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Pro-apoptotic signalling upon toll-like receptor (TLR) stimulation in myeloid cells is normally antagonized by the simultaneous activation of anti-apoptotic pathways. We have previously reported that IFN-alpha can sensitize human monocytes to apoptosis induction by lipopolysaccharide (LPS). Based on these results we investigated whether similarly apoptosis can be cooperatively induced in myeloid tumor cells. When testing established acute myeloid leukemia (AML) cell lines we found the monocytic cell line THP-1 to be sensitive to IFN-alpha plus LPS induced apoptosis, which was partially dependent on caspase-8 and was associated with an enhanced expression of Fas/CD95. We extended our study to 29 short term blast lines from patients with AML and observed additive effects of IFN-alpha and LPS on cell death only with few samples indicating that sensitivity to IFN-alpha plus LPS inducible apoptosis is present in a fraction of AML samples only with no obvious correlation with certain FAB phenotypes.

s / Placenta 36 (2015) 469e521 509 endocytosis blocker) and 1 mmol/L nitrobenzylthioinosine (NBTI, an adenosine transport inhibitor), human equilibrative nucleoside transporter 1 (hENT1) immunofluorescence, protein abundance and mRNA expression levels were measured in primary cultures of HUVEC from normal or GD pregnancies coursing without (N or GD groups, respectively) or with OP (OP and GD+OP groups, respectively). Results: hENT1-mediated adenosine uptake was reduced in OP, GD and GD+OP compared with N, a result that was seen even in the presence of the endocytosis inhibitor. hENT1 expression was increased in OP (~1.7 and ~3fold protein abundance and mRNA expression, respectively), but reduced in GD and GD+OP compared with N. Moreover, hENT1 was unable to reach the plasma membrane in OP, meanwhile in GD and GD+OP, hENT1 accumulated in the plasma membrane. Conclusion: GD and OP are associated with alteration in hENT1 recycling, and GD+OP reduces hENT1 expression in HUVEC. PB.9. HUMAN AMNIOTIC EPITHELIAL CELLS: PROLIFERATION AND APOPTOSIS DURING THEIR HEPATIC DIFFERENTIATION J. Maym o , A. P erez-P erez , R. Riedel , B. Maskin , M. Jaime , O. Parolini , V. S anchez-Margalet , C. Varone . Departamento de Quimica Biol ogica, FCEN, UBA, IQUIBICEN, CONICET, Buenos Aires, Argentina; Departamento de Bioquimica M edica y Biologia Molecular, Universidad de Sevilla, Sevilla, Spain; Hospital Alejandro Posadas, Buenos Aires, Argentina; Centro di Ricerca E. Menni e Fondazione Poliambulanza e Istituto Ospedaliero, Brescia, Italy Objective: The placenta and fetal membranes have recently been proposed as an important stem cell source for regenerative medicine. Gestational cells offer considerable advantages over other stem cells such as bone marrow or embryo-derived cells. The collection and use of such tissues have a virtually unlimited potential, an easy access, and minimal ethical and legal barriers. Epithelial amniotic cells (hAECs) can be isolated from the amnion of the human placenta at term. They express embryonic stem cell markers and have the ability to differentiate toward all three germ layers. hAECs are non-tumorigenic and have immunosuppressive properties. These characteristics would make hAECs ideal candidates for tissue engineering and application in regenerative medicine. Hepatic failure is one of the major causes of morbidity and mortality worldwide. Recently, stem cells have been spotlighted as alternative sources of hepatocytes because of their specific potential for differentiation. The aim of this work was to study the proliferation and apoptosis of hAECs, during early and late hepatic differentiation. We also analyzed expression of hepatic markers expression through this process. Methods and results: Hepatic differentiation was assayed by specific factors (EGF + dexamethasone) or by HepG2 conditioned medium (CM). We found a significant increment of caspase-3 fragmentation, after 72 h of CM treatment, measured by western blot. We observed an augmentation in p53 and p21 genes expression and a decline in cyclin D1, measured by qRT-PCR. The opposite effects were observed with the specific factors treatment. We also determined by 3H-thymidine incorporation that EGF significantly increased cell proliferation while CM diminished it. During hAEC differentiation we observed a significant increment in hepatocyte-related gene expression (a-fetoprotein, a1-AT, albumin, CYP7A1) and in albumin expression, measured by qRT-PCR and Western blot, respectively. Conclusions. Our results begin to unravel the molecular and cellular processes that take place during hepatic differentiation of hAECs. PB.10. CAN MAGNESIUM GLUCONATE IMPROVE THE OSMOTIC FRAGILITY OF RED BLOOD CELLS FROM PREECLAMPTIC PREGNANT WOMEN? Cilia Abad, Cindy Ampueda, Sandy Pi~ nero, Delia I. Chiarello, Reinaldo Marin, Fulgencio Proverbio. Laboratory of Cell Bioenergetics, Center of Biophysics and Biochemistry (CBB), Venezuelan Institute for Scientific Research (IVIC), AP 20632, Caracas 1020A, Venezuela Magnesium gluconate (MgGl2) has been proposed as an oral substitute of magnesium sulfate for the treatment of preeclampsia. Our group has shown that preeclampsia is associated with a rise in lipid peroxidation of the placental plasma membranes and red blood cell ghosts. The osmotic fragility of the intact red blood cell is influenced by its membrane characteristics. Evenwhen the erythrocyte is not thought to contribute per se to the etiology of preeclampsia, it may nevertheless be important in the progression of the disease, particularly in view of the occurrence of oxidative stress. Objectives: To determine the effect of MgGl2 on the osmotic fragility of red blood cells from preeclamptic pregnant women. Methodology: Venous blood was drawn from uncomplicated and preeclamptic pregnant women. Red blood cells were incubated for 24 h in the presence or absence of MgGl2. The red cell osmotic fragility was determined. Results and conclusions: We found an increased level of osmotic fragility in red blood cells from preeclamptic pregnant women, as compared to that from uncomplicated pregnant women. Osmotic fragility from red blood cells from preeclamptic pregnant women returned to normal values after 24 h of incubationwith MgGl2. MgGl2 improves the osmotic fragility in red cells from preeclamptic pregnant women. PB.11. HUMAN TROPHOBLAST CELL MIGRATION AND INVASION ARE INDUCED BY ENDOGENOUS VASOACTIVE INTESTINAL PEPTIDE (VIP) THROUGH AUTOCRINE CIRCUITS AND VPAC2 OVEREXPRESSION Vota Daiana , Hauk Vanesa , Toro Ayelen , Grasso Esteban , Paparini Daniel , Ramhorst Rosanna , Varone Cecilia , P erez Leir os Claudia . 1 Laboratorio de Inmunofarmacologia, Dpto. Quimica Biol ogica, IQUIBICEN e CONICET, Facultad de Ciencias Exactas y Naturales-UBA, Argentina; 2 Laboratorio de Fisiologia Molecular Placentaria, Dpto. Quimica Biol ogica, IQUIBICEN e CONICET, Facultad de Ciencias Exactas y NaturalesUBA, Buenos Aires, Argentina The generation of the human maternal-placental interface requires trophoblast differentiation into phenotypes able to migrate and invade the deciduas. VIP is a pleiotropic polypeptide with immunomodulatory effects through VPAC1 and VPAC2 receptors. Previously, we have reported on its immune homeostasis maintenance role at early pregnancy. The expression of VIP in various cell types is induced by growth factors or neurotransmitters and depends on CRE (cAMP responsive elements) and gp130 cytokine (CyRE) sites on its promoter. Objective: To investigate VIP/VPAC2 expression on first trimester cytotrophoblast cells (Swan-71; HTR-8) and its impact on trophoblast cell migration and invasion. Methods: VIP expression was measured by flow cytometry and RT-qPCR; cell migration was determined by wound healing assays and invasiveness in Matrigel-covered transwells. Trophoblast cells were transfected with XtremeGENE HPDNA reagent with VPAC2 or CRE-Luciferase plasmids for 24h prior to the stimuli, normalized to GFP or beta-gal. Results: Endogenous VIP expression was detected in trophoblast cells and its synthesis was induced after 18 h incubation with 10nM VIP (P

