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The inherited haemoglobin disorders and the clinical laboratory is essential in the diagnosis. Phlebotomy is the therapeutic procedure indicated to reduce blood viscosity and decreasing the risk of thrombotic events in the haemoglobin variants with high affinity for O2 or vaso-occlusive crises in Sickle Cell Disease (SCD) with double heterozygosity HbSC. We describe how therapeutic bleeding remains the treatment in two variant haemoglobinopathies, Haemoglobin Regina and the Haemoglobin Trollhattan, reported for the first time in the Italian population and a double heterozygosity HbSC. In patients with high haematocrit level when other secondary conditions of polyglobulia are excluded, a High-Performance Liquid Chromatography (HPLC) analysis for haemoglobin study should be suggested. Also in children with erythrocytosis frequently, such as the finding of Haemoglobin Okazaky.

Figure S1. Hematologic recovery after NK cell infusion in all treated patients.

Table S1. Percentage and absolute number of alloreactive NK cells in responders (A) and non-responders (B) Table S2. Description of cell processing results in responders (A) and non-responders (B)

Figure S3. Correlation between relapse rate and percentage of alloreactive clones at day 3 after infusion.

Figure S4. Impact of the absolute number of infused donor alloreactive NK cells on the probability of response.

Purpose: In acute myeloid leukemia (AML), alloreactive natural killer (NK) cells are crucial mediators of immune responses after haploidentical stem cell transplantation. Allogeneic NK cell infusions have been adoptively transferred with promising clinical results. We aimed at determining whether the composition of NK graft in terms of frequency of alloreactive NK cells influence the clinical response in a group of elderly AML patients undergoing NK immunotherapy.Experimental Design: Seventeen AML patients, in first complete remission (CR; median age 64 years, range 53–73) received NK cells from haploidentical KIR-ligand–mismatched donors after fludarabine/cyclophosphamide chemotherapy, followed by IL2. To correlate donor NK cell activity with clinical response, donor NK cells were assessed before and after infusion.Results: Toxicity was moderate, although 1 patient died due to bacterial pneumonia and was censored for clinical follow-up. With a median follow-up of 22.5 months (range, 6–68 months), 9 of 16 evaluable patients (0.56) are alive disease-free, whereas 7 of 16 (0.44) relapsed with a median time to relapse of 9 months (range, 3–51 months). All patients treated with molecular disease achieved molecular CR. A significantly higher number of donor alloreactive NK cell clones was observed in responders over nonresponders. The infusion of higher number of alloreactive NK cells was associated with prolonged disease-free survival (0.81 vs. 0.14, respectively; P = 0.03).Conclusions: Infusion of purified NK cells is feasible in elderly AML patients as post-CR consolidation strategy. The clinical efficacy of adoptively transferred haploidentical NK cells may be improved by infusing high numbers of alloreactive NK cells. Clin Cancer Res; 22(8); 1914–21. ©2016 AACR.See related commentary by Muntasell and López-Botet, p. 1831

Figure S2. Immunofluorescence analyses of 2 representative donor alloreactive NK cell clones expressing the KIR for which there is no class I ligand in the recipient as their only inhibitory receptor for self.

supplemental figure legend

Supplementary Figure 1 from Synergistic Proapoptotic Activity of Recombinant TRAIL Plus the Akt Inhibitor Perifosine in Acute Myelogenous Leukemia Cells

Supplementary Figure 2 from Synergistic Proapoptotic Activity of Recombinant TRAIL Plus the Akt Inhibitor Perifosine in Acute Myelogenous Leukemia Cells

Purpose The purpose of this paper is to provide an early assessment of India’s South-South cooperation for trade and technology (SSTT) with East Africa, focusing on Ethiopia, Rwanda, Kenya, Uganda and Tanzania. It aims to analyse the role of SSTT in providing support to targeted sectors. Design/methodology/approach The paper examines SSTT, focusing on India and East Africa over a specific period (2000–2016) of its emergence, and extends the public sponsorship literature in international business (IB) to better understand the relationship between SSTT and value addition – applying to a particular case study of SSTT interventions in spices. Findings The paper highlights SSTT as a pathway to support value addition in global value chains (GVCs). Trade between India and East African countries has grown, with three developments over the period of analysis in particular: shifting trade patterns, growing share of intermediate goods trade and differences in GVC insertion. However, East African exports are largely of lower value. Capacity building to support processing capability and thriving markets can encourage greater value addition. Preliminary findings suggest early gains at the margins, as SSTT interventions have been focusing on capacity boosting with buffering and bridging mechanisms for increased volume of trade. Moving up the value chain however requires that specific value-enhancing activities continue to be targeted, building on regional capacities. Our high-level case study for spices suggests that activities are starting to have a positive effect; however, more focus is needed to specifically target value creation before export and in particular higher levels of processing. Practical implications While findings are preliminary, policy implications emerge to guide SSTT interventions. There is capacity for building higher value-added supply chains as is evident among East African countries that trade with each other – future SSTT programmes could tap into this and help build capacity in these higher-value value chains. Future SSTT programmes can take a comprehensive approach by aiming at interventions at key points of the value chain, and especially at points that facilitate higher value addition than initial processing. An example is that Ethiopia and Rwanda are likely to benefit from an expanded spice industry, but the next phase should be towards building processing for value-addition components of the value chain, such as through trade policies, incentivising exporters to add value to items before export. From a development perspective, more analysis needs to be done on the value chain itself – for instance, trade facilitation measures to help processers engage in value chains and to access investments for increasing value add activities. (iv), Future research should examine more closely the development impacts of SSTT, namely, the connection between increased trade, local job creation and sustained innovation, as it is these tangible benefits that will help countries in the Global South realise the benefits of increased trade. Originality/value The paper underlines how the SSTT approach can contribute to the critical IB and GVCs literature using a theoretical grounded approach from public sponsorship theory, and with a unique lens of development cooperation between countries in the global south and its emerging impact on development outcomes in these countries.

