ALBERT

Description: Background Utilization of intravenous immune globulin (IVIG) is increasing in Canada and worldwide despite few and no new labeled indications. In 2007, Canadian Blood Services in collaboration with the National Advisory Committee on Blood and Blood Products convened a panel of solid organ transplantation (SOT) experts (kidney, heart, lung, and liver) and methodologists to develop an evidence-based practice guideline for the use of IVIG in patients undergoing SOT. The objectives of this guideline are to examine the evidence for the use of IVIG in patients who are candidates for SOT and are sensitized to HLA or ABO antigens, to provide guidance for Canadian practitioners involved in the care of these patients and transfusion medicine specialists on the use of IVIG. Methods: The panel identified clinical areas of SOT that would benefit from treatment with IVIG and generated key clinical questions. A systematic, expert and bibliography literature search up to July 2008 was conducted to ensure all relevant publications were included. The panel generated recommendations based on the evidence. The levels of evidence and grading of recommendations were adapted from the Canadian Task Force on Preventative Health Care. To validate conclusions and recommendations, the practice guideline will be sent to physicians involved in solid organ transplantation in Canada and a patient representative. Recommendations from practitioner feedback will be incorporated, and the guideline will be disseminated to all physicians involved in the care of patients receiving solid organ transplantation in Canada to aid implementation of the guideline. The National Advisory Committee of Blood and Blood Products in Canada will subsequently assess the performance of the guideline and will renew the guideline at timely intervals. Results and Conclusions: The research questions developed by the panel were: Is there evidence that the use of IVIG reduces morbidity and mortality for patients undergoing SOT who are sensitized (HLA or ABO) in the perioperative setting and are sensitized experiencing acute graft rejection or experiencing chronic graft rejection? 791 citations were retrieved, and panel members identified 3 additional citations. 51 reports and a systematic review were used for this guideline. These reports were limited by inconsistent definitions of sensitization, inconsistent reporting of the type and titre of the antibody, the assays used to detect HLA antibodies, the response criteria and dosing schedules for IVIG. Thus, a consensus process was used to account for the poor evidence. The use of IVIG was associated with decreased sensitization and acceptable morbidity and mortality in living donor kidney transplantation. IVIG has been used with several other modalities for ABO-incompatible kidney transplantation and it was difficult from the existing literature to separate outcomes based on a single modality. There was also limited data on the perioperative use of IVIG in renal transplantation. IVIG was shown to be effective in combination with plasmapheresis for acute antibody mediated rejection of the kidney; however the role of IVIG was not clear for other forms of rejection. There were also several methodological limitations in the literature assessing IVIG for cardiac transplantation and only limited data were available to assess the use of IVIG for lung or liver transplantation. Future studies are needed to define the role of IVIG in solid organ transplantation and should capture the following elements: impact on antibody (specificity and titres), transplant rates, time to transplantation, graft function, graft survival, and rejection (cellular and antibody mediated).

Description: Although well characterized in the mouse, the role of Notch signaling in the human T-cell receptor αβ (TCR-αβ) versus TCR-γδ lineage decision is still unclear. Although it is clear in the mouse that TCR-γδ development is less Notch dependent compared with TCR-αβ differentiation, retroviral overexpression studies in human have suggested an opposing role for Notch during human T-cell development. Using the OP9-coculture system, we demonstrate that changes in Notch activation are differentially required during human T-cell development. High Notch activation promotes the generation of T-lineage precursors and γδ T cells but inhibits differentiation toward the αβ lineage. Reducing the amount of Notch activation rescues αβ-lineage differentiation, also at the single-cell level. Gene expression analysis suggests that this is mediated by differential sensitivities of Notch target genes in response to changes in Notch activation. High Notch activity increases DTX1, NRARP, and RUNX3 expression, genes that are down-regulated during αβ-lineage differentiation. Furthermore, increased interleukin-7 levels cannot compensate for the Notch dependent TCR-γδ development. Our results reveal stage-dependent molecular changes in Notch signaling that are critical for normal human T-cell development and reveal fundamental molecular differences between mouse and human.