The soy isoflavone genistein targets adipose tissue and elicits physiological effects that may vary based on dietary intake. We hypothesized that the adipose effects of genistein are dose and gender dependent. Four-week-old C57BL/6 male and female mice received daily oral doses of genistein (50-200,000 microg/kg.d) or 17beta-estradiol (E2) (5 microg/kg.d) for 15 d or a diet containing 800 ppm genistein. Genistein increased epididymal and renal fat pad and adipocyte size at doses up to 50,000 microg/kg.d or at 800 ppm in the diet in males but not in females. The alteration in adipocity correlated with changes in peripheral insulin resistance. These treatments increased genistein serum concentrations from 35+/-6 to 103+/-26 nM 12 h after treatment and lowered plasma triglycerides and cholesterol levels. The 200,000 microg/kg.d genistein dose decreased adipose tissue weight similarly to E2. This genistein dose down-regulated estrogen receptor (beta more than alpha) and progesterone receptor expression and induced estrogen-dependent adipose differentiation factors; it did not change expression of the minimal consensus estrogen-responsive element in ERE-tK-LUC mice, which was positively modulated in other tissues (e.g. the lung). E2 down-regulated almost all examined adipogenic factors. Gene microarray analysis identified factors in fat metabolism and obesity-related phenotypes differentially regulated by low and high doses of genistein, uncovering its adipogenic and antiadipogenic actions. The lower dose induced the phospholipase A2 group 7 and the phospholipid transfer protein genes; the 200,000 microg/kg.d dose inhibited them. The antiadipogenic action of genistein and down-regulation of adipogenic genes required the expression of ERbeta. In conclusion, nutritional doses of genistein are adipogenic in a gender-specific manner, whereas pharmacological doses inhibited adipose deposition.

Immunosuppression, myeloablation, and the use of immunologically immature tissue can overcome major histocompatibility complex barriers by inducing tolerance. With the goal of inducing tolerance to BALB/c-derived murine hybridoma cells producing the 4C6 monoclonal antibody (mAb), we transplanted BALB/c fetal tissue into neonatal pigs during a regimen of low-dose conditioning with busulfan and cyclophosphamide. After the tolerance induction phase, animals received intraperitoneal injections of 4C6 mAb hybridoma cells. Evidence of persistence of injected cells over time was sought by molecular analysis of peripheral blood for the presence of mouse genomic sequences and circulating 4C6 mAb. Persistence of donor polymerase chain reaction signal during the entire duration of the study, detectable mAb titer for 6 weeks, and a twofold increase of mAb concentration after a boost hybridoma infusion was observed in one animal receiving six consecutive administrations of the conditioning regimen. Our model has the distinctive advantage of allowing functional monitoring of engrafted cells for studying tolerance induction strategies. In addition, this model could be the basis for approaches aimed at producing mAbs in tolerized large animals.