This paper provides empirical evidence on the relationship between patterns of integration in global value chains (GVCs), the ‘quality’ of such integration in terms of technological capabilities and the structure of employment in European countries and sectors. We study employment shares in fabrication and headquarter occupations in terms of functional specialisation and employment upgrading, making two specific contributions. First, we explicitly account for pre-existing asymmetries in employment structure. Second, we account for the complex role of technology both in terms of country-industries’ own initial technological intensity and that of their GVC partners. To achieve this we blend data on employment, trade in value added, patents and intangible asset stocks, for the period 2000-2014. We find that such pre-existing asymmetries are highly persistent over time with little sign of convergence in employment structure over our observed period. Furthermore GVC participation is not related, in and of itself, to changes in employment structure. However, this relationship appears to be mediated by country-industries’ initial technological position. Technological leaders exhibit greater shares of employment in headquarter functions as they increase their integration in GVCs; in contrast country-industries that start off as technological laggards see further integration in GVCs alongside an increase in the share of employment in fabrication functions.

The chapter selectively reviews the theoretical and empirical literatures on the potential sources of countries’ asymmetries in their integration in global value chains (GVCs), with the aim of identifying the effects of such processes on the dynamics and composition of employment. We consider asymmetries related to the positioning of countries in: (i) the new international division of labor; (ii) the global technology landscape; and (iii) their initial production and employment structure. We conclude by proposing avenues of research to account for the complex factors that affect how countries position within GVCs and what are the opportunities for technological, functional, and employment upgrading.

open 17 no We thank Rita Bertoni and Antonella Orlando for the technical assistance. This work has been supported by Bologna AIL (Italian Association against Leukemia, Lymphoma and Myeloma), Roberto and Cornelia Pallotti Legacy for Cancer Research (University of Bologna) to M.B., and REUSE with Love ONLUS, Bologna, Italy. Umbilical Cord Blood (UCB) represents a valid option for patients with hematopoietic malignancies lacking an HLA matched donor. To overcome the limitation of the low stem cell dose of UCB, the intrabone (IB) route has been proposed. We report the results of a prospective study on a poor-prognosis cohort of 23 patients receiving intrabone single UCB transplant (Clinicaltrials.gov NCT00886522). Cumulative incidence of hematological recovery at day 90 was 82 ± 9% (ANC > 0.5 × 109/L) and 70 ± 10% (platelet > 50 × 109/L) and correlated with CD34 + cells in the graft. NRM was 20 ± 9%. No severe aGVHD and only one extensive cGVHD occurred, with fast immune reconstitution. To test the hypothesis that the direct IB injection could affect the expression of stem cells regulatory pathways, CD34 + cells from BM aspirates at day + 10, + 20, + 30, processed in hypoxic conditions mimicking the BM-microenvironment (7%pO2), were studied for the expression of c-Mpl, Notch1 and CXCR4. We found that the expression of c-Mpl in CD34 + cells at day + 10 significantly correlated with hematological recovery. In conclusion, IB-UCB transplant success is associated with low incidence of GVHD and high-speed platelet recovery; intrabone route may preserve full hematopoietic stemness by direct delivery of UCB stem cells into the hypoxic HSC niche. none none Bonifazi, Francesca; Dan, Elisa; Labopin, Myriam; Sessa, Mariarosaria; Guadagnuolo, Viviana; Ferioli, Martina; Rizzi, Simonetta; De Carolis, Sabrina; Sinigaglia, Barbara; Motta, Maria Rosa; Bontadini, Andrea; Giudice, Valeria; Martinelli, Giovanni; Arpinati, Mario; Cavo, Michele; Bonafé, Massimiliano; Storci, Gianluca Bonifazi, Francesca; Dan, Elisa; Labopin, Myriam; Sessa, Mariarosaria; Guadagnuolo, Viviana; Ferioli, Martina; Rizzi, Simonetta; De Carolis, Sabrina; Sinigaglia, Barbara; Motta, Maria Rosa; Bontadini, Andrea; Giudice, Valeria; Martinelli, Giovanni; Arpinati, Mario; Cavo, Michele; Bonafé, Massimiliano; Storci, Gianluca

This paper puts forward and empirically tests the conjecture that specialisation in Natural Resource Industries (NRI) might not necessarily be a ‘curse’ for (developing) countries, if it provides opportunities for export diversification in backward linked sectors a la Hirschman. We first revisit the evolution of the debate around the NRI ‘curse’, including those from scholars sceptical of diversification based on beneficiation from NRI. We then empirically test whether NRI might represent a sufficient “domestic representative demand” a la Linder for backward linked sectors such as Knowledge Intensive Business Services (KIBS) or high tech manufacturing that might provide new opportunities for export diversification led by virtuous pathways of domestic structural change. We find empirical support for this conjecture and discuss some implications that revisit the NRI curse debate.

This paper investigates the association between buyer-supplier international trade relationships and supplier’s product upgrading. We proxy the suppliers’ upgrading with a measure of product sophistication. We first propose a measure of power in the trade relationship, combining the dependence of each firm on the trading partner and their market shares. Using transaction data from Colombia, we next estimate if the measure of power relationship predicts a supplier’s export sophistication, the probability of adding a new product in the trading relationship, and that of increasing export sophistication. We find that suppliers that are highly dependent on buyer’s imports are more likely to fall into a specialisation trap in low sophistication products. Buyers with large market shares trade in sophisticated products, therefore with little margin for upgrading; suppliers with large market shares are more likely to introduce new products, but trade pairs where the buyer depends on the supplier are more likely to upgrade. We further test whether these relationships hold across different destination countries, finding in particular that buyers dominating the market in the US tend to import low-sophistication products and make it harder for suppliers to upgrade. We contribute to the recent literature on buyer-supplier relationships by explicitly including a measure of power into our analysis. In doing this, we also offer further support and complement the qualitative evidence put forward by the literature on governance in global value chains (GVCs).

