Platelet aggregation in whole blood is not impaired by a platelet-sparing leukoreduction filter and instead depends upon the presence of leukocytes.

BACKGROUND: Recent studies characterizing in vitro hemostatic properties of whole blood (WB) leukoreduced (LR) with a platelet-sparing filter have described subtle, if any, changes to viscoelastic clotting; however, reductions in platelet (PLT) content and impedance aggregometry (IA) responses have been noted. The effects of filtration of WB (i.e., filter-contact effects, reduction in platelet and leukocyte count) have not been rigorously investigated as to their individual impacts on platelet IA responses. STUDY DESIGN AND METHODS: WB units from healthy donors were collected and characterized to assess the effects of platelet-sparing leukoreduction (LR) upon the in vitro hemostatic measures of platelet IA and thromboelastometry. Further characterization of platelet IA responses was carried out in WB samples to delineate the effects of platelet count and leukocyte presence/absence upon the response. RESULTS: WB filtration reduced the platelet count and IA responses but had no impact on viscoelastic clotting measures in fresh WB. Experiments revealed that IA responses have a linear correlation with platelet count in both apheresis platelets and WB and that passage of platelets through the WB-LR filter has no impact upon the strength of this response. Further experiments in LR WB showed that addition of autologous leukocytes back to the platelets fully restored the platelet aggregation response to pre-filtration levels. CONCLUSION: WB filtration results in platelet count reduction and leukocyte removal; however, platelet IA is not degraded by passage through the filter. Apparent declines in platelet IA responses can be fully attributed to the reduction in platelet count and the removal of leukocytes.

Continuous CD34+ cell collection by a new device is safe and more efficient than by a standard collection procedure: results of a two-center, crossover, randomized trial.

BACKGROUND: Peripheral blood stem cells (PBSCs) collected from granulocyte-colony-stimulating factor (G-CSF)-mobilized donors are routinely used for hematopoietic stem cell transplantation. Most PBSC collections worldwide have used the COBE Spectra (COBE) platform that is being replaced by the Spectra Optia (OPTIA). This study compared the PBSC collection performance and safety of the OPTIA using a single-step, continuous mononuclear cell (CMNC) collection procedure and the standard COBE MNC procedure. STUDY DESIGN AND METHODS: A prospective, noninferiority, randomized, open-label, crossover, multicenter study was conducted in G-CSF-mobilized donors randomized to undergo MNC collection on Days 5 and 6. The primary endpoint was CD34+ cell collection efficiency (CE1%) with a noninferiority margin of 10%. The secondary endpoint was CD34+ cell CE2%. Product purity and safety were also assessed. RESULTS: Twenty-three healthy donors (87% male) participated in the study. Mean (±SD) CD34+ CE1% was 84.4% (±16.4%) and 66.2% (±15.3%) for the OPTIA and COBE, respectively (p < 0.001 for noninferiority and superiority). Mean (±SD) CD34+ CE2% was 62.4 (±11.6) and 48.4 (±11.2) for the OPTIA and COBE, respectively (p < 0.001 for superiority). Granulocyte and platelet (PLT) contamination were lower in OPTIA-collected products. There were no unexpected adverse events (AEs) and no significant differences in the incidence of AEs between study arms. PLT loss was less with the OPTIA than with the COBE. CONCLUSION: The OPTIA CMNC collection procedure is safe and effective for the collection of CD34+ cells in G-CSF-mobilized donors and was superior to the COBE for CE1% and CE2%, collecting approximately 19 and 16% higher, respectively.

Yeast surface display of a noncovalent MHC class II heterodimer complexed with antigenic peptide.