BACKGROUND: Fetal membranes are tissues of particular interest for several reasons, including their role in preventing rejection of the fetus and their early embryologic origin. which may entail progenitor potential. The immunologic reactivity and the transplantation potential of amnion and chorion cells, however, remain to be elucidated. METHODS: Amnion and chorion cells were isolated from human term placenta and characterized by immunohistochemistry, flow cytometric analysis, and expression profile of relevant genes. The immunomodulatory characteristics of these cells were studied in allogeneic and xenogeneic mixed lymphocyte reactions and their engraftment potential analyzed by transplantation into neonatal swine and rats. Posttransplant chimerism was determined by polymerase chain reaction analysis with probes specific for human DNA. RESULTS: Phenotypic and gene expression studies indicated mesenchymal stem cell-like profiles in both amnion and chorion cells that were positive for neuronal, pulmonary, adhesion, and migration markers. In addition, cells isolated both from amnion and chorion did not induce allogeneic nor xenogeneic lymphocyte proliferation responses and were able to actively suppress lymphocyte responsiveness. Transplantation in neonatal swine and rats resulted in human microchimerism in various organs and tissues. CONCLUSIONS: Human amnion and chorion cells from term placenta can successfully engraft neonatal swine and rats. These results may be explained by the peculiar immunologic characteristics and mesenchymal stem cell-like phenotype of these cells. These findings suggest that amnion and chorion cells may represent an advantageous source of progenitor cells with potential applications in a variety of cell therapy and transplantation procedures.

Organochlorines are lipophylic molecules that accumulate in the fat where they remain for years. During weight loss, they are mobilized and their concentration increases in blood. The present work tests, in transgenic estrogen-reporter mice (ERE-tK-LUC), whether this increase is sufficient to modulate the estrogen receptors (ERs) in the whole body. Three weak estrogens were studied: p,p'DDT [1,1,1-trichloro2,2-bis(p-chlorophenyl) ethane], p,p'DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene], and betaBHC [beta-benzene-hexachloride]. Dose-dependent analysis of reporter expression (luciferase) were performed in tissues of acutely treated mice. A body map of ER activation was obtained. All these chemicals modulated the reporter, although with a different efficiency and depending upon the tissue analyzed. Induction was confirmed in the liver by determining the expression of the endogenous progesterone receptor (PR) gene, at the dose and time point at which the luciferase gene was maximally induced. After experimental accumulation in the fat tissue, followed by a 48-h period of fasting, we tested whether these compounds could be mobilized to reach sufficient levels to activate the ERs in selected reproductive and nonreproductive tissues (testicle, prostate, liver, and lung). This experimental setting produced results that were different than those obtained following acute treatments. In loaded mice, fasting induced betaBHC mobilization resulted in strong ER activation in the liver and the lung, which was blocked by ICI-182780. p,p'DDT mobilization had no effect in these tissues, but it acted efficiently in the prostate and testis. betaBHC inhibited the ERE-mediated reporter in the testicle and induced the reporter in the prostate. In this tissue, betaBHC action was not inhibited by the anti-estrogen ICI-182780. During fasting, betaBHC, p,p'DDT, and metabolite p,p'DDE increased in blood concentration, from 2.25 +/- 0.25, 0.51 +/- 0.09, and 0.38 +/- 0.06 microg/ml to 8.24 +/- 0.95, 4.52 +/- 0.68, and 5.06 +/- 0.57 microg/ml, respectively. The effect produced by these organochlorines in the liver correlates with the modulation of the ERalpha protein. We conclude that these organochlorines modulate differently the expression of estrogen-regulated genes in male mice. Their effect is tissue- and compound-specific and is dependent on the energetic balance.

SH2D1A, the X-linked lymphoproliferative disease (XLP) gene, encodes a cytoplasmic protein that plays an essential role in controlling Epstein-Barr virus infection. It is expressed in T and NK cells, but not in B cells or in granulocytes. The promoter, the regulatory regions, as well as the mechanisms controlling its tissue-specific expression, are still unknown. We tested the hypothesis that DNA methylation might contribute to tissue-specific SH2D1A gene expression and analyzed the methylation status of 2,300 bp upstream of the ATG starting codon, the coding region and part of intron 1. By bisulfite sequencing and methylation-sensitive restriction enzyme digestion, we show that a differential methylation pattern of CpG-rich regions in the 5′ region and the adjacent exon 1 of the SH2D1A gene indeed correlates with the tissue-specific gene transcription.

Mutations or deletions in the SH2D1A (src homology 2 domain protein 1A) gene result in a severe immunodeficiency called X-linked lymphoproliferative (XLP) disease. XLP is primarily characterized by a defective immune response against the Epstein-Barr virus (EBV), resulting in an unusually severe and often fatal clinical course following EBV infection. The second major cause of death is the development of B cell lymphomas, both in EBV-infected and EBV-negative patients. To study whether the clinical manifestation of XLP gene defects and/or polymorphisms extends beyond the classically recognized phenotype, we analyzed patients for the presence of SH2D1A gene alterations who presented with fatal or nonfatal, yet unusually severe or chronic EBV infections, and other possibly EBV-associated diseases, such as Hodgkin's lymphomas or nonendemic Burkitt's lymphomas and Burkitt-type leukemias. We identified mutations of the SH2D1A gene only in the majority of patients presenting with fatal mononucleosis or an XLP family history, but not in any of the other patients studied. The only alteration determined was a polymorphism in the 5' region of the SH2D1A gene both in patient groups as well as in controls.

Within the complex scenario of regenerative medicine and the sophisticated field of stem cell research and tissue engineering, it is interesting that biological waste as human placenta raises up and tells us its ancient application story and underlies that it is not essential only during the fetal development but also for future therapeutic application. Even more the amniotic membrane that constitutes the inner part of the amniotic sac has a long story of therapeutic applications. This Special Issue of the journal Cell and Tissue Banking indeed is dedicated to this versatile tissue and collects some contributions dealing with amniotic membrane cells or rather the membrane itself. We span from the most well established use of the amniotic membrane in ophthalmology to the possible future use in regeneration of several tissues as well as to the understanding of the mechanisms of action of the cells derived from the amniotic membrane and to the envisaged possible use already for prenatal diseases. From all these evidences it is demanding to learn more about the characteristics and biological properties of the cells that constitutes the amniotic membrane. Furthermore, it is fundamental to identify the best strategies to better preserve and test this tissue and/or its derived cells for further clinical use. Definitively, we still have to learn more about the properties of this versatile tissue and its cells and subpopulations, about their phenotype, their characteristics and potentiality; not to forget additional formal challenges which need to be met, like GMPcompliance as well as national and international requirements for tissues and ATMPs. Of course the therapeutic potential still needs to be unraveled but we believe that in a few years amniotic membrane will be a very valuable, easy available source in regenerative medicine and tissue engineering leaving all ethical issues behind.