This chapter aims at revisiting the empirical evidence on the recent trends of countries’ integration in global value chains in Europe. It investigates two potential sources of unbalances that these processes might relate to: (i) the sectoral specialization of the patterns of international fragmentation, whether high technology manufacturing or knowledge intensive services (KIBS); (ii) the occupational categories that have benefited or been penalized by these trends. A rich empirical mapping of these trends in the European countries is provided, based on OECD ICIO and EU ISCO data. The results on the overall and sectoral-specific trends of integration in GVCs and the associated changes in the shares of managers and manual workers show a dual-speed and qualitatively different integration patterns in Europe, with Eastern European (EE) countries rapidly integrating in high tech manufacturing, and the core of western countries strengthening their mutual integration in the KIBS area. Despite the relatively “good quality” integration of EE countries, the evidence does not seem to reveal a mirroring upgrading of employment structures. While this empirical contribution does not attempt to identify causal relationships, the picture provided in the chapter shows that, overall, integration in GVC seems to reproduce and perhaps exacerbate the initial asymmetries in the sectoral and employment structure, with manual workers occupation reducing overall and knowledge intensive occupations concentrating in western Europe.

This paper investigates whether trade specialisation explains economies’ trade performance within a Global Value Chain (GVC) context. We consider trade specialisation in natural resources, high and low tech manufacturing and business services, before and after the financial crisis. The aimed contribution of this paper is to shed light on the effects of trade specialisation as measured in domestic value added embodied in exports rather than gross exports. We add to the literature on GVCs by: (i) studying the role of the domestic productive structure in countries’ trade specialisation and performance, (ii) accounting for the rate of changes in trade specialisation as affecting GVC performance. We employ Balassa indexes based on value added flows in a GMM dynamic panel framework. We find that trade specialisation in low-tech manufacturing and natural resources have a negative impact on value added exported by countries. High-tech manufacturing and knowledge intensive services exhibit a positive effect during the crisis period. We discuss these findings in relation to the recent debates on the role of manufacturing and premature de-industrialisation in developing countries.

BACKGROUND: Numerous studies have established the importance of innate immunity, particularly natural killer (NK) cells, in transplantation tolerance. NK cells express killer cell immunoglobulin-like receptors (KIRs) on their surface. By recognizing and binding major histocompatibility complex class I antigens, KIRs prevent autologous cell killing and promote lysis of non-self antigen-presenting cells. This study investigated the role of 16 KIR genes and donor-recipient KIR/HLA combinations on 5-year outcomes in a population of deceased donor kidney transplant recipients. MATERIAL/METHODS: We genotyped 126 renal transplant patients and their donors for HLA A, B, C, DR, and KIR genes. Patients underwent standardized transplantation and immunosuppressive protocols and were followed-up for 5 years. Graft function was evaluated by serum creatinine level and glomerular filtration rate calculated using the 4-variable modification of diet in renal disease (MDRD) equation. RESULTS: The presence of KIR2DS3 in the recipients was associated with better graft function indexes over time (p

This multicenter case-controlled pilot study evaluated myocardial inflammatory burden (IB) and phenotype in endomyocardial biopsies (EMBs) with and without pathologic antibody-mediated rejection (pAMR). Sixty-five EMBs from five European heart transplant centers were centrally reviewed as positive (grade 2, n = 28), suspicious (grade 1, n = 7) or negative (n = 30) for pAMR. Absolute counts of total, intravascular (IV) and extravascular (EV) immunophenotyped mononuclear cells were correlated with pAMR grade, capillary C4d deposition, donor specific antibody (DSA) status and acute cellular rejection (ACR). In pAMR+ biopsies, equivalent number of IV CD3+ T lymphocytes (23 ± 4/0.225 mm(2) ) and CD68+ macrophages (21 ± 4/0.225 mm(2) ) were seen. IB and cell phenotype correlated with pAMR grade, C4d positivity and DSA positivity (p < 0.0001). High numbers of IV T lymphocytes were associated with low grade ACR (p = 0.002). In late-occurring AMR EV plasma cells occurring in 34% of pAMR+ EMBs were associated with higher IB. The IB in AMR correlated with pAMR+, C4d positivity and DSA positivity. In pAMR+ equivalent numbers of IV T lymphocytes and macrophages were found. The presence of plasma cells was associated with a higher IB and occurrence of pAMR late after transplantation.

Several studies have shown that chronic GVHD (cGVHD) is more frequent in patients receiving transplants from PBSC than in those receiving BM. In the setting of PBSC-unrelated transplants, the addition of anti-T-cell globulin (ATG) has shown a significant decrease in incidence/severity of cGVHD, without an increase in relapses or infections. However, no prospective data are yet available in the sibling setting. We retrospectively analyzed the effects of intensification of standard GVHD prophylaxis (CsA+MTX) by the addition of low-dose ATG in 245 patients receiving a transplant from HLA-identical sibling. From 1996 to 2001, patients received PBSC as the preferred source (group 2), and then ATG was added before transplant (group 3) because of a high cGVHD rate. Patients receiving BM in the same time period were analyzed as a control group (group 1). The incidence of grade III-IV acute GVHD and cGVHD was not significantly different in the three groups, but extensive cGVHD was highest in group 2 (38%) compared with group 3 (21%) or group 1 (28%; P=0.03). OS, TRM and time to relapse/progression were similar in the three groups. Our analysis shows that adding ATG to PBSC sibling allogeneic transplants can lower cGVHD, without an increase of relapse. Further prospective studies are needed to confirm these findings.

