Miniaturized and High-Throughput Assays for Analysis of T-Cell Immunity Specific for Opportunistic Pathogens and HIV

Monitoring of antigen-specific T-cell responses is valuable in numerous conditions that include infectious diseases, vaccinations, and opportunistic infections associated with acquired or congenital immune defects. A variety of assays that make use of peripheral lymphocytes to test activation markers, T-cell receptor expression, or functional responses are currently available. The last group of assays calls for large numbers of functional lymphocytes. The number of cells increases with the number of antigens to be tested. Consequently, cells may be the limiting factor, particularly in lymphopenic subjects and in children, the groups that more often require immune monitoring. We have developed immunochemical assays that measure secreted cytokines in the same wells in which peripheral blood mononuclear cells (PBMC) are cultured. This procedure lent itself to miniaturization and automation. Lymphoproliferation and the enzyme-linked immunosorbent spot (ELISPOT) assay have been adapted to a miniaturized format. Here we provide examples of immune profiles and describe a comparison between miniaturized assays based on cytokine secretion or proliferation. We also demonstrate that these assays are convenient for use in testing antigen specificity in established T-cell lines, in addition to analysis of PBMC. In summary, the applicabilities of miniaturization to save cells and reagents and of automation to save time and increase accuracy were demonstrated in this study using different methodological approaches valuable in the clinical immunology laboratory

T-cell depleted HLA-haploidentical HSCT in a child with neuromyelitis optica

Neuromyelitis optica is an immune-mediated disease characterized by a relapsing course, resulting in progressive disability. In children, given the long life expectancy, a disease-modifying treatment could be particularly desirable. Unfortunately, the currently available treatment strategies with this potential are scarce. Very limited data are available about the use of allogeneic hematopoietic stem cell transplantation (HSCT) for autoimmune neurological diseases. In this report, we present a pediatric case successfully treated with allogeneic HSCT from an HLA-haploidentical donor, after ex vivo TCR/CD19-depletion of the graft. To the best of our knowledge, this is the first case of a pediatric patient to benefit from such a treatment

Selective Depletion of αβ T Cells and B Cells for Human Leukocyte Antigen–Haploidentical Hematopoietic Stem Cell Transplantation. A Three-Year Follow-Up of Procedure Efficiency

HLA-haploidentical family donors represent a valuable option for children requiring allogeneic hematopoietic stem cell transplantation (HSCT). Because graft-versus-host diseases (GVHD) is a major complication of HLA-haploidentical HSCT because of alloreactive T cells in the graft, different methods have been used for ex vivo T cell depletion. Removal of donor αβ T cells, the subset responsible for GVHD, and of B cells, responsible for post-transplantation lymphoproliferative disorders, have been recently developed for HLA-haploidentical HSCT. This manipulation preserves, in addition to CD34+ progenitors, natural killer, γδ T, and monocytes/dendritic cells, contributing to anti-leukemia activity and protection against infections. We analyzed depletion efficiency and cell yield in 200 procedures performed in the last 3 years at our center. Donors underwent CD34+  hematopoietic stem cell (HSC) peripheral blood mobilization with granulocyte colony–stimulating factor (G-CSF). Poor CD34+ cell mobilizers (48 of 189, 25%) received plerixafor in addition to G-CSF. Aphereses containing a median of 52.5 × 109 nucleated cells and 494 × 106 CD34+ HSC were manipulated using the CliniMACS device. In comparison to the initial product, αβ T cell depletion produced a median 4.1-log reduction (range, 3.1 to 5.5) and B cell depletion led to a median 3.4-log reduction (range, 2.0 to 4.7). Graft products contained a median of 18.5 × 106 CD34+ HSC/kg recipient body weight, with median values of residual αβ T cells and B cells of 29 × 103/kg and 33 × 103/kg, respectively. Depletion efficiency monitored at 6-month intervals demonstrated steady performance, while improved recovery of CD34+ cells was observed after the first year (P = .0005). These data indicate that αβ T cell and B cell depletion of HSC grafts from HLA-haploidentical donors was efficient and reproducible

Optical and structural properties of sol-gel derived materials embedded in porous anodic alumina

Structure composing a xerogel, doped with lanthanide ions (erbium, terbium and europium), embedded in porous anodic alumina (PAA) have been fabricated and their optical and electrical characterisitics have been studied. Erbium photoluminescence at 1.53 µm from titania xerogel/PAA was found to increase with the number of xerogel layers and erbium concnetration for the excitation wavelength 532 nm, matching the area of transparency of both titania xerogel and PAA. Visible green and red electroluminescence was observed for terbium- and europium-doped IN2O3 and SnO2 xerogels embedded in porous anodic alumina. The improvement of the electrical properties of the xerogel/PAA cell is discussed, taking into account the observed ability of conducting In2O3:Sn (ITO) nanoparticles to penetrate into the anodic alumina pores

Outcome of children with acute leukemia given HLA-haploidentical HSCT after ab T-cell and B-cell depletion