X-linked severe combined immunodeficiency (SCIDX1) is an inherited disease characterized by profound abnormalities of cell-mediated and humoral immunity. Patients with SCIDX1 have defects in the common cytokine receptor gamma chain gene (IL2RG) that encodes a shared, essential component of the receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9 and IL-15. We have characterized nine SCIDX1 families by using a DNA-based, non-radioactive screening method and DNA sequencing. Nine different mutations were found, scattered from exon 1 to exon 5 of the IL2RG gene. Two of these mutations have been previously identified in other unrelated patients; the other seven are novel mutations that differ from all of the 95 already reported in the IL2RG mutation data base. In addition to describing novel mutations in the IL2RG gene, this study shows that the knowledge of the genetic defect and the use of an efficient, non-radioactive, and rapid screening approach have important implications for prenatal and postnatal diagnosis, carrier female identification, and possibly prenatal therapy.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder described as a clinical triad of thrombocytopenia, eczema, and immunodeficiency. The gene responsible for WAS encodes a 502-amino acid proline-rich protein (WASp) that is likely to play a role in the cytoskeleton reorganization and/or in signal transduction of hematopoietic cells. However, the function and the regulation of the WAS gene (WASP) have not yet been clearly defined. We have studied WASP expression at the transcriptional level in freshly isolated mature peripheral blood cells and during hematopoietic development. For this purpose, we have isolated CD34+ hematopoietic precursor cells from cord blood. These cells were cultured in vitro with various growth factors to generate committed or mature cells belonging to different hematopoietic differentiation pathways, such as granulocytic (CD15+) cells, monocytic (CD14+) cells, dendritic (CD1a+) cells, erythroid lineage (glycophorin A+) cells, and megakaryocytic cells (CD41+). We have shown by reverse transcriptase polymerase chain reaction analysis that the WASP transcript is ubiquitously detectable throughout differentiation from early hematopoietic progenitors, including CD34+CD45RA− and CD34+CD45RA+ cells, to cells belonging to different hematopoietic lineages, including erythroid-committed and dendritic cells. In addition, Northern blot analysis showed that peripheral blood circulating lymphocytes (CD3+ and CD19+ cells) and monocytes express WASP mRNA. Several hematopoietic cell lines were tested and higher levels of expression were consistently detected in myelomonocytic cell types. By contrast, primary nonhematopoietic cells, including fibroblasts, endothelial cells, and keratinocytes, were consistently negative for WASP mRNA.

BTK, the gene that is defective in X-linked agammaglobulinemia, encodes a cytoplasmic tyrosine kinase that is critical for B-cell proliferation, or survival. To identify regulatory elements that control the expression of BTK we evaluated the methylation pattern of this gene in cell lines and in freshly isolated cells. An Hpa II site that was specifically demethylated in mature B cells but not in pre-B cells, T cells, neutrophils, or nonhematopoietic cells was identified in the tenth intron of BTK. In a 40 kilobase (kb) segment of DNA spanning the entire coding region of BTK plus 3 kb upstream of the first exon there were no other sites that demonstrated lineage-specific demethylation. The B-cell-specific demethylation site in intron 10, which falls within the SH2 domain, 26 kb distal to the first exon, occurs in a region rich in regulatory elements including two E2 boxes, two AP-2 sites, and a cAMP response element. It is likely that this site plays a role in maintaining BTK transcription in mature B cells.

Mutations in the gene for Bruton's tyrosine kinase (Btk) are responsible for X-linked agammaglobulinemia (XLA). Thus far, mutations in this gene have been identified based on alterations in Southern or Northern blot analysis or cDNA sequence. To permit detection of mutations in genomic DNA, we designed PCR primers to flank each of the 19 exons of Btk with splice sites. Two overlapping PCR products were employed for exons longer than 230 base pairs. Single strand conformation polymorphism (SSCP) analysis was used to screen PCR products from 30 unrelated families presumed to carry a Btk mutation. It was possible to amplify DNA in every reaction from every patient, indicating that large deletions in Btk are uncommon. Twenty three different mutations were found in 25 unrelated families, including one family in whom DNA was available from a carrier but not an affected patient. Seven mutations were single base pair substitutions resulting in premature stop codons scattered throughout the gene. Small insertions or deletions causing frameshifts and secondary premature stop codons constituted an additional seven mutations. One patient had a point mutation in the start codon and one patient had a mutation in a splice donor site. Point mutations resulting in amino acid substitutions were seen in nine patients. Northern blot analysis of RNA from three patients with premature stop codons showed an absence of Btk transcript whereas four patients with amino acid substitutions had normal amounts of transcript of normal size. These studies document the considerable variability in the Btk mutations causing XLA and they demonstrate an approach that will be useful for carrier detection as well as mutation identification.

