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Background aims Natural killer (NK) cells have the potential to become a successful immunotherapy as they can target malignant cells without being direct effectors of graft-versus-host disease. Our group has previously shown that large numbers of functional NK cells can be differentiated in vitro from umbilical cord blood (CB) CD34 + cells. To produce a clinically relevant and effective immunotherapy, we hypothesized that it is essential that the NK cells are able to proliferate and persist in vivo while maintaining an optimal activation status and killing capacity. Methods We evaluated the proliferation capacity, telomere length and terminal differentiation markers expressed by NK cells differentiated in vitro . We also determined how their cytotoxicity compared with peripheral blood (PB) NK cells and CBNK cells when targeting patient acute myeloid leukemia (AML) blasts and solid tumor cell lines. Results We found that the differentiated NK cells could respond to interleukin-2 and proliferate in vitro. Telomere length was significantly increased, whereas CD57 expression was significantly reduced compared with PBNK cells. The cytotoxicity of the differentiated NK cells was equivalent to that of the PBNK and CBNK cell controls, and priming consistently led to higher levels of killing of patient leukemic blasts and solid tumor cell lines in vitro . Interestingly, this activation step was not required to observe killing of patient AML blasts in vivo. Conclusion We are able to generate NK cells from CBCD34 + cells in high numbers, allowing for multiple infusions of highly cytotoxic NK cells that have potential to further proliferate in vivo , making them a desirable product for application as an immunotherapy in the clinic.

Background Allogeneic haematopoietic cell transplantation (HCT) is a curative therapy for severe haematological disorders, including acute leukaemia. However, it carries significant morbidity and mortality due to infection, graft-versus-host disease and relapse. The cellular content of HCT grafts, in particular the ratio between regulatory T cells (Treg) and conventional T cells, is an important factor influencing the severity of these complications. Whilst previous studies have been performed in fresh adult peripheral blood mobilised grafts, it has not been applied in umbilical cord blood (CB) HCTs. CB enumerations are more challenging as they must be performed from small cryopreserved segments stored alongside the CB units used for HCT. Study and design methods Fresh CB units and thawed segments were analysed for their Treg and T cell content using both flow cytometry (the benchmark technique) and an epigenetic, DNA-based methodology. The two methods were compared for their agreement, consistency and susceptibility to error when assessing Treg and CD3+ cell numbers in both fresh and cryopreserved samples. Results Epigenetic enumeration gave consistent results in both fresh and frozen samples, providing Treg/CD3 estimates that were similar. Assessment of Tregs and CD3+ cells by flow cytometry and epigenetic assessments in fresh samples showed that these two methods were correlated. Conversely, significant cell death was observed in the thawed segments which affected Treg and CD3 cell estimates by flow cytometry. Conclusion Epigenetic assessments offer significant advantages over flow cytometry for analysing cryopreserved CB. Unlike with flow cytometry assessments of thawed segments, similar cell numbers were observed in fresh and frozen samples, with material requirements not being limiting and being unaffected by high cell death. With this method, multiple epigenetic assessments can be performed from extracted DNA, to provide statistical confidence and confirm observed results. Finally, the method raises the possibility of retrospective studies of historical samples where only DNA is available.

The high-energy evolution in perturbative QCD suffers from a severe lack-of-convergence problem, due to higher order corrections enhanced by double and single transverse logarithms. We resum double logarithms to all orders within the non-linear Balitsky-Kovchegov equation, by taking into account successive soft gluon emissions strongly ordered in lifetime. We further resum single logarithms generated by the first non-singular part of the splitting functions and by the one-loop running of the coupling. The resummed BK equation admits stable solutions, which are used to successfully fit the HERA data at small $x$ for physically acceptable initial conditions and reasonable values of the fit parameters., Comment: 4 pages, based on a talk presented at Quark Matter 2015, 27 Sep-3 Oct 2015, Kobe, Japan