Description: Background: Erythropoiesis-stimulating proteins (ESPs) are commonly used to treat anemia in patients with cancer receiving chemotherapy. While it is well established that these agents increase hemoglobin levels and reduce the incidence of red blood cell (RBC) transfusions, the effects of ESPs on overall survival are inconclusive, with reports of both positive and negative impacts on survival. A recent Cochrane meta-analysis of double-blind randomized placebo-controlled trials (Bohlius et al, 2005) determined that the hazard ratio for overall survival associated with recombinant human erythropoietin (rHuEPO) in 19 trials with 2865 patients was 0.81 (95% CI: 0.67 to 0.99) for adjusted data and 0.84 (95% CI: 0.69, 1.02) for unadjusted data, suggesting no negative impact on survival. The objective of the present investigation was to conduct an analysis with identical methodology to that reported by Bohlius et al, but including trials of darbepoetin alfa, which were not part of the Cochrane meta-analysis. Trial inclusion was limited to those that addressed patient populations with chemotherapy-induced anemia (studies by Henke et al and Leyland-Jones et al, which reported decreased survival in patients treated with rHuEPO, were not included in this analysis as they were not conducted in anemic patients). Methods: Data from 4 randomized, placebo-controlled clinical trials of darbepoetin alfa in chemotherapy-induced anemia were analyzed using meta-analysis methodology as performed in the Cochrane analysis. The primary outcomes included hematological response (hemoglobin increase of ≥ 2 g/dL); change in hemoglobin from baseline; RBC transfusions; and overall survival. Survival was also analyzed by combining the 4 darbepoetin alfa trials with the rHuEPO trials used in the Cochrane analysis. Results: In the meta-analysis of 4 trials (N=759), darbepoetin alfa significantly reduced the risk of receiving a RBC transfusion (RR 0.69; 95% CI 0.59–0.81; p

Description: Between 1995 and 2004, two NIH-sponsored studies (STOP/STOP II) showed that children with sickle cell disease (SCD) and abnormal transcranial Doppler blood flow measurements (high stroke risk) are protected from stroke with regular blood transfusions. Iron overload, which may lead to complications and requires iron removal therapy, was monitored by serum ferritin (SF). Liver iron concentration (LIC) measurement was not mandated by protocol and was performed at investigator discretion. Biopsy dates and lab values were captured during STOP/STOP II, providing an opportunity to validate SF against LIC. 75 LICs on 36 patients (19 female, 17 male) at 8 centers were obtained. No liver biopsy complications were reported. LICs were correlated with STOP/STOP II core laboratory SF and alanine aminotransferase (ALT) obtained within 180 days of LICs. Median age at first biopsy was 11.1 years (range, 4.5–17.8), median time from start of transfusion was 36 months (range, 2–100). Iron removal treatment was initiated a median 23 months (range, 4–108) from start of transfusion, with deferoxamine (n=27), and/or exchange transfusion (n=9). 21 pts (58%) had multiple LIC measures: 2 (n=9), 3 (n=8), 4 (n=2), 5 (n=2). Last LICs on iron removal therapy were obtained a median 72 months (range, 35–124) from start of transfusion. Correlation between SFs and LICs were r=-0.06 (n=18) for first LICs obtained prior to iron removal therapy, r=0.50 (n=17) for last LICs obtained on iron removal therapy, and r=0.51 for all LICs (n=60). Pts with single/last LIC 〉=15 mg/gram dry liver were significantly more likely to have ALTs 〉=45 IU/L compared to those with LICs =15 mg/gram and ALT 〉=45 IU/L tended to have higher SFs then those with normal ALT (mean SF 4927 ng/ml, 95% CI 1739–8115 vs. mean SF 2255 ng/ml, 95% CI 1599–2912). 37% (7/19) of pts with LIC 〉=15 mg/gram had SFs