The identification of a cytoplasmic tyrosine kinase, Btk, as the defective protein in human XLA and xid in the mouse, supports the hypothesis that both disorders are due to defects in B-cell activation or differentiation. Phenotypic analysis of B-lineage cells and studies on X-chromosome inactivation patterns in both mice and human patients suggest that mutations in Bth do not affect entry of stem cells into the B-lineage pathway but they do inhibit progression at multiple steps along that pathway. Although the exact function of Btk in signal transduction is not yet known, it is probable that studies which correlate specific mutations in different patients with alterations in Btk function will provide clues about critical sites in the molecule. Diagnosis and genetic counseling for families at risk of carrying the gene for XLA will be improved almost immediately by the identification of the responsible gene. Improvements in therapy may come more slowly. The possibility of curative gene therapy is attractive; however, there are several features of Btk that suggest that this will be a challenging undertaking. Overexpression or expression in inappropriate cell lineages may carry unacceptable risks. Mutant proteins may interfere with the function of wild-type proteins provided by gene therapy. However, it is likely that a better understanding of Btk function and regulation will benefit not only patients with XLA but also other patients with defects in B-cell function.

Coinheritance of X-linked agammaglobulinemia and growth hormone deficiency (XLA/GHD) has been classified as an independent primary immune deficiency. We evaluated the pattern of growth and endocrine function in seven XLA subjects (ages 10.9-20.1 years); four belonged to two different XLA pedigrees and three represented sporadic XLA cases. Three had reached adulthood (final stature 176.0, 173.5 and 165.0 cm, respectively) and their retrospective growth showed delay in growth and puberty during adolescence. In the other four subjects, growth hormone production was measured by growth hormone pharmacological stimulation tests (clonidine, arginine): three of four patients had insufficient growth hormone responses (peak growth hormone10 micrograms/l); all three had delayed puberty; their growth hormone responses increased after "priming" with testosterone, reaching values10 micrograms/l in two of them and allowing diagnosis of "true" growth hormone deficiency in the third. The fourth was a normally growing subject who showed a normal growth hormone response both before and after testosterone priming. Six out of the seven subjects showed a growth pattern consistent with delay in growth and puberty. Our results suggest that true XLA/GHD is rarer than previously supposed and that subnormal responses to growth hormone stimulation tests may be found without sex steroid priming of the test in adolescence. The most probable growth pattern in XLA appears to be delay in growth and puberty, as has already been described for other chronic diseases.

It has recently been demonstrated that mutations in the gene for Bruton's tyrosine kinase (BTK) are responsible for X-linked agammaglobulinemia. Southern blot analysis and sequencing of cDNA were used to document deletions, insertions, and single base pair substitutions. To facilitate analysis of BTK regulation and to permit the development of assays that could be used to screen genomic DNA for mutations in BTK, we determined the genomic organization of this gene. Subcloning of a cosmid and a yeast artificial chromosome showed that BTK is divided into 19 exons spanning 37 kilobases of genomic DNA. Analysis of the region 5' to the first untranslated exon revealed no consensus TATAA or CAAT boxes; however, three retinoic acid binding sites were identified in this region. Comparison of the structure of BTK with that of other nonreceptor tyrosine kinases, including SRC, FES, and CSK, demonstrated a lack of conservation of exon borders. Information obtained in this study will contribute to our understanding of the evolution of nonreceptor tyrosine kinases. It will also be useful in diagnostic studies, including carrier detection, and in studies directed towards gene therapy or gene replacement.