Background/Aims Orthotopic liver transplantation (OLT) is an important therapeutic option for HBV-related end-stage-liver disease, yet it is often hampered by a scarcity of organ availability. One option to increase organ availability is the use of virologically compromised organs from HBV-infected donors. Transplantation of anti-HBcore positive grafts has been associated with a low risk of HBV recurrence if adequately treated with nucleoside analogs, irrespective of concomitant HBV-specific immunoglobulin therapy. Experience using HBsAg positive grafts is, however, very limited. Methods Here, the analysis of the cellular and humoral HBV-specific immunity of a subject with past HBV infection (anti-HBs and anti-HBc positive) receiving an HBsAg positive liver graft is reported. Results Nine months post-OLT, the patient experienced a spontaneous anti-HBs re-seroconversion allowing the discontinuation of HBIG. The data show a concurrent increase in the cellular and humoral immunity at times of reduced viral antigenemia, demonstrating effective immune control of HBV post-OLT. Conclusions These data support the use of marginal organs in this setting, providing a potential strategy to further alleviate organ shortage.

HLA testing is an essential part of the process to identify a donor who may be a good match for the patients who need haematopoietic stem cells from bone marrow, peripheral blood or cord blood and the DNA typing in high resolution is now recommended as the Scientific Societies also describe in their standards. Recently the new PCR-Luminex HLA typing method, based on the reverse sequence specific oligonucleotide probes coupled with a microsphere beads in an array platform, has been well established. We report the data from 146 samples previously typed to a four digits level and used to evaluate the accuracy, sensitivity and performance of the new high definition DRB1 by PCR-Luminex kit. One hundred and forty-six samples from unrelated healthy donors, haematological patients or external proficiency tests were used in this study. The Luminex high definition DRB1 typing represents a versatile method and may be easily introduced in the routine, particularly when the technical team has already acquired experience on the technique. Only few HLA allelic combinations need an additional typing by PCR-SSP or SBT to solve the ambiguous results thus reducing the time necessary to produce a final report.

Human umbilical cord blood units (UCBs) are an alternative source in allogeneic-stem-cell transplantation. Human leukocyte antigen (HLA)-G is a tolerogenic molecule with a possible implication in UCB immunoregulatory effect. HLA-G expression was observed in UCB myeloid and plasmacytoid dendritic cells; in contrast, CD34(+) cells did not produce this molecule. CD34(+) cells are primitive hematopoietic progenitor cells that are present in UCB and are necessary for long-term engraftment via production of immunoregulatory molecules and a hematopoietic progeny that supports cellular recovery. The role of these cells in UCB transplantation needs further evaluation of HLA-G expression in CD34(+) cells and their hematopoietic progeny. We confirmed the absence of HLA-G expression in CD34(+) cells, whereas CD34(+)-derived progeny secreted HLA-G molecules and expressed HLA-G mRNA in in vitro cultures. Furthermore, soluble HLA (sHLA)-G molecules purified from the culture supernatants of CD34(+)-derived progeny were able to suppress lymphoproliferative response in an HLA-G dose-dependent manner. Overall these results identify CD34(+)-derived hematopoietic progeny as producers of HLA-G molecules and support a role of this antigen as an immuno-modulatory factor in UCB.

Background & Aims The main limitation of orthotopic liver transplantation (OLT) is the scarcity of available donor organs. A possibility to increase the organ pool is to use grafts from hepatitis B virus surface antigen (HBsAg) positive donors, but few data are currently available in this setting. We assessed the clinical, serovirological, and immunological outcomes of liver transplant from HBsAg positive donors in a single centre study. Methods From 2005 to 2009 10 patients underwent OLT from HBsAg positive donors, for HBV-related disease (n=6) or HBV-unrelated disease (n=4). The median follow-up was 42months (range 12–60). All recipients were HBcAb positive and were given antiviral prophylaxis. Results Patients transplanted for HBV-related disease never cleared HBsAg. Two HBsAg negative patients never tested positive for HBsAg, whereas the others experienced an HBsAg appearance, followed by spontaneous production of anti-HBs, allowing HBsAg clearance. No patient ever had any sign of HBV hepatitis. HBV replication was effectively controlled by antiviral therapy. The immunologic sub-study showed that a most robust anti-HBV specific T cell response was associated with the control of HBV infection. Conclusions OLT from HBsAg positive donors seems to be a safe procedure in the era of highly effective antiviral therapy.

Background. Persistent mixed chimerism represents a state wherein recipient and donor cells stably co-exist after haematopoietic stem cell transplantation. However, since in mostly of the studies reported in literature the engraftment state was observed in the nucleated cells, in this paper we determined the donor origin in the mature erythrocytes of patients with persistent mixed chimerism after transplantation for haemoglobinopathies. Results were compared with the engraftment state observed in singularly picked-up burst-forming unit-erythroid colonies and in the nucleated cells collected from the peripheral blood and from the marrow. Design and Methods. The donor origin of the erythrocytes was determined analyzing differences on the surface antigens of the erythrocytes suspension after incubation with anti-ABO and/or anti-C, -c, -D, -E and -e monoclonal antibodies by a flow cytometer. Short tandem repeats analysis was used to determine the donor origin of nucleated cells and burst-forming unit-erythroid colonies singularly picked up after 14 days incubation. Results. A proportion of donor-derived nucleated cells of 71%, 46%, 15% and 25% was observed at day 1364, 1385, 1314 and 932 respectively, in four transplanted patients affected by haemoglobinopathies. Similar results were also obtained in the erythroid precursors, analyzing the donor/recipient origin of the burst-forming unit-erythroid colonies, while on the contrary, at the same days of observation, a proportion of 100%, 100%, 73% and 90% donor-derived erythrocytes was observed in the four patients with persistent mixed chimerism. Conclusions. Our results showed that mostly of the erythrocytes present in four long-term transplanted patients affected by haemoglobinopathies and characterized by the presence of few donor engrafted nucleated cells were of donor origin. The indication that small proportions of donor engrafted cells might be sufficient to clinical control the disease in patients affected by haemoglobinopathies is relevant, although the biological mechanisms underlying these observations need to be further investigated.

