., Higgins, D.G., Gibson, T.J., CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequences weighting, positions-specific gap penalties and weight matrix choice (1994) Nucleic Acids Res., 22, pp. 4673-4680Torsoni, M.A., Ogo, S.H., Oxygenation properties of hemoglobin from the turtle Geochelone carbonaria (1995) Braz. J. Med. Biol. Res., 28, pp. 1129-1131Torsoni, M.A., Viana, R.I., Stoppa, G.R., Barros, B.F., Cesquini, M., Ogo, S.H.J., Effect of thiol reagents on functional properties and heme oxidation in the hemoglobin of Geochelone carbonaria (1996) Biochem. Mol. Biol. Int., 40, pp. 355-364Torsoni, M.A., Souza-Torsoni, A., Ogo, S.H., Involvement of available SH groups in the heterogeneity of hemoglobin from the tortoise Geochelone carbonaria (1998) Biochem. Mol. Biol. Int., 44, pp. 851-860Torsoni, M.A., Ogo, S.H., Hemoglobin-sulfhydryls from tortoise (Geochelone carbonaria) can reduce oxidative damage induced by organic hydroperoxide in erythrocyte membrane (2000) Comp. Biochem. Physiol. B. Biochem. Mol. Biol., 126, pp. 571-577Zhang, Y., Frohman, M.A., CDNA library protocols (1997) Methods in Molecular Biology, vol. 69, pp. 61-87. , I.G. Cowell, & C.A. Austin. Totowa, NJ: Humana Press In

Mesenchymal stromal cells from adipose tissue attached to suture material enhance the closure of enterocutaneous fistulas in a rat model

Surgical treatment for enterocutaneous fistulas (EF) frequently fails. Cell therapy may represent a new approach to treatment. Mesenchymal stromal cells (MSCs) have high proliferative and differentiation capacity. This study aimed to investigate whether M161217091719sem informaçãosem informaçã

Imatinib restores vasp activity and its interaction with zyxin in bcr-abl leukemic cells

Vasodilator-stimulated phosphoprotein (VASP) and Zyxin are interacting proteins involved in cellular adhesion and motility. PKA phosphorylates VASP at serine 157, regulating VASP cellular functions. VASP interacts with ABL and is a substrate of the BCR-ABL oncoprotein. The presence of BCR-ABL protein drives oncogenesis in patients with chronic myeloid leukemia (CML) due to a constitutive activation of tyrosine kinase activity. However, the function of VASP and Zyxin in BCR-ABL pathway and the role of VASP in CML cells remain unknown. In vitro experiments using K562 cells showed the involvement of VASP in BCR-ABL signaling. VASP and Zyxin inhibition decreased the expression of anti-apoptotic proteins, BCL2 and BCL-XL. Imatinib induced an increase in phosphorylation at Ser157 of VASP and decreased VASP and BCR-ABL interaction. VASP did not interact with Zyxin in K562 cells; however, after Imatinib treatment, this interaction was restored. Corroborating our data, we demonstrated the absence of phosphorylation at Ser157 in VASP in the bone marrow of CML patients, in contrast to healthy donors. Phosphorylation of VASP on Ser157 was restored in Imatinib responsive patients though not in the resistant patients. Therefore, we herein identified a possible role of VASP in CML pathogenesis, through the regulation of BCR-ABL effector proteins or the absence of phosphorylation at Ser157 in VASP.Vasodilator-stimulated phosphoprotein (VASP) and Zyxin are interacting proteins involved in cellular adhesion and motility. PKA phosphorylates VASP at serine 157, regulating VASP cellular functions. VASP interacts with ABL and is a substrate of the BCR-AB18532388395CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOsem informaçãosem informaçã

Dnaase I Hypersensitive Site 3′ To The β-globin Gene Cluster Contains A Taa Insertion Specific For βs-benin Haplotype

