Immunomagnetic t-lymphocyte depletion (ITLD) of rat bone marrow using OX-19 monoclonal antibody

Graft versus host disease (GVHD) may be abrogated and host survival prolonged by in vitro depletion of T lymphocytes from bone marrow (BM) prior to allotransplantation. Using a mouse anti-rat pan T-lymphocyte monoclonal antibody (0×19) bound to monosized, magnetic, polymer beads, T lymphocytes were removed in vitro from normal bone marrow. The removal of the T lymphocytes was confirmed by flow cytometry. Injection of the T-lymphocyte-depleted bone marrow into fully allogeneic rats prevents the induction of GVHD and prolongs host survival. A highly efficient technique of T-lymphocyte depletion using rat bone marrow is described. It involves the binding of OX-19, a MoAb directed against all rat thy-mocytes and mature peripheral T lymphocytes, to monosized, magnetic polymer spheres. Magnetic separation of T lymphocytes after mixing the allogeneic bone marrow with the bead/OX-19 complex provides for a simple, rapid depletion of T lymphocytes from the bone marrow. In vitro studies using flow cytometry and the prevention of GVHD in a fully allogeneic rat bone marrow model have been used to demonstrate the effectiveness of the depletion procedure. © 1989 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted

JACIE accreditation for blood and marrow transplantation: past, present and future directions of an international model for healthcare quality improvement.

Blood and marrow transplantation (BMT) is a complex and evolving medical speciality that makes substantial demands on healthcare resources. To meet a professional responsibility to both patients and public health services, the European Society for Blood and Marrow Transplantation (EBMT) initiated and developed the Joint Accreditation Committee of the International Society for Cellular Therapy and EBMT-better known by the acronym, JACIE. Since its inception, JACIE has performed over 530 voluntary accreditation inspections (62% first time; 38% reaccreditation) in 25 countries, representing 40% of transplant centres in Europe. As well as widespread professional acceptance, JACIE has become incorporated into the regulatory framework for delivery of BMT and other haematopoietic cellular therapies in several countries. In recent years, JACIE has been validated using the EBMT registry as an effective means of quality improvement with a substantial positive impact on survival outcomes. Future directions include development of Europe-wide risk-adjusted outcome benchmarking through the EBMT registry and further extension beyond Europe, including goals to faciliate access for BMT programmes in in low- and middle-income economies (LMIEs) via a 'first-step' process

A full scale comparative study of methods for generation of functional Dendritic cells for use as cancer vaccines

<p/> <p>Background</p> <p>Dendritic cells (DCs) are professional antigen-presenting cells with the ability to induce primary T-cell responses and are commonly produced by culturing monocytes in the presence of IL-4 and GM-CSF for 5–7 days (Standard DC). Recently, Dauer and co-workers presented a modified protocol for differentiation of human monocytes into mature DCs within 48 hours (Fast DC). Here we report a functional comparison of the two strategies for generation of DCs from human monocytes with adaptions for large-scale clinical use.</p> <p>Methods</p> <p>The Elutra Cell Selection System was used to isolate monocytes after collection of leukapheresis product. The enriched monocytes were cultured in gas permeable Teflon bags with IL-4 and GM-CSF for 24 hours (Fast DC) or 5 days (Standard DC) to obtain immature DCs. The cells were then transfected with mRNA from the leukemia cell line Jurkat E6 by electroporation and incubated for additional 24 h or 2 days in the presence of pro-inflammatory cytokines (TNFα, IL-1β, IL-6 and PGE₂) to obtain mature DCs.</p> <p>Results</p> <p>Mature Fast DC and Standard DC displayed comparable levels of many markers expressed on DC, including HLA-DR, CD83, CD86, CD208 and CCR7. However, compared to Standard DC, mature Fast DC was CD14^highCD209^low. Fast DC and Standard DC transfected with Jurkat E6-cell mRNA were equally able to elicit T cell specifically recognizing transfected DCs in vitro. IFNγ-secreting T cells were observed in both the CD4+ and CD8+ subsets.</p> <p>Conclusion</p> <p>Our results indicate that mature Fast DC are functional antigen presenting cells (APCs) capable of inducing primary T-cell responses, and suggest that these cells may be valuable for generation of anti-tumor vaccines.</p

Immunotherapy with allotumour mRNA-transfected dendritic cells in androgen-resistant prostate cancer patients

Here, we present results from a clinical trial employing a new vaccination method using dendritic cells (DCs) transfected with mRNA from allogeneic prostate cancer cell lines (DU145, LNCaP and PC-3). In all, 20 patients were enrolled and 19 have completed vaccination. Each patient received at least four weekly injections with 2 × 107 transfected DCs either intranodally or intradermally. Safety and feasibility of vaccination were determined. Immune responses were measured as delayed-type hypersensitivity and by in vitro immunoassays including ELISPOT and T-cell proliferation in pre- and postvaccination peripheral blood samples. Serum prostate-specific antigen (PSA) levels and bone scans were monitored. No toxicity or serious adverse events related to vaccinations were observed. A total of 12 patients developed a specific immune response to tumour mRNA-transfected DCs. In total, 13 patients showed a decrease in log slope PSA. This effect was strengthened by booster vaccinations. Clinical outcome was significantly related to immune responses (n=19, P=0.002, r=0.68). Vaccination with mRNA-transfected DCs is safe and results in cellular immune responses specific for antigens encoded by mRNA derived from the prostate cancer cell lines. The observation that in some patients vaccination affected the PSA level suggests that this approach may become useful as a treatment modality for prostate cancer patients