Background and objectives Packed red blood cells (pRBCs) contain apoptotic white cells. We studied apoptotic cells in pRBCs after filtration and at various time-points during storage. Materials and methods To maintain the same subset of cells, seven pRBC units were pooled in a single bag and divided equally into seven aliquots. Two series of five experiments were performed: in the first we utilized the Biofil R01 Max filter, and in the second the Pall BPF4 filter was used. One aliquot was immediately leucodepleted while the others were stored at 4 degrees C and filtered on days 3, 7, 10, 14, 21 and 42 of storage. The postfiltration leucocyte counts and apoptotic evaluations were performed by using the Nageotte chamber and flow cytometry. Results The absolute number of residual leucocytes was always less than 0.5 x 106 in each experiment. Nageotte chamber counts showed a greater number of white blood cells than flow cytometry during the 42 days of storage. On day 0, the percentage of apoptotic cells in non-leucodepleted pRBCs was 1.1 +/- 0.4 and 1.2 +/- 0.4, while in filtered pRBCs it was high from day 0, at 53.5 +/- 16.3 and 52 +/- 18.5, respectively, with Biofil and Pall filters. On day 10 of storage, apoptotic cells reached a percentage of 42.5 +/- 15.8 and 41.6 +/- 18.6 in non-leucodepleted pRBCs, while in filtered units an average value of approximately 90% was found with both filters. Conclusions The percentage of apoptotic cells was higher in leucodepleted than in non-leucodepleted pRBCs. After filtration, the degree of apoptosis was already high on day 0, and reached a mean of approximately 90% by day 10. The difference in residual WBC counts between the Nageotte chamber and flow cytometry could be related to the presence of a high percentage of apoptotic cells in filtered blood components, and to the method used to distinguish viable from apoptotic cells.

Background Thalassemia major can be cured with allogeneic hematopoietic stem cell transplantation. Persistent mixed chimerism develops in around 10% of transplanted thalassemic patients, but the biological mechanisms underlying this phenomenon are poorly understood.Design and Methods The presence of interleukin-10-producing T cells in the peripheral blood of eight patients with persistent mixed chimerism and five with full donor chimerism was investigated. A detailed characterization was then performed, by T-cell cloning, of the effector and regulatory T-cell repertoire of one patient with persistent mixed chimerism, who developed stable split erythroid/lymphoid chimerism after a hematopoietic stem cell transplant from an HLA-matched unrelated donor.Results Higher levels of interleukin-10 were produced by peripheral blood mononuclear cells from patients with persistent mixed chimerism than by the same cells from patients with complete donor chimerism or normal donors. T-cell clones of both host and donor origin could be isolated from the peripheral blood of one, selected patient with persistent mixed chimerism. Together with effector T-cell clones reactive against host or donor alloantigens, regulatory T-cell clones with a cytokine secretion profile typical of type 1 regulatory cells were identified at high frequencies. Type 1 regulatory cell clones, of both donor and host origin, were able to inhibit the function of effector T cells of either donor or host origin in vitro.Conclusions Overall these results suggest that interleukin-10 and type 1 regulatory cells are associated with persistent mixed chimerism and may play an important role in sustaining long-term tolerance in vivo. These data provide new insights into the mechanisms of peripheral tolerance in chimeric patients and support the use of cellular therapy with regulatory T cells following hematopoietic stem cell transplantation.

A new human leukocyte antigen-B allele was found in an unrelated Italian donor.

BACKGROUND: Overall survival of adult acute myeloid leukemia (AML) remains poor due to the lack of novel and effective therapies. The cancer cell death induced by some chemotherapeutic agents, especially anthracyclines, such as daunorubicin (DNR), named immunogenic cell death (ICD), is characterized by intra- and peri-cellular modifications, which favor the induction of anti-tumor T-cell immune response. Among them, the extracellular release of adenosine triphosphate (extracellular ATP, eATP) from dying tumor cells primes dendritic cells (DCs) by activating purinergic P2X7 receptors, thus eliciting the presentation of tumor antigens to T cell. DCs are key regulators of adaptive immunity, promoting or suppressing T-cell responses. One of the suppressive mechanisms involves the expression of indoleamine 2,3-dioxygenase 1 (IDO1), which plays a major role in the induction of T-cell tolerance through the expansion of regulatory T cells (Tregs). The present study aimed at evaluating the involvement of IDO-1 during ATP-driven ICD in AML. METHODS: AML patients were analyzed at diagnosis and after DNR-based chemotherapy. Ex vivo T cells were characterized by FACS and tested for their capacity to produce IFN-γ in response to autologous blasts. Then, CD8+IFN-γ-producing T cells were expanded and further characterized. ATP was used as an ICD representative model. In vitro, murine WEHI-3B and human HL-60 leukemic cell lines and primary blasts were tested for ATP release after DNR treatment. To in vivo investigate DNR-induced ICD, WEHI-3B cells stable transfected with luciferase PmeLUC were inoculated subcutaneously in BALB/c mice to measure ATP release directly from tumor mass. Tumor infiltrating DCs and T cells were characterized by FACS and immunohistochemistry after chemotherapy and plasma levels of cytokines were measured. In vitro DNR-treated AML cells were pulsed into immature DCs, previously generated from healthy donors. DCs maturation and IDO1 expression were examined (by FACS and western blot, respectively) and correlated with the presence of ATP in culture medium. IDO-driven Tregs induction was assessed. Finally, functional immunological tests were performed in vitro to test the ability of Tregs to inhibit leukemia antigen-specific IFN-γ production (FACS analysis) by ICD-activated T cells. RESULTS: After chemotherapy, 15/23 AML patients had an increase in leukemia-specific IFN-γ producing CD4+ and CD8+ T cells. Also an increase of Tregs was observed with a peak at day 21. CD8+ IFN-γ-producing T cells, which resulted in a skewing toward an effector memory phenotype, were activated and cytotoxic against autologous AML blasts but showed features of exhaustion and were defective in perforin production. In vitro and in vivo DNR induced ATP release from AML cells. In vivo the analysis of tumor-infiltrating T cells after treatment has shown an exhausted phenotype of cytotoxic CD8+ cells, increased IFN-γ+ Tregs and decreased TNF-α+ effector T cells. DNR treatment also increased in vivo plasma levels of cytokines IFN-γ, IL-1β, TNF-α, IL-12. Moreover, in DNR-treated mice we observed a significant increase of CD11c+ mature DCs which express IDO1 in tumor infiltrate. In vitro, loading of DNR-treated AML cells into DCs resulted in increased maturation, but also in IDO1 induction. Interestingly, extracellular ATP was directly involved in DCs maturation and IDO1 expression via purinergic receptor P2Y11. ICD-driven DCs were able to expand Tregs in an IDO-dependent manner. Finally, ICD both triggers a leukemia-specific IFN-γ production by CD8+T cells and induces Tregs, via IDO1-expressing DCs, which in turn inhibit leukemia-specific T cell. CONCLUSIONS: Overall, our data indicate that in AML chemotherapy-induced ICD has contrasting, and not fully elucidated, effects on T-cell immune response, resulting in the induction of leukemia-specific CTLs, albeit with defective features, and Tregs. In this scenario, the effects of ATP release from dying leukemia cells on DCs may be pivotal, as indicated by its capacity to concomitantly induce DC maturation and activation as well as tolerogenic function via IDO1. The combination of novel immunological drugs, such as IDO1 and/or checkpoint inhibitors, with conventional chemotherapy may represent an interesting approach to contrast tolerance induction and, then, fully exploit the immunogenic effect of chemotherapy. Disclosures Martinelli: Genentech: Consultancy; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; MSD: Consultancy; BMS: Speakers Bureau. Cavo:Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