Allogeneic hematopoietic stem cell transplantation (HSCT) from an HLA-haploidentical relative (haplo-HSCT) is a suitable option for children with acute leukemia (AL) either relapsed or at high-risk of treatment failure. We developed a novel method of graft manipulation based on negative depletion of ab T and B cells and conducted a prospective trial evaluating the outcome of children with AL transplanted with this approach. Eighty AL children, transplanted between September 2011 and September 2014, were enrolled in the trial. All children were given a fully myeloablative preparative regimen. Anti–T-lymphocyte globulin from day 25 to 23 was used for preventing graft rejection and graft-versus-host disease (GVHD); no patient received any posttransplantation GVHD prophylaxis. Two children experienced primary graft failure. The cumulative incidence of skin-only, grade 1-2 acute GVHD was 30%; no patient developed extensive chronic GVHD. Four patients died, the cumulative incidence of nonrelapse mortality being 5%, whereas 19 relapsed, resulting in a 24% cumulative incidence of relapse. With a median follow-up of 46 months for surviving patients, the 5-year probability of chronic GVHD-free, relapse-free survival (GRFS) is 71%. Total body irradiation–containing preparative regimen was the only variable favorably influencing relapse incidence and GRFS. The outcomes of these 80 patients are comparable to those of 41 and 51 children given transplantation from an HLA-identical sibling or a 10/10 allelic-matched unrelated donor in the same period. These data indicate that haplo-HSCT after ab T- and B-cell depletion represents a competitive alternative for children with AL in need of urgent allograft. This trial was registered at www.clinicaltrials.gov as #NCT01810120

CUORE and beyond: bolometric techniques to explore inverted neutrino mass hierarchy

The CUORE (Cryogenic Underground Observatory for Rare Events) experiment will search for neutrinoless double beta decay of $^{130}$Te. With 741 kg of TeO$_2$ crystals and an excellent energy resolution of 5 keV (0.2%) at the region of interest, CUORE will be one of the most competitive neutrinoless double beta decay experiments on the horizon. With five years of live time, CUORE projected neutrinoless double beta decay half-life sensitivity is $1.6\times 10^{26}$ y at $1\sigma$ ($9.5\times10^{25}$ y at the 90% confidence level), which corresponds to an upper limit on the effective Majorana mass in the range 40--100 meV (50--130 meV). Further background rejection with auxiliary light detector can significantly improve the search sensitivity and competitiveness of bolometric detectors to fully explore the inverted neutrino mass hierarchy with $^{130}$Te and possibly other double beta decay candidate nuclei.Comment: Submitted to the Proceedings of TAUP 2013 Conferenc

Exploring the Neutrinoless Double Beta Decay in the Inverted Neutrino Hierarchy with Bolometric Detectors

Neutrinoless double beta decay (0nubb) is one of the most sensitive probes for physics beyond the Standard Model, providing unique information on the nature of neutrinos. In this paper we review the status and outlook for bolometric 0nubb decay searches. We summarize recent advances in background suppression demonstrated using bolometers with simultaneous readout of heat and light signals. We simulate several configurations of a future CUORE-like bolometer array which would utilize these improvements and present the sensitivity reach of a hypothetical next-generation bolometric 0nubb experiment. We demonstrate that a bolometric experiment with the isotope mass of about 1 ton is capable of reaching the sensitivity to the effective Majorana neutrino mass (|mee|) of order 10-20 meV, thus completely exploring the so-called inverted neutrino mass hierarchy region. We highlight the main challenges and identify priorities for an R&D program addressing them.Comment: 22 pages, 15 figures, submitted to EPJ

Status of the CUORE and results from the CUORE-0 neutrinoless double beta decay experiments

CUORE is a 741 kg array of TeO2 bolometers for the search of neutrinoless double beta decay of 130Te. The detector is being constructed at the Laboratori Nazionali del Gran Sasso, Italy, where it will start taking data in 2015. If the target background of 0.01 counts/keV/kg/y will be reached, in five years of data taking CUORE will have a 1 sigma half life sensitivity of 10E26 y. CUORE-0 is a smaller experiment constructed to test and demonstrate the performances expected for CUORE. The detector is a single tower of 52 CUORE-like bolometers that started taking data in spring 2013. The status and perspectives of CUORE will be discussed, and the first CUORE-0 data will be presented.Comment: 7 pages, 4 figures, to be published in the proceedings of ICHEP 2014, 37th International Conference on High Energy Physics, Valencia (Spain) 2-9 July 201

CUORE-0 results and prospects for the CUORE experiment

With 741 kg of TeO2 crystals and an excellent energy resolution of 5 keV (0.2%) at the region of interest, the CUORE (Cryogenic Underground Observatory for Rare Events) experiment aims at searching for neutrinoless double beta decay of 130Te with unprecedented sensitivity. Expected to start data taking in 2015, CUORE is currently in an advanced construction phase at LNGS. CUORE projected neutrinoless double beta decay half-life sensitivity is 1.6E26 y at 1 sigma (9.5E25 y at the 90% confidence level), in five years of live time, corresponding to an upper limit on the effective Majorana mass in the range 40-100 meV (50-130 meV). Further background rejection with auxiliary bolometric detectors could improve CUORE sensitivity and competitiveness of bolometric detectors towards a full analysis of the inverted neutrino mass hierarchy. CUORE-0 was built to test and demonstrate the performance of the upcoming CUORE experiment. It consists of a single CUORE tower (52 TeO2 bolometers of 750 g each, arranged in a 13 floor structure) constructed strictly following CUORE recipes both for materials and assembly procedures. An experiment its own, CUORE-0 is expected to reach a sensitivity to the neutrinoless double beta decay half-life of 130Te around 3E24 y in one year of live time. We present an update of the data, corresponding to an exposure of 18.1 kg y. An analysis of the background indicates that the CUORE performance goal is satisfied while the sensitivity goal is within reach.Comment: 10 pages, 3 figures, to appear in the proceedings of NEUTRINO 2014, 26th International Conference on Neutrino Physics and Astrophysics, 2-7 June 2014, held at Boston, Massachusetts, US