Background aims Natural killer (NK) cells offer the potential for a powerful cellular immunotherapy because they can target malignant cells without being direct effectors of graft-versus-host disease. We have previously shown that high numbers of functional NK cells can be differentiated in vitro from umbilical cord blood (CB) CD34 + cells. To develop a readily available, off-the-shelf cellular product, it is essential that NK cells differentiated in vitro can be frozen and thawed while maintaining the same phenotype and functions. Methods We evaluated the phenotype and function of fresh and frozen NK cells differentiated in vitro . We also assessed whether the concentration of NK cells at the time of freezing had an impact on cell viability. Results We found that cell concentration of NK cells at the time of freezing did not have an impact on their viability and on cell recovery post-thaw. Moreover, freezing of differentiated NK cells in vitro did not affect their phenotype, cytotoxicity and degranulation capacity toward K562 cells, cytokine production and proliferation. Conclusions We are therefore able to generate large numbers of functional NK cells from CB CD34 + cells that maintain the same phenotype and function post-cryopreservation, which will allow for multiple infusions of a highly cytotoxic NK cell product.

A total of 125 Costa Ricans of Amerindian descent were genotyped at high-resolution for the human leukocyte antigen loci HLA-A, -B, -C, and -DRB1 using sequence-based typing methods. The respective allele and extended haplotype frequencies, as well as Hardy-Weinberg proportions were calculated. The most frequent extended haplotype identified was A*24:02:01-B*40:02:01-C*03:05-DRB1*04:07:01G, with an estimated frequency of 8.26%. A deviation from Hardy-Weinberg Equilibrium was detected at the DRB1 locus (p = 0.099). The HLA genotypic data of the population sample reported here are available publicly in the Allele Frequencies Net Database under the population name “Costa Rica Amerindians” and the identifier AFN3608.

A total of 221 Costa Rican Mestizos from the Central Valley were genotyped at high-resolution for the human leukocyte antigen loci HLA-A, -B, -C, -DRB1, and -DQB1 using sequence-based typing methods. The respective allele and extended haplotype frequencies, as well as Hardy-Weinberg proportions were calculated. The most frequent extended haplotype identified was A*24:02:01-B*40:02:01-C*03:05-DRB1*08:02:01-DQB1*04:02:01, with an estimated frequency of 2.04%. No deviation from Hardy-Weinberg Equilibrium was detected at any of the loci studied. The HLA genotypic data of the population sample reported here are available publicly in the Allele Frequencies Net Database under the population name “Costa Rica Central Valley Mestizo” and the identifier AFN3606.

Even when kidney allografts are well matched for human leukocyte antigen (HLA) and anti-HLA antibodies are not detected, graft rejection can still occur. There is evidence that some patients who lose their graft have antibodies specific for major histocompatibility complex (MHC) class I-related chain A (MICA) antigens. We investigated whether mismatching MICA alleles associates with MICA antibody production and graft rejection or dysfunction. MICA and HLA antibody screening in 442 recipients was performed, and specificities were confirmed in a subgroup of 227 recipients using single-antigen multiplex technology. For assignment of MICA antibody specificity, we used three independent assays. In addition, MICA alleles of 227 recipients and donors were determined by DNA sequencing. In all, 17 patients (7.5%) had MICA antibodies, and 13 patients (6%) developed MICA donor-specific antibodies (DSA). Multivariate analysis revealed MICA mismatching, as an independent significant factor associated with the presence of MICA antibodies (p = 0.009), and 14 mismatched MICA residues significantly correlated with MICA antibody production. MICA and HLA antibodies significantly associated with acute rejection (AR) and MICA DSA and HLA DSA correlated with decreased graft function by univariate and multivariate analysis. We conclude that mismatching for MICA epitopes in renal transplantation is a mechanism leading to production of MICA antibodies that associate with AR and graft dysfunction.