Human intestinal infections by Shiga toxin (Stx)-producing Escherichia coli cause hemorrhagic colitis and hemolytic uremic syndrome (HUS), which represents the main cause of acute renal failure in early childhood. In HUS, Stx released in the gut enter the bloodstream and are targeted to renal endothelium. The mechanism of toxin delivery is still a matter of debate, although the role of polymorphonuclear leukocytes (PMN) as a Stx carrier has been indicated. The aim of this paper was to better define the interactions between Stx and human PMN. Direct and indirect flow cytometric analysis and binding experiments with radiolabeled toxins demonstrated that Stx bind to the surface of human mature PMN but not to immature PMN from G-CSF-treated donors. The use of the human myeloid leukemia cell (HL-60) model for inducible cell differentiation confirmed that the toxin binding occurs only after granulocytic differentiation. Stx binding caused a delay of the spontaneous apoptosis of PMN, as shown by the delayed appearance of apoptotic nuclei and activation of caspase 3 and by the higher number of cells negative to the annexin V-binding assay after 48 h. Moreover, flow cytometric analysis of mixed Stx-positive and Stx-negative PMN populations showed that the toxins were transferred from positive to negative PMN. The delayed, spontaneous apoptosis and the passage of the toxic ligand from older PMN to new, mature cells entering the circulation from the bone marrow may explain the previously reported persistence of Stx in the blood of children with HUS.

BACKGROUND: The aim of the present study was the evaluation of the apoptosis in residual white cells (WBCs) contained in platelet concentrates (PCs) and of the relationship of this apoptosis with the concentration of inflammatory cytokines in the medium and with platelet activation. STUDY DESIGN AND METHODS: Three independent methods were used to evaluated apoptosis in WBCs present in 9 PCs, either from single donors by apheresis (SD-PCs) or from pooled buffy coats (BC-PCs). All PCs were divided in two parts, one of which was irradiated. PCs were stored up to 4 days at room temperature, and samples were withdrawn daily for analysis of apoptosis, of platelet activation (surface and soluble CD62P), and of cytokine concentration (interleukin [IL]-1α , IL-1β, IL-6, IL-8, and tumor necrosis factor α ). RESULTS: Apoptosis was found to occur with storage in both irradiated and nonirradiated units. Platelet activation increased with storage time and was higher in BCPCs. The amount of released cytokines was rather variable among PC units. Only IL-8 was consistently found to increase with storage time. CONCLUSIONS: Apoptosis of residual WBCs occurred in PC units as a function of storage time. The amount and the time course of apoptosis seem to correlate with IL-8 release rather than with platelet activation or with the occurrence of febrile nonhemolytic transfusion reactions.

Background: The efficiency of prestorage leukodepletion of whole blood on the production of proinflammatory cytokines (IL-β, IL-6, IL-8 and TNF-α) was compared i

Background: Interferon may trigger autoimmune disorders, including autoimmune hepatitis, in immunocompetent patients. To date, no such disorders have been described in liver transplanted patients. Methods: 9 of 44 liver transplanted patients who had been receiving pegylated-interferon alpha-2b and ribavirin for at least 6 months for hepatitis C virus (HCV) recurrence, developed graft dysfunction despite on-treatment HCV-RNA clearance in all but one case. Laboratory, microbiological, imaging and histological evaluations were performed to identify the origin of graft dysfunction. The International Autoimmune Hepatitis scoring system was also applied. Results: In all cases infections, anastomoses complications and rejection were excluded, whereas the autoimmune hepatitis score suggested a “probable autoimmune hepatitis” (score from 10 to 14). Three patients developed other definite autoimmune disorders (overlap anti-mitochondrial antibodies (AMA)-positive cholangitis, autoimmune thyroiditis and systemic lupus erythematosus, respectively). In all cases, pre-existing autoimmune hepatitis was excluded. Anti-lymphocyte antibodies in immunosuppressive induction treatment correlated with the development of the disorder, whereas the use of granulocyte colony-stimulating factor to treat interferon-induced neutropenia showed a protective role. Withdrawal of antiviral treatment and treatment with prednisone resulted in different outcomes (five remissions and four graft failures with two deaths). Conclusions: De novo autoimmune hepatitis should be considered in differential diagnosis along with rejection in liver transplanted patients developing graft dysfunction while on treatment with interferon.