Description: INTRODUCTION: Normal hematopoietic stem cells (HSC) are characterized by their ability to self-renew, to generate multiple cell-lineages, and show slow divisional kinetics. Leukemic stem cells (LSC) have been reported to show similar characteristics but their identification has been elusive. We have studied various methods and have identified aldehyde dehydrogenase (ALDH) staining as an optimal method for the enrichment of primary human LSC. MATERIAL&METHODS: Bone marrow samples were obtained from patients with newly diagnosed AML after informed consent. Mononuclear cells were stained with Aldefluor and sorted by flow cytometry according to their forward/side scatter characteristics and ALDH activity (ALDH+/ALDH−). Alternatively, primary AML samples were being enriched for CD34+ cells by magnetic column, then double-stained with CD34-antibodies and Aldefluor and sorted for the co-expression of CD34+ and ALDH+, respectively for CD34+ alone. Human Mesenchymal Stromal Cells (MSC), isolated from human bone marrow, were used as a surrogate model for the cellular microenvironment of the hematopoietic niche. Adhesion of various AML cell lines and subpopulations of primary leukemic cells (ALDH+, ALDH−, CD34+, CD34+/ALDH+, all blasts) to MSC was tested in the adhesion chamber assay. Semi-quantitative RT-PCR was used to analyze the gene expression of various adhesion molecules and Western- Blot analysis was performed to validate the PCR-results on protein level. The generation of secondary leukemic colonies was evaluated in a semi-solid methylcellulose medium, as well as in a long term co-culture system (LSC-IC assay; in analogy to the LTC-IC assay). RESULTS: The percentage of ALDH+ cells ranged from 0.01% to 13.2% with a median of 1.47% (n=55). Adhesion significantly differed in the ALDH+ and ALDH− subpopulations: 85±4% of ALDH+ cells but only 61±8% of ALDH− cells were adherent (n=11, p

Description: Introduction: An insufficient production of hepcidin, the master regulator of iron metabolism, is recognized as the key pathogenetic feature of HFE-related hereditary hemochromatosis (HH). There is a growing interest in measuring the hepcidin levels, which may improve diagnosis, prognostic evaluation and clinical management of HH. Nevertheless, few investigative tools are available: an immunodot method for urinary hepcidin developed by a single centre (UCLA), not yet ready for large-scale diffusion, and mass spectrometry (MS) based assays, such as surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF-MS). The latter is well suited to small peptides like hepcidin, and can rapidly analyze crude samples with high throughput. Until now, urinary hepcidin has been measured by SELDI-TOF-MS only in small groups of C282Y homozygous patients, the majority of them under phlebotomy treatment. No data are available on C282Y/H63D compound heterozygotes, that can develop a milder clinical form of HH. This study was aimed to measure urinary hepcidin levels by SELDI-TOF-MS in a large group of HH patients. Methods: We used a protocol based on PBSIIc mass spectromer and Normal Phase chips similar to that recently proven successful for semi-quantitative detection of urinary hepcidin. Urinary samples from 30 control subjects were compared to those obtained from 80 HH patients (57 C282Y homozygotes, 23 C282Y/H63D compound heterozygotes). Eighteen C282Y homozygotes and 11 C282Y/H63D compound heterozygotes were analyzed at diagnosis, the remainder during maintenance phlebotomy (at least 30 days from last phlebotomy). Results: C282Y homozygotes had significantly lower urinary hepcidin levels vs. controls either at diagnosis, or after phlebotomy (P 〈 0.05). C282Y/H63D compound heterozygotes had hepcidin levels at diagnosis similar to controls, while the hepcidin:ferritin ratio was significantly decreased (P 〈 0.001) suggesting a relatively inappropriate hepcidin production. Moreover, also in this group means hepcidin levels after phlebotomy were significantly lower than in controls (P 〈 0.001). Samples from 12 randomly selected control subjects were sent to UCLA for duplicate measurement by the immunodot method, yielding a good correlation (r= 0.77; P