The human leukocyte antigen (HLA) system is the most polymorphic in humans. Its allele, genotype, and haplotype frequencies vary significantly among different populations. Molecular typing data on HLA are necessary for the development of stem cell donor registries, cord blood banks, HLA-disease association studies, and anthropology studies. The Costa Rica Central Valley Population (CCVP) is the major population in this country. No previous study has characterized HLA frequencies in this population. Allele group and haplotype frequencies of HLA genes in the CCVP were determined by means of molecular typing in a sample of 130 unrelated blood donors from one of the country's major hospitals. A comparison between these frequencies and those of 126 populations worldwide was also carried out. A minimum variance dendrogram based on squared Euclidean distances was constructed to assess the relationship between the CCVP sample and populations from all over the world. Allele group and haplotype frequencies observed in this study are consistent with a profile of a dynamic and diverse population, with a hybrid ethnic origin, predominantly Caucasian-Amerindian. Results showed that populations genetically closest to the CCVP are a Mestizo urban population from Venezuela, and another one from Guadalajara, Mexico.

Resumen Antecedentes La seguridad de los farmacos inhibidores selectivos de la ciclooxigenasa 2 (COX-2), especialmente cuando se usan en hipertensos o en pacientes de alto riesgo, ha sido cuestionada. Esto puede deberse a los efectos de estos medicamentos en la funcion endotelial. Objetivo Evaluar los efectos de la inhibicion selectiva y no selectiva de la COX en la vasodilatacion dependiente de NO y prostaciclina (PGI 2 ) en pacientes hipertensos. Metodos Se incluyo a 24 pacientes hipertensos en un estudio prospectivo, controlado, aleatorizado, a doble ciego en el cual se evaluaron los efectos de la indometacina y el rofecoxib en la vasodilatacion dependiente de flujo y el efecto hipotensor agudo del captopril. Se midio la vasodilatacion de la arteria humeral en respuesta a 5 min de isquemia mediante ultrasonido Doppler de alta resolucion con transductores arteriales de 10 MHz. El efecto hipotensor agudo del captopril se evaluo con un esfigmomanometro electronico validado a las 0, 1, 2, 3, 4, 5, 12 y 24 h tras los tratamientos. Resultados Ni la indometacina, el rofecoxib, el captopril, el captopril mas indometacina o el captopril mas rofecoxib tuvieron efecto en la vasodilatacion dependiente de flujo. La indometacina, pero no el rofecoxib, inhibio el efecto hipotensor agudo del captopril. Conclusiones Esto indica que la vasodilatacion aguda inducida por captopril depende en buena medida de PGI 2 cuya liberacion es mediada por COX-1 y que el riesgo cardiovascular inducido por los inhibidores selectivos de COX-2 pudiera reducirse con la administracion concomitante de captopril u otros inhibidores de la enzima de conversion de angiotensina.

The Nucleotide-binding Oligomerisation Domain (NOD) 2 protein is encoded by the Caspase Recruitment Domain (CARD) 15 gene and has a critical role in innate immunity. Recent studies have implicated Single Nucleotide Polymorphisms (SNPs) of the NOD2/CARD15 gene with the onset of several Inflammatory Bowel Disorders (Crohn's Disease, Blau syndrome) and the progression of several malignant diseases. The identification of SNPs in the genotypes of donor and recipient pairs prior to haematopoietic stem cell transplantation have also been shown to predict for a worse outcome, specifically causing increases in the incidence and severity of acute Graft-versus-Host disease, disease relapse and mortality. In light of these widespread areas of interest, we have developed a Polymerase Chain Reaction assay using Sequence Specific Primers (PCR-SSP) to identify the three SNPs that have been implicated, (SNPs 8, 12 and 13). The assay has proven to be a rapid and accurate method of performing NOD2/CARD15 genotyping when compared to other techniques described to date.

