Functional immunologic assays using cryopreserved peripheral blood mononuclear cells (PBMC) are influenced by blood processing, storage and shipment. The objective of this study was to compare the viability, recovery and ELISPOT results of PBMC stored and shipped in liquid nitrogen (LN/LN) or stored in LN and shipped on dry ice (LN/DI) or stored at −70°C for 3 to 12 weeks and shipped on DI (70/DI 3 to 12); and to assess the effect of donor HIV infection status on the interaction between storage/shipment and the outcome measures. PBMC from 12 HIV-infected and 12 uninfected donors showed that LN/LN conferred higher viability and recovery than LN/DI or 70/DI 3, 6, 9 or 12. LN/DI PBMC had higher viability than any 70/DI PBMC. The PBMC viability and recovery linearly decreased with the duration of storage at −70°C from 3 to 12 weeks. This effect was more pronounced in samples from HIV-infected than uninfected donors. Results of ELISPOT assays using CMV pp65, CEF and Candida albicans antigens were qualitatively and quantitatively similar across LN/LN, LN/DI and 70/DI 3. However, ELISPOT values significantly decreased with the duration of storage at −70°C both in HIV-infected and uninfected donors. ELISPOT results also decreased with PBMC viability <70%.

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Virus specific CD8+ T cells expand dramatically during acute Epstein Barr virus (EBV) infection, and their persistence is important for lifelong control of EBV-related disease. To better define the generation and maintenance of these effective CD8+ T cell responses, we used microarrays to characterize gene expression in total and EBV-specific CD8+ T cells isolated from the peripheral blood of ten individuals followed from acute infectious mononucleosis (AIM) into convalescence (CONV). In total CD8+ T cells, differential expression of genes in AIM and CONV was most pronounced among those encoding proteins important in T cell activation/differentiation, cell division/metabolism, chemokines/cytokines and receptors, signaling and transcription factors (TF), immune effector functions, and negative regulators. Within these categories, we identified 28 genes that correlated with CD8+ T cell expansion in response to an acute EBV infection. In EBV-specific CD8+ T cells, we identified 33 genes that were differentially expressed in AIM and CONV. Two important TF, T-bet and Eomesodermin (Eomes), were upregulated and maintained at similar levels in both AIM and CONV; by contrast, protein expression declined from AIM to CONV. Expression of these TF varied among cells with different epitope specificities. Altogether, gene and protein expression patterns suggest that a large proportion, if not a majority of CD8+ T cells in AIM are virus-specific, activated, dividing, and primed to exert effector activities. High expression of T-bet and Eomes may help to maintain effector mechanisms in activated cells, and to enable proliferation and transition to earlier differentiation states in CONV.

Solution grade and extruded grade Biomer (SB and EB, respectively) are polyurethanes that have been suggested for use in biomedical applications. The bulk materials were examined by elemental analysis, differential scanning calorimetry, thermomechanical testing, and stress-strain testing. The extruded grade material has a lower soft segment molecular weight (650 g/mol) than the solution grade material (2000 g/mol). As a result of its higher molecular weight, the soft segment phase of SB is semicrystalline in the solid state. The hard segments of the extruded grade material are chain extended with water yielding a lower urea concentration than in the solution grade material in which the hard segments are chain extended with diamines. Chemical structures for the two materials consistent with elemental analysis, urea/urethane ratios and thermal and mechanical data, are proposed. X-ray photoelectron spectroscopy (ESCA) was used to analyze the surfaces of extruded grade Biomer, solution grade Biomer cast on the inner surface of polyethylene tubing, and extruded grade Biomer dissolved in DMA and similarly cast on polyethylene (CB). Soft segment concentrations were highest on the EB surface and lowest on the SB surface. Soft segment concentrations on the EB surface were higher than on the CB surface, indicating that the method of fabrication affected the composition of the surface layer. The three materials were tested for blood tolerance in a canine femoral arteriovenous shunt configuration. Blood compatibility was correlated with increasing concentration of polyether soft segments on the surface.