s S131 visits. A novel approach for quantifying dd-cfDNA using targeted amplification and next-generation sequencing was used to quantify dd-cfDNA on 109 plasma samples from these 24 patients. 3 to 10 samples were collected between 2 months and 1 year post-transplantation for each patient. Most plasma samples had endomyocardial biopsy rejection grades and geneexpression profiling (GEP) test scores available from the same clinic visits. Results: Median dd-cfDNA was 0.79% (interquartile range [0.38%, 1.30%]). Total between-patient variability (51% CV) was similar to within-patient variability (45% CV). Analytical variability was 17%. The ratio of betweenpatient variability to within-patient variability was 1.6 times lower for ddcfDNA than for GEP. No effect of recipient gender, age, CMV serologic status, or time post-transplantation was seen on % dd-cfDNA measurements. Conclusion: In this longitudinal study of non-rejecting heart transplant recipients, the withinand between-patient variabilities of dd-cfDNA were shown to be of similar size, and significantly larger than the analytical variability of the assay. The ratio of between-patient variability to within-patient variability was significantly less than in GEP, suggesting that dd-cfDNA does not depend as strongly on patient-specific factors as does GEP. dd-cfDNA showed no dependence on patient age, gender, CMV serologic status, or time since transplant in this cohort. Taken together, these characteristics indicate that dd-cfDNA is a stable analyte in non-rejecting patients. (Study sponsored by CareDx, Inc.)

Engraftment by recipient's (R) cells has been already demonstrated in gender mismatched liver grafts using fluorescence in situ hybridization (FISH), with contrasting results concerning epithelial cells. Mismatch for human leukocyte antigen (HLA) class I (HLA-I) is quite common in patients with orthotopic liver transplantation (OLT). We thus aimed to assess whether monoclonal antibodies (MoAbs), currently employed in the HLA typing process, could be used to study the dynamics of R cells in liver grafts. A total of 50 frozen liver biopsies from 37 patients receiving a HLA mismatch liver were tested. Biopsies were obtained from 3 days to more than 360 days after OLT. Frozen sections of graft biopsies were stained using an immunoperoxidase technique with the proper MoAbs. In selected cases, a double immunofluorescence was also performed. Circulating R blood cells and sinusoidal cells were occasionally observed in liver biopsies obtained within 10 days after OLT and were commonly detected after 1 month. The number of sinusoidal cells continued to increase up to 6 months, as shown on serial biopsies. On the whole, R blood cells and R sinusoidal cells were detected in 86% and 82% of the biopsies, respectively. R hepatocytes and biliary cells were detected after 40 and 60 days after OLT, respectively, in 14% (hepatocytes), 8% (bile ducts), and 12% (proliferating bile ducts) of the biopsies. R hepatocytes presented as single cells or groups of few cells; their number was lower than 1% and apparently did not increase with time after OLT. In conclusion, it is possible to detect R cells in liver graft using MoAbs to specific mismatched HLA-I alleles. R sinusoidal cells start to appear after 10 days and are commonly observed after 1 month; bile duct cells and hepatocytes appear later and their number does not increase with time. Engraftment by R epithelial cells seems to be less important than previously reported.

The recent development of new filters used for leucocyte reduction aims at restricting the number of leucocytes to a threshold where their undesirable effects can be minimized or excluded. In this paper we describe the performance of a new filter named BIO R01 MAX and claimed by the manufacturer to perform 5 Log10 depletion. The results show that the efficiency of the filter reached 5 Log10 depletion and the absolute number of white blood cells in the post-filtration units is always less than 2 x 10(4) with considerable safety in the prevention of transfusion reactions.

Pre-operative donation of autologous blood is a practice that is now being abandoned. Alternative methods of transfusing autologous blood, other than predeposited blood, do however play a role in limiting the need for transfusion of allogeneic blood. This survey of autologous blood transfusion practices, promoted by the Italian Society of Transfusion Medicine and Immunohaematology more than 2 years after the publication of national recommendations on the subject, was intended to acquire information on the indications for predeposit in Italy and on some organisational aspects of the alternative techniques of autotransfusion.A structured questionnaire consisting of 22 questions on the indications and organisational methods of autologous blood transfusion was made available on a web platform from 15 January to 15 March, 2013. The 232 Transfusion Services in Italy were invited by e-mail to complete the online survey.Of the 232 transfusion structures contacted, 160 (69%) responded to the survey, with the response rate decreasing from the North towards the South and the Islands. The use of predeposit has decreased considerably in Italy and about 50% of the units collected are discarded because of lack of use. Alternative techniques (acute isovolaemic haemodilution and peri-operative blood salvage) are used at different frequencies across the country.The data collected in this survey can be considered representative of national practice; they show that the already very limited indications for predeposit autologous blood transfusion must be adhered to even more scrupulously, also to avoid the notable waste of resources due to unused units.Users of alternative autotransfusion techniques must be involved in order to gain a full picture of the degree of use of such techniques; multidisciplinary agreement on the indications for their use is essential in order for these indications to have an effective role in "patient blood management" programmes.

We report the identification of an HLA-DRB1*01 nucleotide sequence variant in three members of a Caucasian Italian family by using sequence -based typing. The nucleotide sequence of exon 2 observed in the new allele is identical to that of HLA-DRB1*010201 except in position 189 (codon 34) where the adenine of the consensus was replaced by a guanine and it was designated officially as HLA-DRB1*010203* by the WHO Nomenclature Committee. The nucleotide sequence is available at the ebml database under accession number AJ430382.1.

We performed a flow-cytometric analysis of the expression of D antigen in D+W samples (previously termed Du). We also analysed a series of D-positive and D-negative (cde phenotype) samples to obtain positive and negative controls, respectively. The evaluation was carried out by immunofluorescence and the intensity of positivity was expressed as mean channel value (MCV) of fluorescence. Results demonstrated that D+W samples have lower expression (less than 1 log) than D-positive cases (p > 0.001, Student t test), while cde samples show the same MCVs as negative controls. Moreover, it was also possible to set a grading of D antigen expression and to analyze cases difficult to assess by agglutination only.