The introduction of in vitro T cell expansion and assay methods that are robust and easy to use would be welcome in cancer vaccine and infectious disease research. By coating HLA class I −ve B cells with recombinant HLA class I peptide complexes, we are able to produce antigen presenting cells and target cells expressing a single defined antigen in the context of costimulatory and adhesion molecules. HLA class I mono-specific cells promoted the in vitro expansion of CMV epitope specific CD8+ T cells from 0.03% to 30.6% in 2 weeks, which was comparable to using peptide-loaded dendritic cells. The HLA class I mono-specific cells were also shown to promote in vitro antigen specific T cell function in assays based on measuring cytokine production and cytolytic activity. HLA class I mono-specific cells are simple to prepare, can be used with any recombinant HLA class I allele/peptide combination and should provide a useful system for in vitro T cell expansion and functional analysis.

Lithium insertion mechanism in some antimony-based compounds: SnSb, CoSb3, CrSb2, TiSb2 has been studied by means of 121 Sb Mossbauer spectrometry which gives valuable information about the local electronic structure of the probed element (Sb). The structural and electronic modifications induced by insertion of lithium have been characterized. For these Sb-based materials the lithium insertion mechanisms involve Li3Sb formation and composite multi-phase separations with one component displaced from the pristine compound.

Human leukocyte antigen (HLA) class I polymorphism was studied within a population of 70 unrelated Kolla Amerindians from the far northwest of Argentina close to the Bolivian border. The results indicate that the HLA-A, -B, and -C alleles typical of other Amerindian populations also predominate in the Kolla. These alleles belong to the following allele groups: HLA-A*02, *68, *31, *24, HLA-B*35, *15, *51, *39, *40, *48, and Cw*01, *03, *04, *07, *08, and *15. For the HLA-A locus, heterogeneity was seen for HLA-A*02 with A*0201, *0211, and *0222; and for A*68 with *68012 and *6817, the latter being a novel allele identified in this population. Analysis of HLA-B identified heterogeneity for all Amerindian allele groups except HLA-B*48, including the identification of the novel B*5113 allele. For HLA-C heterogeneity was identified within the Cw*07, *04, and *08 groups with Cw*0701/06, *0702, *04011, *0404, *0803, and *0809 identified. The most frequent "probable" haplotype found in this population was B*3505-Cw*04011. This study supports previous studies, which demonstrate increased diversity at HLA-B compared with HLA-A and -C. The polymorphism identified within the Kolla HLA-A, -B, and -C alleles supports the hypothesis that HLA evolution is subject to positive selection for diversity within the peptide binding site.

We studied the reduction mechanism of SnO in lithium cells by X-ray absorption near OK and SnL I edge spectroscopy, 119 Sn Mossbauer spectroscopy and X-ray diffraction. The reduction mechanism is complex, involving mixed valence intermediate compounds. In the interval 0≤Li/Sn≤2 the main reaction corresponds to a partial reduction of Sn II , which is partially reversible, regenerating the tin oxide during the charge. For Li/Sn greater than two, LiSn bonds are formed, but SnO interactions are still present. The charge within this interval also partially regenerates tin oxide, but the reversibility does not extend to Li/Sn lower than two. At low voltage and very large depth of discharge, the formation of LiSn alloys and Li 2 O clearly takes place, with negligible SnO interactions.

We wanted to determine whether naive T cells could make the Types 1, 2 and 0 defining cytokines Interleukin (IL)-4 and Interferon (IFN)gamma. We show that stimulation of naive T cells (CD3+ CD45RA+) derived from cord blood by phorbol ester (phorbol-12-myristate-13-acetate: PMA) plus lonomycin induced detection of Types 1, 2 and 0 cells. Conversely, when we stimulated the naive T cells through the T cell receptor (with anti-CD3 monoclonal antibody alone) there was no detection of IFNgamma or IL-4 producing cells. Stimulation with PMA and CD3 induced detection of only Type 2 cells. This unexpected finding shows that there is a high frequency of Type 2 cells within the naive T cell population, contrary to previously published reports. The highest percentage of Type 2 naive cells (10.5%) was obtained with 50 ng/ml PMA plus 50 microg/ml anti-CD3. Thus, we have shown that naive T cells derived from cord blood have the capacity to make both Types 1 and 2 cytokines and the frequency of cells producing these cytokines can be greater than 20%, depending on the stimulus used.