Background and Objectives. Analysis of DNA polymorphic sites is a powerful tool for detection of gene flow in human evolutionary studies and to trace genetic background associated with abnormal genes. The β-globin locus contains more than 20 single-base restriction fragment length polymorphism (RFLP) sites spanning over 80 kb on chromosome 11. Far downstream of the expressed genes, there is a hypersensitive site (HS). The function of the 3′-HS remains unknown. As an approach to the understanding of the 3′-HS region in sickle cell anemia we searched for sequence polymorphism in the AT-rich region, using a non-radioactive polymerase chain reaction (PCR)-single strand conformational polymorphism (SSCP) technique. Design and Methods. A 460 bp fragment located at the 3′ of the β globin gene was amplified from patients (with sickle cell anemia and HbSC disease), and from AS individuals. Standard RFLP-haplotyping was performed and compared with the PCR-SSCP screening strategy. Results. Two distinct band patterns were revealed by SSCP testing, each one in strict linkage disequilibrium with either Benin or Bantu haplotypes. Direct sequencing of the amplified segment revealed a TAA insertion in the AT-rich region, in all 121 βs Benin chromosomes tested, but not in other βs haplotypes from the total of 380 βs chromosomes typed. Interpretation and Conclusions. SSCP analysis could easily distinguish sequence variations in the 3′AT-rich region of the β-globin cluster, and a TAA insertion in this region seems to be specific for the Benin-βs chromosome. ©2002, Ferrata Storti Foundation.873246249Cavalli-Sforza, L.L., Menozzi, P., Piazza, A., Genetic history of world populations. Area and time of origin of major mutants, with special attention to hemoglobins (1994) The history and geography of human genes, pp. 145-154. , L. Cavalli-Sforza, P. Menozzi, A Piazza, eds. Princeton: Princeton University PressLabie, D., Elion, J., Sequence polymorphisms of potential functional relevance in the β-globin gene locus (1996) Hemoglobin, 20, pp. 85-101Felsenfeld, G., Boyes, J., Chung, J., Clark, D., Studitsky, V., Chromatin structure and gene expression (1996) Proc Natl Acad Sci USA, 93, pp. 9384-9388Fleenor, D.E., Kaufman, R.E., Characterization of the DNase I hypersensitive site 3′of the human β globin gene domain (1993) Blood, 81, pp. 2781-2790Gonçalves, M.S., Nechtman, J.F., Figueiredo, M.S., Kerbauy, J., Arruda, V.R., Sonati, M.F., Sickle cell disease in a Brazilian population from Sao Paulo: A study of the βs haplotypes (1994) Hum Hered, 44, pp. 322-327Arruda, V.R., Von Zuben, P.M., Annichino-Bizzacehi, J.M., Costa, F.F., Rapid detection of factor V Leiden (FVQ506) by non-radioactive single strand conformation polymorphism (SSCP) (1996) Sangre, 41, pp. 379-381Gerhard, D.S., Kidd, K.K., Kidd, J.R., Egeland, J.A., Housman, D.E., Identification of a recent recombination event within the human β-globin gene cluster (1984) Proc Natl Acad Sci USA, 81, pp. 7875-7879Ofori-Acquah, S.F., Lalloz, M.R.A., Layton, D.M., Localization of cis-active determinants of fetal hemoglobin level in sickle cell anemia (1996) Blood, 88 (SUPPL. 1), p. 493. , abstractBordin, S., Crespi, V.G., Bassères, D.S., Different rates of recombination among polymorphic short tandem repeats of the β-globin gene cluster in βs chromosomes (1997) Blood, 90 (SUPPL. 1), p. 444. , abstractZago, M.A., Silva Jr., W.A., Dalle, B., Gualandro, S., Hutz, M.H., Lapoumeroulie, C., Atypical β(s) haplotypes are generated by diverse genetic mechanisms (2000) Am J Hematol, 63, pp. 79-84Zago, M.A., Silva Jr., W.A., Gualandro, S., Rearrangements of the beta-globin gene cluster in apparently typical betaS haplotypes (2001) Haematologica, 86, pp. 142-145Nagel, R.L., Steinberg, M.H., Genetics of the βs gene: Origins, genetic epidemiology, epistasis in sickle cell anemia (2001) Disorders of hemoglobin, , Steinberg MH, Forget BC, Higgs DR, Nagel RL, editors. Cambridge, UK, Cambridge University Pres

Useful properties of undifferentiated mesenchymal stromal cells and adipose tissue as the source in liver-regenerative therapy studied in an animal model of severe acute fulminant hepatitis

End-stage liver diseases frequently require liver transplantation. Cell therapy could be an alternative. This study aimed to analyze whether undifferentiated mesenchymal stromal cells (U-MSCs) or MSC-derived hepatocyte-like cells (DHLCs) from adipose tissue (AT), umbilical cord blood (UCB) and bone marrow (BM) would better restore damaged liver. AT was obtained from lipo-aspiration, UCB from an Umbilical Cord Blood Bank and BM from a BM Transplantation Unit. AT (collagenase digestion), UCB and BM (Ficoll gradient) were cultured (Dulbecco's modified Eagle's medium, low glucose, FBS) for 3 days. Detached adherent cells, at passage 4, were characterized as MSCs. Genetic stability was investigated by means of telomerase enzyme activity and karyotype. Hepatocyte differentiation protocol was performed with the use of Dulbecco's modified Eagle's medium, hepatocyte growth factor, basic fibroblast growth factor and nicotinamide (7 days); maturation medium (oncostatin, dexamethasone, insulin, transferrin and selenium) was added at 36 days. Hepatogenesis analyses were performed by use of morphology and albumin, AF, tyrosine-aminotransferase and glutamine synthetase gene expression and quantitative reverse transcription-polymerase chain reaction on days 9, 18, 25 and 36. Functionality was assessed through glycogen storage detection, indocyanine green absorption and transplantation procedure. U-MSCs and DHLCs were injected 48 h after induced fulminant hepatitis (intraperitoneal injection of carbon tetrachloride) in SCID/BALB-c mice. Histopathologic analyses were performed on days 7 and 15. Human origin included albumin and CK19 human markers. All MSCs differentiated into functional hepatocyte-like cells, stored glycogen and absorbed indocyanine green. AT-MSC DHLC gene expression was more consistent with a normal hepatogenic-differentiation profile. UCB-MSCs expanded weakly, impairing their use for the transplantation procedure. AT and BM U-MSCs and DHLCs regenerated liver injury equally. Regenerated hepatocytes exhibited human origin. AT might be the source and U-MSCS the stem cells useful for liver-regenerative therapy.End-stage liver diseases frequently require liver transplantation. Cell therapy could be an alternative. This study aimed to analyze whether undifferentiated mesenchymal stromal cells (U-MSCs) or MSC-derived hepatocyte-like cells (DHLCs) from adipose tiss17810521065sem informaçãosem informaçã