Description: The CACCC box is duplicated in the β globin gene promoter of humans and other mammals. While the function of the proximal element as a binding site for EKLF has already been well established, the role of the distal element remains unclear The distal CACCC box has been previously reported not to bind EKLF in vitro. A minor role of the distal CACCC element in β globin gene promoter function is suggested by the observation that naturally occurring β thalassemia mutations affecting the proximal CACCC box are far more severe than those affecting the distal element. Nevertheless recent evidences demonstrate: that EKLF does indeed bind to the distal CACCC motif, although with low affinity. that the CCTCACCC is required for maximal stimulation of the β-globin gene by EKLF and that silent β-thalassemia due to mutations of the distal CACCC box affects the binding and responsiveness to EKLF of the β-globin gene promoter. Our interest in the function of the distal CACCC element springs from the observation that β thalassemia mutation affecting the distal box show an age related pattern of expression being more severe in the childhood than in the adulthood. In order to get light inside the role of this element in the function of the β globin gene and in the γ to β hemoglobin switching we have analyzed the effect of mutations at the proximal and distal element “in vivo”. We have engineered, by site specific mutagenesis, the β-101 (distal CACCC element) and β-87 (proximal CACCC element) mutations inside the “minilocus “ γ-β construct. The minilocus construct has been widely used to study hemoglobin switching in vivo. This construct contains the full β-globin Locus Control Region (LCR), the Aγ globin gene, the β-globin gene and the 3′ hypersensitive site (HS) of the β-globin cluster. Three mice transgenic lines have been produced. The pattern of g versus β-globin switching has been analyzed during the development by S1 analysis and real time PCR. We have dissected the yolk sac at 10 days post conception (pc) to asses the embryonic stage of erythopoiesis; the fetal liver at 12, 14 and 16 days pc to asses the fetal stage or erythropoiesis when the g to b competitive switching take place; and the adult blood. Our results indicated that neither the β-101 nor the β-87 thalassemia mutations affect the competitive silencing of the b-globin gene in the yolk sac. During the fetal liver stage of erythropoiesis, were both human g and b human transgenes are expressed, the pattern of γ-β hemoglobin switching is striking different for the two different constructs. The b-87 minilocus γ-β construct shows a delayed switching patter mainly due to the low activation of the mutated β globin gene. The impairment of the expression of the β-87 globin gene became more severe during the fetal development compared to the control line. On the other hand the β-101 minilocus γ-β construct shows a γ-β hemoglobin switching pattern which is anticipated respect to the control line. In addition the effect of the β-101 mutation became less severe during the fetal development. These results highlight a possible role of the distal CACCC element in hemoglobin switching and in particular in the early stage of β-globin activation.

Description: Erythopoietin (EPO) and erythropoietin receptor (EPOR) regulate survival, proliferation, differentiation and viability of erythroid progenitor cells. Beyond erythropoiesis, we have observed that human vascular endothelial cells respond to EPO stimulation by inducing EPOR and endothelial nitric oxide synthase (eNOS) expression, increasing NO production and cGMP, particularly under low oxygen tension. In this study, we investigate the response of vascular smooth muscle (VSM) to EPO stimulation and the contribution of blood vessel to relaxation/contraction. We found that VSM cells express EPOR and that treatment with EPO (5 U/ml) at reduced oxygen tension increased EPOR mRNA by 2 fold. This increased EPO responsiveness at low oxygen tension is accompanied by increased cell proliferation at 2% O2 more than 2 fold. Unlike endothelial cells, EPO did not induce eNOS or NO production in VSM cells. PI-3 kinase was involved in EPO stimulation with no change in MAP kinase. In an isolated blood vessel model system, we checked EPO responsiveness. EPO produced contractions (0.32 ± 0.3 g) of rat renal artery, and pretreatment with LY294002 (10 mM), an inhibitor of PI-3 kinase, statistically significantly inhibited this contraction (58.7 ± 7%). This response was also observed with the endothelium layer removed. In preparations of human internal mammary artery (HIMA) and human saphenous vein (HSV) from patients undergoing coronary artery bypass, EPO did not affect basal vascular tone of HIMA and HSV with the endothelium layer removed, but EPO (5 U/ml) potentated noradrenalin-induced contraction by up to 2 fold in HSV and HIMA. Also, pretreatment with EPO significantly increase angiotensin-evoked contractions of these blood vessels (50 ± 8%, 113 ± 17%, respectively P 〈 0.01), suggesting that EPO has synergistic effects on angiotensin or noradrenalin-induced [Ca2+] mobilization, particularly on intracellular Ca2+ release. These data suggest that EPO stimulation of vascular smooth muscle may act to modulate or balance the vasodilatory effects of increased NO production by EPO stimulated endothelium. Under oxygen stress vascular smooth muscle can respond to EPO by proliferation and PI-3 kinase activation as a protective effect and in conjunction with Ca2+ release likely contributes to the overall vascular response.