To date, over 1000 cord blood (CB) transplants have been reported from different centers world-wide and it is generally agreed that CB represents an encouraging alternative to bone marrow (BM) transplantation. There are a variety of reasons for this, however, possibly the two most controversial aspects are (a) whether there is less graft versus host disease (GVHD) with CB compared to BM transplantation, and (b) whether we can use more HLA mismatches with CB transplantation. The major theory regarding the reduced immunological response of CB lymphocytes is that CB T and NK cells are naive and, therefore, not primed for activation. However, the naive phenomena that has been noted in vitro may be bypassed in vivo by unforeseen factors. We show evidence that there are differences in the soluble factors present in CB and adult serum and that these differences play a role in T cell function. Thus, adult serum will enhance both mitogen and IL-2 specific T cell growth whereas CB serum has no effect, suggesting that there is an activation/growth factor present in adult sera, which is absent in CB sera. This work could enable us to identify the molecular mechanisms which are associated with a lower GVHD in CB compared to BM transplanted individuals.

Mesocarbon microbeads (MCMB) derived from petroleum residua and heat treated under different experimental conditions were characterised by X-ray and electron diffraction, proton magnetic resonance (PMR), Fourier transform infrared spectroscopy (FTIR) and electron paramagnetic resonance (EPR). The presence of two different forms of hydrogen is retained after heating to 750°C under vacuum. Graphitisation to 3000°C leads to graphite ribbon-like particles surrounding microbeads of a few microns in size. The crystalline graphite monodomains are with a small band gap or are semimetallic as observed by EPR. Heat treatment even at 3000°C does not eliminate completely the localised paramagnetic defects in the microbeads. These properties condition the aptitude of these materials toward their use in lithium and sodium electrochemical cells. The samples prepared at 750°C have a reversible intercalation behaviour, while samples prepared at 3000°C evidence solvent decomposition resulting in a non-reversible extended discharge plateau when using sodium perchlorate electrolyte dissolved in pure propylene carbonate (PC).

Human soluble CD8 (sCD8) is secreted by activated CD8 + cytotoxic T lymphocytes (CTLs). The immunological role of sCD8 is poorly defined, however. We have studied the influence of sCD8 on HLA class I interactions by real-time analysis. Using an optical biosensor we demonstrated that the binding of sCD8 to HLA-A2 promotes exchange of β 2 -microglobulin (β 2 m) in order to stabilize the complex. Kinetic analysis showed that sCD8 significantly increased the affinity (K A ) of HLA-A2 for immobilized human β 2 m; from 1.14 ± 0.04 × 10 9 M −1 in its absence, to 2.18 ± 0.21 × 10 9 M −1 following preincubation with sCD8. This suggests that the sCD8:HLA class I complex is unlikely to be degraded at the cell surface. Even in the presence of exogenous peptide (HLA-A2 specific or nonspecific), sCD8 has a stabilizing influence on the HLA class I molecule. These findings point to an immunosuppressive role for sCD8, because the binding of sCD8 to HLA class I would block the binding site for CTL-bound CD8 and, therefore, interfere with T cell activation and proliferation. This may have particular significance in pathological situations where elevated levels of sCD8 are found in extracellular fluids, and sCD8 may provide an alternative approach for immunosuppressive therapy.