s / Placenta 32 (2011) S326–S340 S336 the new TH-expressing neurons suggestive of the dopaminergic neurogenesis. Infusion of fluorogold (FG) into striatum confirmed that these newborn neurons have successfully restored the nigrostriatal circuit that was disrupted by the HI insult. Although, the implanted hPLC-NPs were matured in the recipient's brain, we propose that the recovery was mainly mediated by the resident progenitors because nestin was induced in the SNc of the recipient animals. Possibility of the migration of the transplanted cells into the host substantia nigra can be safely ruled out since TH-expressing cells expressed neither human nuclear antigen nor human mitochondrial antigen. Relevance of these results to other dopaminergic degenerative disease needs to be confirmed, however, we propose that progenitors can induce regeneration of the diseased tissues by stimulating the resident tissuespecific progenitors as well as supplementing the lost tissues. doi:10.1016/j.placenta.2011.07.062 AMNIOTIC MESENCHYMAL TISSUE CELLS INHIBIT TUMOR CELL LINE PROLIFERATION M. Magatti, S. De Munari, E. Vertua, S. Acali, O. Parolini Centro di Ricerca E. Menni, Fondazione Poliambulanza-Istituto Ospedaliero, Brescia, Italy We and others have demonstrated that amniotic membrane-derived cells strongly inhibit in vitro lymphocyte proliferation, abolish the production of inflammatory cytokines and suppress the generation and maturation of monocyte-derived dendritic cells. In this study, we aimed to investigate whether these cells may also exert suppressive effects on the proliferation of different tumor cell lines. We have isolated cells from the mesenchymal layer of amniotic membrane (AMTC) and cultured themwith KG1, KG1a, Jurkat, U937, Girardi heart, Hela and Saos cells. Tumor cell line proliferationwas then assessed by measuring [3H]-Thymidine incorporation. We demonstrated that AMTC are able to inhibit the proliferation of different tumorcell lines of lymphoid andmyeloid origin, as well as tumor cell lines of non-hematopoietic origin, both when cultured in contact and transwell settings, demonstrating the involvement of inhibitory soluble factor(s) in this suppression. This inhibition does not seem to be mediated by tumor cell line apoptosis, but likely to cell cycle arrest of the tumor cells in G0/G1 phase, as revealed by combined analysis with Annexin-V and IP staining, and BrdU and 7-AAD staining, respectively. These findings reveal further interesting features of AMTC and suggest a possible application of these cells in controlling tumor cell proliferation. doi:10.1016/j.placenta.2011.07.063 IN VITRO AND IN VIVO DIFFERENTIATION OF AMNIOTIC EPITHELIAL STEM CELLS INTO HEPATOCYTE-LIKE CELLS F. Marongiu , R. Gramignoli , S. Doratiotto , M. Serra , M. Sini , S. Sharma , T. Sellaro , K. Dorko , E. Laconi , S. Strom a Department of Pathology University of Pittsburgh; Department of Biomedical Sciences and Technologies, Universita degli Studi di Cagliari The use of cell transplantation as an alternative to Orthotopic Liver Transplant for the treatment of liver diseases is still limited by the availability of useful cells. Stem cell-derived liver cells could be useful if they expressed the enzymes and functions needed for liver support. We investigated the differentiation of human Amniotic Epithelial cells (hAECs) into hepatocytes in vitro. We also tested the ability of rat-derived Amniotic Epithelial cells (rAECs) to integrate and differentiate into hepatic cells upon transplantation into a rat model of liver repopulation. In vitro differentiation: The expression of mature liver genes was strongly increased in hAECs after culture on a Pig Liver-derived extracellular matrix sandwich system in the presence of EGF, HGF, bFGF, OSM, Dexamethasone and ITS. Differentiated cells were able tometabolize TE, HPC and ammonia, confirming the expression of functional liver enzymes. In vivo transplant: After transplantation of rAECs into Retrorsine-treated animals, clusters of donor cells were present into the liver of recipient animals. Two, 6 and 12 months following transplants, donor cells had morphological appearance of mature hepatocytes with normal growth pattern. They expressed major liver proteins such as CYP2E1, 3A1 and Albumin. Taken together, these data indicate that hAECs and rAECs have the potential to differentiate into mature hepatocytes both in vitro and in vivo, thus suggesting that these cells will be a useful source of cells for regeneration of liver tissue. doi:10.1016/j.placenta.2011.07.064 THE KEY ROLE OF DECIDUALIZATION IN THE IMMUNOMODULATORY ACTIVITIES OF HUMAN DECIDUAL STROMAL CELLS R. Munoz-Fernandez , E. Leno-Duran , A. Prados , A. Abadia-Molina , M. Ruiz Ruiz , M. Delgado , E. Olivares b a Instituto de Biomedicina “Lopez Neyra”, Consejo Superior de Investigaciones Cientificas, Armilla, Granada; b Instituto de Biopatologia y Medicina Regenerativa, Centro de Investigacion Biomedica, Universidad de Granada, Armilla, Granada Decidual stromal cells (DSC) are the main cellular component of the decidua, the maternal tissue which is in close contact with the fetal trophoblast. We have isolated and maintained human DSC lines in culture and used them to characterize the antigen phenotype and properties of these cells. We previously reported that DSC are closely related to mesenchymal stem cells, have contractile activity and exert different immunological activities thatmaybe involved inmaternal–fetal cross-talk. Decidual stromal cells differentiate (decidualize) under the effect of the progesterone, changing their morphology to a rounder shape and secreting prolactin. Decidualization led DSC to a more immunoregulatory profile: it inhibited their phagocytic activity and secretion of IL-6, while increasing their secretion of IL-10 and expression of HLA-G. Decidualization also decreased the expression of CD21, CD54 and BAFFbyDSC, and althoughDSC are resistant to many apoptosis-inducing factors, it induced apoptosis in these cells. The potential therapeutic use of DSC was supported by the finding that these cells survived in mice weeks after injection. However, in mice with trinitrobenzene sulfonic acid-induced colitis, decidualized DSC but not undifferentiated DSC showed a therapeutic effect. We also found that endometrial stromal cells, the cellular counterpart of DSC in the nongestating uterus, exhibited an antigen phenotype equivalent to that of DSC, but ESC were less sensitive to the effects of progesterone. doi:10.1016/j.placenta.2011.07.065 TRANSPLANTATION OF PLACENTA-DERIVED MESENCHYMAL STEM CELLS IN IMMUNOCOMPETENT MICE SUBMITTED TO MYOCARDIAL INFARCTION J. Passipieri , G.D. Suhett , G.V. Brasil , T.H. Kasai-Brunswick , A.B. Martins , D.C. Rodrigues , N. Rocha , J. Nascimento-Silva , B.B. Christie , B.J.S. Mendes , B.L.B. Esporcatte , R.C.S. Goldenberg , A.B. Carvalho , A.C.C. Carvalho a,b a Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; b Instituto Nacional de Cardiologia, Rio de Janeiro, RJ, Brazil; c Universidade Federal Fluminense, Niteroi, RJ, Brazil Death of cardiomyocytes due to myocardial infarction (MI) causes wall thinning and ventricular dilatation leading to heart failure. This study aims to evaluate the role placenta-derived mesenchymal stem cells (pMSCs) in the treatment of cardiac failure in immunocompetent mice. pMSCs were characterized as plastic-adherent and multipotent cells. They did not express hematopoietic or endothelial cells markers (CD45, CD34

The Wiskott-Aldrich syndrome (WAS) is a severe X-linked, recessive disorder, with a high mortality rate at early age due to hemorrhages, infections, and lymphoid malignancies. The molecular pathogenesis of the disease is unknown. Carrier females of WAS are clinically and immunologically normal, thus precluding carrier detection by simple laboratory tests. Major advances in molecular genetics have allowed mapping of the WAS gene to the pericentromeric short arm of the X chromosome, and have made carrier detection and prenatal diagnosis feasible by segregation analysis with closely linked polymorphic DNA markers. Furthermore, the observation that carriers of WAS exhibit a unilateral inactivation of the X chromosome in hematopoietic cells has provided a new tool for carrier detection. However, critical interpretation of molecular analysis data is essential to provide accurate genetic counseling to WAS families.