Microbial protein display technologies have enabled directed molecular evolution of binding and stability properties in numerous protein systems. In particular, dramatic improvements to antibody binding affinity and kinetics have been accomplished using these tools in recent years. Examples of successful application of display technologies to other immunological proteins have been limited to date. Herein, we describe the expression of human class II major histocompatibility complex allele (MHCII) HLA-DR4 on the surface of Saccharomyces cerevisiae as a noncovalently associated heterodimer. The yeast-displayed MHCII is fully native as assessed by binding of conformationally specific monoclonal antibodies; failure of antibodies specific for empty HLA-DR4 to bind yeast-displayed protein indicates antigenic peptide is bound. This report represents the first example of a noncovalent protein dimer displayed on yeast and of successful display of wild-type MHCII. Results further point to the potential for using yeast surface display for engineering and analyzing the antigen binding properties of MHCII.

Beryllium presentation to CD4+ T cells is dependent on a single amino acid residue of the MHC class II beta-chain.

Chronic beryllium disease (CBD) is characterized by a CD4+ T cell alveolitis and granulomatous inflammation in the lung. Genetic susceptibility to this disease has been linked with HLA-DP alleles, particularly those possessing a glutamic acid at position 69 (Glu69) of the beta-chain. However, 15% of CBD patients do not possess a Glu69-containing HLA-DP allele, suggesting that other MHC class II alleles may be involved in disease susceptibility. In CBD patients without a Glu69-containing HLA-DP allele, an increased frequency of HLA-DR13 alleles has been described, and these alleles possess a glutamic acid at position 71 of the beta-chain (which corresponds to position 69 of HLA-DP). Thus, we hypothesized that beryllium presentation to CD4+ T cells was dependent on a glutamic acid residue at the identical position of both HLA-DP and -DR. The results show that HLA-DP Glu69- and HLA-DR Glu71-expressing molecules are capable of inducing beryllium-specific proliferation and IFN-gamma expression by lung CD4+ T cells. Using fibroblasts expressing mutated HLA-DP2 and -DR13 molecules, beryllium recognition was dependent on the glutamic acid at position 69 of HLA-DP and 71 of HLA-DR, suggesting a critical role for this amino acid in beryllium presentation to Ag-specific CD4+ T cells. Thus, these results demonstrate that a single amino acid residue of the MHC class II beta-chain dictates beryllium presentation and potentially, disease susceptibility.

Use of soluble MHC class II/peptide multimers to detect antigen-specific T cells in human disease.

Most techniques that identify antigen-specific T cells are dependent on the response of these cells to the relevant antigen in culture. Soluble multimers of MHC molecules, when occupied with the same peptide, will bind selectively to T cells specific for that MHC/peptide complex. Techniques to produce fluorescent MHC class II/peptide multimers have recently been developed. These reagents provide a method to facilitate detection and isolation of antigen-specific CD4+ T cells and they represent a new research tool to study these cells in patients with immune-mediated diseases.

Excess apoptosis of mononuclear cells contributes to the depressed cytomegalovirus-specific immunity in HIV-infected patients on HAART.

HIV-infected patients on highly active antiretroviral therapy (HAART) have persistently decreased cytomegalovirus (CMV)-specific proliferative responses [lymphocyte proliferation assay (LPA)] in spite of increases in CD4+ T cell counts. Here we demonstrate an association between apoptosis of unstimulated peripheral blood mononuclear cells (uPBMC) and decreased CMV-LPA. HAART recipients had more apoptosis of uPBMC than controls when measured by caspases 3, 8, and 9 activities and by annexin V binding. Patients with undetectable HIV replication maintained significantly higher apoptosis of CD4+ and CD14+ cells compared to controls. CMV-LPA decreased with higher apoptosis of uPBMC in patients only. This association was independent of CD4+ cell counts or HIV replication. Furthermore, rescuing PBMC from apoptosis with crmA, but not with TRAIL- or Fas-pathway blocking agents or with other caspase inhibitors, increased CMV-LPA in HAART recipients. This effect was not observed in uninfected controls, further indicating that the down regulatory effect of apoptosis on cell-mediated immunity (CMI) was specifically associated with the HIV-infected status.