To date, over 400 human umbilical cord blood cord blood (CB) transplants have been reported from different centres world-wide and it is generally agreed that CB represents an encouraging alternative to bone marrow (BM) transplantation. There are a variety of reasons for this which include the wider availability and easier access of CB compared to BM. In addition it has been suggested that there is a reduced graft-versus-host-disease (GvHD) with CB compared to BM transplantation. The explanations for this implied benefit are numerous, but research into this area is only just beginning. Nevertheless, it is clear that both T cells and natural killer (NK) cells have reduced function when isolated from CB compared to adult and both these cell types have been implicated in GvHD pathogenesis. How and why the function is reduced is yet to be determined. Many laboratories have tried to answer these questions and the majority have done this by comparing the function of lymphocytes obtained from adult blood with those compared with CB. Since cytokine production by a cell is an indication of the cells function it is important to determine the differences between adult and CB with respect to production of these soluble factors. Here, we have reviewed the current research regarding these CB and adult cell comparisons with an emphasise on cytokine production. Our aim is to obtain a clearer understanding of the mechanisms which may be involved in causing a reduced GvHD in CB compared to BM transplantation.

We have shown that both demographic and immunogenetic factors are involved in the immune responses of Hepagene vaccinated individuals who were persistent nonresponders to 'S' containing hepatitis B vaccines. The HLA-DRB1 0701; DQB1 0202 genotype was found to be associated with a decline of anti-HBs antibodies (anti-HBs) and were frequent in those individuals who remained nonresponders following booster vaccination. Contrary to previously published 'S' vaccination data, Hepagene stimulated T-cell responses showed a lack of correlation with the humoral responses. Limiting dilution analysis demonstrated that the cellular immune response is associated with the kinetics of exposure to Hepagene rather than magnitude of the anti-HBs response. It remains that despite the inclusion of the pre-S proteins 74% nonresponder vaccinated individuals failed to produce > 100 IU/l of anti-HBs. However, these were persistent nonresponders and it was therefore encouraging that two doses of Hepagene did seroconvert (> 10 IU/L) 61% of this difficult group.

Aim To sequence entire HLA class I genes of 45 DNA samples in a single sequencing reaction using DNA-barcode-labelled primers and Single Molecule, Real-Time (SMRT®) DNA sequencing technology. Method Our previous experience of sequencing full-length HLA class I genes using SMRT® technology has shown that it has read length capabilities and accuracy amenable for definitive allele-level resolution HLA typing. Further experiments to determine multiplexing using DNA barcodes have shown that multiplexing up to 20 DNA samples for a single HLA locus or 8 DNA samples for multiple loci, is possible. Forty-five DNA samples were selected for HLA class I typing and were d derived from different DNA sources (blood, saliva and umbilical cord blood). Nine of these DNA samples had previously been identified as having novel alleles at one of their HLA class I loci using standard HLA typing methods. Whole gene amplicons were generated for the HLA class I genes. Each of the forty-five DNA samples were amplified for HLA-A, -B and -C using generic primers tagged with a DNA barcode unique to that sample resulting in 135 barcoded amplicons. Results Sufficient numbers of reads were obtained for each locus of each sample to enable HLA allele calling (mean 145; range 47–385) with mean a QV > 75. 252 HLA class I alleles were expected from the 45 DNA samples when homozygous loci were considered. The HLA types of 219 of these alleles immediately matched the types observed previously. The consensus sequences also confirmed the presence and phasing of the variants within the nine novel alleles. The consensus sequences on the 24 remaining alleles contained novel differences when compared to existing reference sequences in the IMGT/HLA Database. Sanger sequencing is being used to verify these novel positions. Conclusion SMRT DNA sequencing has the capability and the accuracy to facilitate definitive allele-level resolution HLA typing. We have shown for the first time that it is possible to use this method to sequence multiple DNA samples for multiple HLA class I genes using DNA barcode technology, to a level that makes it viable for routine use as a high-throughput methodology. The identification of previously unknown mismatches between recipients and their donors could significantly improve stem cell transplant outcome. S. Ranade: Employee; Company/Organization; Pacific Biosciences . K. Eng: Employee; Company/Organization; Pacific Biosciences. B. Bowman: Employee; Company/Organization; Pacific Biosciences . L. Hepler: Employee; Company/Organization; Pacific Biosciences.