The gene for X-linked agammaglobulinemia (XLA) has been mapped to Xq22. No recombinations have been reported between the gene and the probe p212 at DXS178; however, this probe is informative in only 30-40% of women and the reported flanking markers, DXS3 and DXS94, are 10-15 cM apart. To identify additional probes that might be useful in genetic counseling, we examined 11 polymorphisms that have been mapped to the Xq21.3-q22 region in 13 families with XLA. In addition, pulsed-field gel electrophoresis and yeast artificial chromosomes (YACs) were used to further characterize the segment of DNA within which the gene for XLA must lie. The results demonstrated that DXS366 and DXS442, which share a 430-kb pulsed-field fragment, could replace DXS3 as proximal flanking markers. Probes at DXS178 and DXS265 identified the same 145-kb pulsed-field fragment, and both loci were contained within a 200-kb YAC identified with the probe p212. A highly polymorphic CA repeat (DXS178CA) was isolated from one end of this YAC and used in linkage analysis. Probes at DXS101 and DXS328 shared several pulsed-field fragments, the smallest of which was 250 kb. No recombinations were seen between XLA and the DXS178-DXS265-DXSq178CA complex. DXS101, DXS328, DXS87, or the gene for proteolipid protein (PLP). Key crossovers, when combined with the linkage data from families with Alport syndrome, suggested the following order of loci: cen-DXS3-DXS366-DXS442-(PLP, DXS101, DXS328, DXS178-DXS265-DXS178CA complex, XLA)-(DXS87, DXS94)-DXS327-(DXS350, DXS-362)-tel. The strongest linkage (multipoint lod score of 15.2 at θ = 0) was found between XLA and the DXS178-DXS265-DXS178CA complex. When these data are combined with those from other published studies, one can estimate that the cumulative lod scores for DXS178 and XLA are approximately 31.4 at θ = 0. Our studies also limit the segment of DNA within which the XLA gene must lie to the 3- to 4-cM distance between DXS442 and DXS94 and they identify and orient polymorphisms that can be used in genetic counseling not only for XLA but also for Pelizaeus-Merzbacher disease (PLP deficiency), Alport syndrome (COL4A5 deficiency), and Fabry disease (α-galactosidase A deficiency).

X-linked severe combined immunodeficiency (XSCID) is characterized by hypogammaglobulinemia, markedly reduced numbers of T cells, absent mitogen responses, decreased numbers of NK cells, and normal or elevated numbers of B cells. The abnormalities in the NK cell and B cell lineages could be attributed to dependence of these cell lineages on T cells or T cell-derived factors, or to expression of the XSCID gene defect in these cell lineages. In past experiments, we have examined X chromosome inactivation patterns in T cells and cultured B cells from female obligate carriers of XSCID and have found that both cell lineages demonstrate nonrandom X chromosome inactivation. This indicates that the gene defect is intrinsic to both of these cell lineages. In the present experiments, a polymerase chain reaction technique was used to evaluate X chromosome inactivation patterns in highly purified populations of freshly isolated NK cells, B cells, CD4+ cells, and CD8+ cells from three obligate carriers of XSCID. All four lymphoid cell populations from these three women exhibited exclusive use of a single X as the active X. In contrast, both X chromosomes were used as the active X in neutrophils and monocytes. These findings indicate that the XSCID gene is expressed in the NK cell lineage as well as in T cells and B cells. This observation makes it highly unlikely that the XSCID gene is involved in Ag receptor gene rearrangements.

X-linked agammaglobulinemia (XLA), was one of the first immunodeficiencies described (Bruton 1952) and it is often thought of as a prototype immunodeficiency because the clinical and laboratory findings are felt to be distinctive. Affected boys have the onset of recurrent bacterial infections in the first few years of life. The serum immunoglobulins are profoundly decreased and the number of B cells is markedly reduced; although T cell number and function appears to be normal. Similarly affected male are seen in the maternal lineage (Ochs 1989; Lederman 1985). However, the prototype is evolving. Over the years, there has been a gradual change in the criteria required to make the diagnosis of XLA, and in the approach to identifying the underlying genetic defect.

The effects of the antitumor drugs daunorubicin, doxorubicin and their complexes with Fe(III) on phosphoinositide hydrolysis, lipid peroxidation and protein kinase C (PKC) activation were measured in intact human platelets. Doxorubicin and the Fe(III) complexes of both doxorubicin and daunorubicin quickly induced lipid peroxidation [as measured by the thiobarbituric acid (TBA) assay], phosphorylation of the 40 K substrate of PKC, and increased levels of phosphatidic acid and inositol phosphates. Fe(III) alone or complexed to acetohydroxamic acid induced high levels of TBA-reactive material but did not affect either PKC activation or phosphoinositide turnover. In contrast, daunorubicin, which was ineffective per se , inhibited all these doxorubicin- and anthracyclines/Fe(III)-induced biochemical events. We suggest that phosphoinositide hydrolysis determined by anthracyclines, and consequently PKC activation, could be due to lipid peroxidation, thus triggering the activity of phospholipase C.

Wiskott-Aldrich syndrome (WAS) is a fully penetrant X-linked recessive disorder characterized by thrombocytopenia with small platelets, eczema, and defects of both T-cell and B-cell immunity. Obligate carriers of this disorder show no signs of the gene defect because in the cell lineages primarily affected by the disorder they demonstrate preferential use of the normal, nonmutant X as the active X. This can be explained by the selective disadvantage in proliferation and/or survival experienced by the cells with the mutant X as the active X. We have recently evaluated an 8-year-old girl with a disorder phenotypically identical to WAS. Cytogenetic studies did not show any structural abnormalities of the X chromosome and X chromosome inactivation analysis showed that both of her X chromosomes could function as the active X. These findings suggest that there is an autosomal recessive disorder that is very similar to classic WAS.