Description: Following hematopoietic stem cell transplantation (HSCT), longterm T-cell reconstitution should be established by thymus-dependent de-novo generation of naïve T-cells (thymopoiesis), which is especially important for generating a naïve T-cell pool with a broad T-cell receptor (TCR) repertoire. However, while erythroid and myeloid hematopoietic cell lineages recover rapidly following HSCT, T-cell development may severely lag behind due to thymic insufficiency. Recent studies in fetal mice have identified common thymic epithelial progenitor cells (TEPC) that were capable to re-establish a thymus in-vivo upon transplantation into a-thymic nude mice. These TEPC are characterized by expression of the transcription factor Foxn1 and by cell surface expression of MTS24. These TEPC arise exclusively from progenitors originating from the anterior foregut endoderm during embryogenesis. Therefore, we hypothesized that common TEPC may be generated in-vitro from embryonic stem (ES) cells that have differentiated towards definitive endoderm. Currently, the mechanisms underlying commitment of definitive endoderm towards a thymic fate are unknown. In order to differentiate murine ES cells towards definitive endoderm and TEPC and to identify the factors involved in the commitment of endoderm towards a thymic fate we investigated the expression of MTS24 and of genes associated with thymic differentiation in ES-cell derived endoderm using a Gcs–GFP/Sox17–huCD25 reporter ES cell line. Culture of these GscgfpSox17huCD25 ES cells in the presence of Activin A resulted in a rapid induction of mesendodermal differentiation. After 6 days of culture the majority of cells differentiated towards mesoderm (Gsc+Sox17−, 60%) and definitive endoderm (GSC+Sox17+, 35%). Apart from the addition of Activin A, the use of low passage number ES-cells and a seeding density between 200–300 cells/cm2 were the most important factors determining efficient differentiation towards definitive endoderm. Addition of insulin or WNT-3a had no significant effect on differentiation, while usage of a high passage number of ES-cells and/or a high seeding density mainly promoted development of visceral endoderm. Real-time quantitative PCR of the definitive endoderm fraction of these cultures not only showed expression of genes associated with definitive endoderm and gut tube formation (i.e. Sox17, Foxa2, Hnf4a and TCF2) but also of genes associated with anterior foregut endoderm (i.e. Hhex, Pax9) and a low, but significant, expression of Foxn1. Analysis of MTS24 expression within these cultures showed the presence of this antigen on all three cell types. The percentage of cells expressing MTS24 was highest in visceral endoderm (30–50%) and lowest in mesoderm (5–10%). The expression was approximately 12% in definitive endoderm. We conclude that murine ES cells cultured in the presence of Activin A can efficiently differentiate towards gut-tube like endoderm, including anterior forgut endoderm, and that a fraction of the generated endoderm also expresses the surface marker MTS24, suggesting the generation of epithelial progenitors with phenotypic characteristics of TEPC.