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The human pentraxin proteins, serum amyloid P component (SAP) and C‐reactive protein (CRP) are important in routine clinical diagnosis, SAP for systemic amyloidosis and CRP for monitoring the non‐specific acute phase response. They are also targets for novel therapies currently in development but their roles in health and disease are controversial. Thus, both for clinical use and to rigorously elucidate their functions, structurally and functionally intact, pharmaceutical grade preparations of the natural, authentic proteins are required. We report here the production from normal human donor plasma and the characterization of the first such preparations. Importantly, we demonstrate that, contrary to reports using recombinant proteins and less well characterized preparations, neither CRP nor SAP stimulate the release by human peripheral blood mononuclear cells in vitro of any TNFα, IL‐6 or IL‐8, nor does SAP cause release of IL‐1β or IL‐10. Furthermore neither of our preparations was pro‐inflammatory in mice in vivo.

Antigen-specific proliferation is a critical function of memory T cells that is often utilised to measure vaccine immunogenicity and T cell function. We proposed that measurement of intracellular expression of the nuclear protein, Ki67, could reliably assess specific T cell proliferation in vitro. Ki67 was expressed in CD4+ and CD8+ T cells that had undergone in vitro proliferation after 6-day culture of human whole blood or PBMC with antigens. T cells cultured with no antigen did not express Ki67. When compared to current flow cytometry based proliferation assays, Ki67 detected proliferating cells with greater sensitivity than BrdU incorporation, whereas its sensitivity was similar to dye dilution of Oregon Green (OG), a CFSE derivative. Overall, the magnitude and cytokine expression profile of proliferating T cells detected by Ki67 expression correlated strongly with T cells detected with BrdU or OG. The intra-assay variability of Ki67 proliferation was 2–3% for CD4+ T cells, and 10–16% for CD8+ T cells. Finally, we demonstrate that the Ki67 assay detects tetanus toxoid-specific CD4+ T cell proliferation after infant vaccination with tetanus toxoid (TT). Overall our data suggest that intracellular Ki67 expression provides a specific, quantitative and reproducible measure of antigen-specific T cell proliferation in vitro.

Cervical cytobrushing, biopsy, or lavages have previously been used to collect mononuclear cells from the female genital tract. Compared with blood, obtaining cells from the female genital tract is more invasive and generally yields few cells for subsequent immune studies. Because of the value of including mucosal sampling in HIV vaccine trials, standardisation of methods for collection, processing, and analysis of immunity from cells derived from the female genital tract is important. The aim of this study was to assess the effect of transport conditions on the viability, recovery and antigenic responsiveness of cervical T cells. This was investigated in cervical cytobrush specimens collected from 215 chronically HIV-infected women. Cytobrushes were either processed immediately, after cryopreservation, or after 24h at 37°C, 4°C or room temperature. CD3+ T cell numbers were quantified using Guava automated cell counting. Viability was assessed using Trypan and Annexin/PI staining. Intracellular cytokine staining was used to evaluate IFN-γ responses to PMA, PHA and CEF peptides in cytobrush-derived T cells ex vivo and after delayed processing. In vitro polyclonal expansion of thawed cervical lymphocytes was conducted for 14days in the presence of anti-CD3 and IL-2. We found that CD3+ T cell recovery and viability was similar in cytobrushes processed immediately or after 24h irrespective of the conditions at which they were maintained. Fifty percent of the CD3+ T cells could be recovered after cryopreservation of cytobrushes and these could be polyclonally expanded in half of the cryopreserved samples. IFN-γ production following mitogenic stimulation was similar in ex vivo and delayed processing cytobrushes. Maintaining cytobrushes at 37°C prior to processing significantly improved the detection of CEF-specific T cell responses compared to ex vivo. We conclude that cervical cytobrush-derived T cells are robust and can preserve their viability, phenotype and function over 24h of mock transport.