BACKGROUND The reduction of white cells in blood components before transfusion by filters with at least 3 log10 depletion may prevent adverse transfusion reactions such as HLA alloimmunization, febrile reactions, transmitted infections, and immunomodulation. A new generation of filters with 4 log10 depletion is now available. STUDY DESIGN AND METHODS The aim of this study is to compare the efficiency of white cell reduction by six commercial filters for packed red cells with 3 and 4 log10 depletion (claimed by manufacturers). The analysis of white cell concentration in the white cell-reduced units was performed by flow cytometry and with a Nageotte chamber. RESULTS The last generation of filters (BPF4, RC400, R01 Plus) show mean residual white cell numbers of 0.18 +/- 0.14, 0.26 +/- 0.21, and 0.25 +/- 0.15 x 10(6), respectively, by flow cytometric analysis and 0.05 +/- 0.04, 0.18 +/- 0.15, and 0.38 +/- 0.23 x 10(6), respectively, by Nageotte chamber evaluation. The 3 log10 depletion filters (R01, Leucostop-4LT-mono, R200) have mean residual white cell numbers of 1.41 +/- 0.92, 2.4 +/- 1.99, and 1.05 +/- 0.64 x 10(6), respectively, by flow cytometric analysis and 3.56 +/- 1.7, 1.67 +/- 1.3, and 3.21 +/- 4.1 x 10(6), respectively, by Nageotte chamber evaluation. The data show that the BPF4, RC400, and R01 Plus filters are likely to be more efficient by 1 log10 reduction than the R01, Leucostop-4LT-mono, and R200 filters. CONCLUSION The most recent generation of filters is able to deplete white cells from packed red cells by 4 log10; in particular, with one of the filters, the residual WBC content was less than 0.5 x 10(6) per unit in all experiments, while two other filters reached that level in 9 of 10 experiments.

The HLA loci are a part of the genetic region known as the major histocompatibility complex (MHC). In the last twenty years there has been an exponential growth in the application of DNA technology to the field of histocompatibility and immunogenetics. Histocompatibility between the patient and donor is a prerequisite for the success of haematopoietic stem cell transplantation. In haematopoietic stem cell transplantation allele-level typing needs to evaluate compatibility for the HLA-A,B,C Class I and DRB1 and DQB1 Class II loci in the average transplant program because it is well established that mismatches at certain HLA loci between donor-recipients are closely linked to the risk of graft versus host disease. Resolution at an antigen level in solid organ transplantation is currently sufficient for HLA-A,B and DR antigens and it could be achieved by serological or molecular biology techniques. In solid organ transplantation the definition of antibodies in the recipient to HLA antigens is more important and it was performed primarily by serological technique and more recently by solid phase immunoassays that are more sensitive and specific.

Interest in biological studies on various cell types for many biomedical applications, from research to patient treatments, is constantly increasing. The ability to discriminate (sort) and/or quantify distinct subpopulations of cells has become increasingly important. For instance, not only detection but also the highest depletion of neoplastic cells from normal cells is an important requisite in the autologous transplantation of lymphocytes for blood cancer treatments. In this work, gravitational field-flow fractionation (GrFFF) is shown to be effective for sorting a heterogeneous mixture of human, living lymphocytes constituted of neoplastic B cells from a Burkitt lymphoma cell line and healthy T and B lymphocytes from blood samples. GrFFF does not require the use of fluorescent immunotags for sorting cells, and the sorted cells can be collected for their further characterization. Flow cytometry was used to assess the viability of the cells collected, and to evaluate the cell fractionation achieved. A low amount of neoplastic B lymphocytes (less than 2%) was found in a specific fraction obtained by GrFFF. The high depletion from neoplastic cells (more than 98%) was confirmed by a clonogenicity test.

Purpose Antibody mediated rejection (AMR) is a serious emerging issue in the management of heart transplant (HT) recipients. Although consensus criteria currently define pathological diagnosis of AMR (pAMR), the clinical drawbacks of the pAMR criteria in term of graft function and prognosis, as well as pAMR relationship with donor specific antibodies, (DSA) are unexplored. Methods and Materials In this study we analyze the correlation between pAMR grading, hemodynamic features, DSA detection, and subsequent prognosis in a cohort of consecutive HT recipients with suspected AMR. Results We included 184 patients, with estimated 7-year graft survival of 84±4%, after biopsy. Luminex assay was available in 86 patients, of whom 24 (28%) positive for DSA. pAMR was diagnosed in 72 (39%) recipients (61, pAMR1 and 11 pAMR2). Capillary wedge pressure was higher in patients with pAMR≥1 than in those with pAMR 0 (P=0.03) but we found no difference in hemodynamic data between pAMR 1 and 2. Patients with DSA had a lower cardiac output (P=0.02). However, DSA did not correlate with pAMR grading, but were more often detected in patients with mixed rejection (cellular and AMR), than in those with pAMR alone (P=0.03). Significant angiographic vasculopathy (CAV) accounted for 30% of total deaths, regardless of any rejection grade, and was associated with 12% yearly mortality. pAMR grading per se, on the other hand, was not associated with graft survival, while mixed rejection accounted for a 3.6 times increased risk of graft-related death, in patients without CAV (P=0.04). Conclusions While showing that pathological signs of AMR were associated with a worse hemodynamic profile, with however no differences between pAMR 1 and 2 grades, this study provide suggestive evidence that DSA co-activate both cellular and antibody-mediated graft injury, and that mixed rejection, not AMR alone, drives poor outcome. These data shed new light on the clinical significance of pAMR grading, and brings attention to the adverse consequences of mixed rejection, as opposed to “pure” AMR.