Diagnosis of X-linked adrenoleukodystrophy is based upon demonstration of high levels of very long chain fatty acids. More recently, in addition to biochemical analysis, closely linked DNA probe St14 has been used for prenatal diagnosis in informative families. Identification of heterozygotes is particularly important, both in order to specifically address only carrier females to prenatal diagnosis, and because appropriate dietary therapy is now available to treat those heterozygotes presenting with neurological symptoms. We report two pedigrees in which carrier detection was performed by a combination of biochemical and molecular genetic analysis. Such approach should allow extremely high accuracy in carrier detection.

The pattern of X-chromosome inactivation was analyzed, by means of two different DNA probes (pSPT-PGK and M27 beta), in several cell lineages derived from females belonging to a pedigree with X-linked immunodeficiency with hyper-IgM (HIGM1). Non-random X-chromosome inactivation was demonstrated in T cells, B cells, and neutrophils, but not in fibroblasts, of obligate carriers, suggesting that different hematopoietic cell lineages are primarily involved in HIGM1. Preferential inactivation of the paternally derived X-chromosome was demonstrated by analysis of segregation of the alleles defined by the pSPT-PGK and M27 beta probes. The possibility that the HIGM1 mutation may confer a proliferative and/or differential advantage to hematopoietic precursors carrying the mutated allele on the active X-chromosome is discussed.

Mesenchymal stromal cells (MSC) isolated from human term placental tissues possess unique characteristics, including their peculiar immunomodulatory properties and their multilineage differentiation potential. The osteogenic differentiation capacity of placental MSC has been widely disputed, and continues to be an issue of debate. This review will briefly discuss the different MSC populations which can be obtained from different regions of human term placenta, along with their unique properties, focusing specifically on their osteogenic differentiation potential. We will present the strategies used to enhance osteogenic differentiation potential in vitro, such as through the selection of subpopulations more prone to differentiate, the modification of the components of osteo-inductive medium, and even mechanical stimulation. Accordingly, the applications of three-dimensional environments in vitro and in vivo, such as non-synthetic, polymer-based, and ceramic scaffolds, will also be discussed, along with results obtained from pre-clinical studies of placental MSC for the regeneration of bone defects and treatment of bone-related diseases.

X-linked severe combined immunodeficiency (XSCID) is characterized by hypogammaglobulinemia, markedly reduced numbers of T cells, absent mitogen responses, decreased numbers of NK cells, and normal or elevated numbers of B cells. The abnormalities in the NK cell and B cell lineages could be attributed to dependence of these cell lineages on T cells or T cell-derived factors, or to expression of the XSCID gene defect in these cell lineages. In past experiments, we have examined X chromosome inactivation patterns in T cells and cultured B cells from female obligate carriers of XSCID and have found that both cell lineages demonstrate nonrandom X chromosome inactivation. This indicates that the gene defect is intrinsic to both of these cell lineages. In the present experiments, a polymerase chain reaction technique was used to evaluate X chromosome inactivation patterns in highly purified populations of freshly isolated NK cells, B cells, CD4+ cells, and CD8+ cells from three obligate carriers of XSCID. All four lymphoid cell populations from these three women exhibited exclusive use of a single X as the active X. In contrast, both X chromosomes were used as the active X in neutrophils and monocytes. These findings indicate that the XSCID gene is expressed in the NK cell lineage as well as in T cells and B cells. This observation makes it highly unlikely that the XSCID gene is involved in Ag receptor gene rearrangements.

The Wiskott–Aldrich syndrome is a life-threatening X-linked recessive disorder. Affected males present with recurrent infections, eczema, and thrombocytopenia with small platelets. The immune defect involves both humoral and cellular immunity and increases in severity with age.1 The gene involved in this disease, located on the short arm of the X chromosome in the region Xp11.22–23, was recently cloned and named the Wiskott–Aldrich syndrome protein (WASP) gene.2,3 Different mutations or deletions within the WASP gene have been described in patients with the Wiskott–Aldrich syndrome and X-linked thrombocytopenia.4 The gene is expressed in early progenitor cells as well as . . .

We report on a thrombocytopenic female belonging to a pedigree with the Wiskott-Aldrich syndrome (WAS). Restriction fragment length polymorphism (RFLP) analysis with probe M27 beta, closely linked to the WAS gene, demonstrated that she is a carrier of WAS. Both small-sized and normal-sized platelets were present, suggesting that, unlike the vast majority of WAS carriers, she does not manifest nonrandom X-chromosome inactivation in the thrombopoietic cell lineage. Study of X-chromosome inactivation by means of RFLP and methylation analysis demonstrated that the pattern of X-chromosome inactivation was nonrandom in T lymphocytes, but random in granulocytes. While this is the first complete report on the occurrence of thrombocytopenia in a carrier female of WAS as the result of atypical lyonization, it also suggests that expression of the WAS gene occurs at (or extends up to) a later stage than the multipotent stem cell along the hematopoietic differentiation pathway.

Wiskott-Aldrich syndrome (WAS) is a fully penetrant X-linked recessive disorder characterized by immunodeficiency, thrombocytopenia, and severe eczema. WAS is a life-threatening disease, with a poor quality of life and high mortality rate in childhood. The gene responsible for the disease has been localized to the proximal short arm of the X- chromosome and recently isolated through positional cloning and named WAS protein (WASP). We have characterized 17 WAS families. We have developed a rapid, nonradioactive screening protocol for identifying WASP gene alterations in genomic DNA. Our method allows simultaneous evaluation of single strand confirmation polymorphism and heteroduplex formation. We have identified 15 novel mutations that involve single basepair changes, or small insertions or deletions, all of which result in premature stop cordon, frame shift with secondary premature stop codon, or splice site defect. These studies document the considerable heterogeneity of the location of mutations in the WASP gene causing full-blown WAS and show the efficiency and rapidity of a screening approach for mutation identification in WAS that will be useful for carrier detection and prenatal diagnosis.