Mycoplasma Contamination Revisited: Mesenchymal Stromal Cells Harboring Mycoplasma hyorhinis Potently Inhibit Lymphocyte Proliferation In Vitro

Mesenchymal stromal cells (MSC) have important immunomodulatory effects that can be exploited in the clinical setting, e.g. in patients suffering from graft-versus-host disease after allogeneic stem cell transplantation. In an experimental animal model, cultures of rat T lymphocytes were stimulated in vitro either with the mitogen Concanavalin A or with irradiated allogeneic cells in mixed lymphocyte reactions, the latter to simulate allo-immunogenic activation of transplanted T cells in vivo. This study investigated the inhibitory effects of rat bone marrow-derived MSC subsequently found to be infected with a common mycoplasma species (Mycoplasma hyorhinis) on T cell activation in vitro and experimental graft-versus-host disease in vivo.We found that M. hyorhinis infection increased the anti-proliferative effect of MSC dramatically, as measured by both radiometric and fluorimetric methods. Inhibition could not be explained solely by the well-known ability of mycoplasmas to degrade tritiated thymidine, but likely was the result of rapid dissemination of M. hyorhinis in the lymphocyte culture.This study demonstrates the potent inhibitory effect exerted by M. hyorhinis in standard lymphocyte proliferation assays in vitro. MSC are efficient vectors of mycoplasma infection, emphasizing the importance of monitoring cell cultures for contamination

Estimation and application of the thermodynamic properties of aqueous phenanthrene and isomers of methylphenanthrene at high temperature

Estimates of standard molal Gibbs energy (ΔGf°) and enthalpy (ΔHf°) of formation, entropy (S°), heat capacity (Cp°) and volume (V°) at 25 °C and 1 bar of aqueous phenanthrene (P) and 1-, 2-, 3-, 4- and 9-methylphenanthrene (1-MP, 2-MP, 3-MP, 4-MP, 9-MP) were made by combining reported standard-state properties of the crystalline compounds, solubilities and enthalpies of phenanthrene and 1-MP, and relative Gibbs energies, enthalpies and entropies of aqueous MP isomers from published quantum chemical simulations. The calculated properties are consistent with greater stabilities of the β isomers (2-MP and 3-MP) relative to the α isomers (1-MP and 9-MP) at 25 °C. However, the metastable equilibrium values of the abundance ratios 2-MP/1-MP (MPR) and (2-MP + 3-MP)/(1-MP + 9-MP) (MPI-3) decrease with temperature, becoming <1 at ~375–455 °C. The thermodynamic model is consistent with observations of reversals of these organic maturity parameters at high temperature in hydrothermal and metamorphic settings. Application of the model to data reported for the Paleoproterozoic Here’s Your Chance (HYC) Pb–Zn–Ag ore deposit (McArthur River, Northern Territory, Australia) indicates a likely effect of high-temperature equilibration on reported values of MPR and MPI-3, but this finding is contingent on the location within the deposit. If metastable equilibrium holds, a third aromatic maturity ratio, 1.5 × (2-MP + 3-MP)/(P + 1-MP + 9-MP) (MPI-1), can be used as a proxy for oxidation potential. Values of log aH2(aq) determined from data reported for HYC and for a sequence of deeply buried source rocks are indicative of more reducing conditions at a given temperature than those inferred from data reported for two sets of samples exposed to contact or regional metamorphism. These results are limiting-case scenarios for the modeled systems that do not account for effects of non-ideal mixing or kinetics, or external sources or transport of the organic matter.Nevertheless, quantifying the temperature dependence of equilibrium constants of organic reactions enables the utilization of organic maturity parameters as relative geothermometers at temperatures higher than the nominal limits of the oil window

Prostate Cancer Cell Lines under Hypoxia Exhibit Greater Stem-Like Properties

Hypoxia is an important environmental change in many cancers. Hypoxic niches can be occupied by cancer stem/progenitor-like cells that are associated with tumor progression and resistance to radiotherapy and chemotherapy. However, it has not yet been fully elucidated how hypoxia influences the stem-like properties of prostate cancer cells. In this report, we investigated the effects of hypoxia on human prostate cancer cell lines, PC-3 and DU145. In comparison to normoxia (20% O2), 7% O2 induced higher expressions of HIF-1α and HIF-2α, which were associated with upregulation of Oct3/4 and Nanog; 1% O2 induced even greater levels of these factors. The upregulated NANOG mRNA expression in hypoxia was confirmed to be predominantly retrogene NANOGP8. Similar growth rates were observed for cells cultivated under hypoxic and normoxic conditions for 48 hours; however, the colony formation assay revealed that 48 hours of hypoxic pretreatment resulted in the formation of more colonies. Treatment with 1% O2 also extended the G0/G1 stage, resulting in more side population cells, and induced CD44 and ABCG2 expressions. Hypoxia also increased the number of cells positive for ABCG2 expression, which were predominantly found to be CD44bright cells. Correspondingly, the sorted CD44bright cells expressed higher levels of ABCG2, Oct3/4, and Nanog than CD44dim cells, and hypoxic pretreatment significantly increased the expressions of these factors. CD44bright cells under normoxia formed significantly more colonies and spheres compared with the CD44dim cells, and hypoxic pretreatment even increased this effect. Our data indicate that prostate cancer cells under hypoxia possess greater stem-like properties