The neotropical, non-human primates (NHP) of the genus Saimiri and Aotus are recommended by the World Health Organization as experimental models for the study of human malaria because these animals can be infected with the same Plasmodium that cause malaria in humans. However, one limitation is the lack of immunological tools to assess the immune response in these models. The present study focuses on the development and comparative use of molecular and immunological methods to evaluate the cellular immune response in Saimiri sciureus.
Blood samples were obtained from nineteen uninfected Saimiri. Peripheral blood mononuclear cells (PBMC) from these animals and splenocytes from one splenectomized animal were cultured for 6, 12, 18, 24, 48, 72 and 96 hrs in the presence of phorbol-12-myristate-13-acetate and ionomycin. The cytokine levels in the supernatant were detected using human and NHP cytometric bead array Th1/Th2 cytokine kits, the Bio-Plex Pro Human Cytokine Th1/Th2 Assay, enzyme-linked immunosorbent assay, enzyme-linked immunospot assays and intracellular cytokine secretion assays. Cytokine gene expression was examined through TaqMan® Gene Expression Real-Time PCR using predesigned human gene-specific primers and probes or primers and probes designed based on published S. sciureus cytokine sequences.
The use of five assays based on monoclonal antibodies specific for human cytokines facilitated the detection of IL-2, IL-4 and/or IFN-γ. TaqMan array plates facilitated the detection of 12 of the 28 cytokines assayed. However, only seven cytokines (IL-1A, IL-2, IL-10, IL-12B, IL-17, IFN-β, and TNF) presented relative expression levels of at least 70% of the gene expression observed in human PBMC. The use of primers and probes specific for S. sciureus cytokines facilitated the detection of transcripts that showed relative expression below the threshold of 70%. The most efficient evaluation of cytokine gene expression, in PBMC and splenocytes, was observed after 6-12 hrs of culture, except for LTA in PBMC, whose expression was best analysed after 24 hrs of culture.
Real-time PCR facilitates the analysis of a large number of cytokines altered during malaria infection, and this technique is considered the best tool for the evaluation of the cellular immune response in S. sciureus.

In the direct pathway, T cells recognize intact donor major histocompatability complexes and allogeneic peptide on the surface of donor antigen presenting cells (APCs). Indirect allorecognition results from the recognition of processed alloantigen by self MHC complexes on self APCs. In this study, we wished to evaluate the relative contribution of different intragraft cells to the alloactivation of nave and memory T cells though the direct and the indirect pathway of allorecognition.
The processing of membrane fragments from IFN-treated single donor endothelial cells (EC), fibroblasts or renal epithelial cells (RPTEC) was evaluated by DiOC labeling of each cell type and flow cytometry following interaction with PBMC. Direct pathway activation of nave CD45RA+ or memory CD45RO+ CD4+ T cells was evaluated following coculture with IFN-treated and MHC class II-expressing EC, fibroblasts or RPTEC. Indirect pathway activation was assessed using CD45RA+ or CD45RO+ CD4+ T cells cocultured with autologous irradiated APCs in the absence or presence of sonicates derived from IFN-treated allogeneic EC, fibroblasts or RPTEC. Activation of T cells was assessed by [3H]thymidine incorporation and by ELISpot assays.
We find that CD14+ APCs readily acquire membrane fragments from fibroblasts and RPTEC, but fail to acquire membrane fragments from intact EC. However, APCs process membranes from EC undergoing apoptosis.There was a notable direct pathway alloproliferative response of CD45RO+ CD4+ T cells to IFN-treated EC, but not to fibroblasts or RPTEC. Also, there was a minimal direct pathway response of CD45RA+ CD4+ T cells to all cell types. In contrast, we found that both CD45RA+ and CD45RO+ CD4+ T cells proliferated following coculture with autologous APCs in the presence of sonicates derived from IFN-treated EC, fibroblasts or RPTEC. By ELISpot, we found that these T cells stimulated via the indirect pathway also produced the cytokines IFN, IL-2, IL-4 and IL-5.
Recipient APCs may readily process membrane fragments from allogeneic intragraft cells, but not from EC unless they are undergoing apoptosis. This processing is sufficient for indirect pathway alloactivation of both CD45RA+ and CD45RO+ CD4+ T cells. Only graft vascular EC mediate direct pathway reactivation of CD4+ T cells.

NKT cells contribute to the modulation of immune responses and are believed to be important in the pathogenesis of autoimmune and infectious diseases, as well as cancer. Variations in the composite NKT cytokine response may determine individual disease susceptibility or severity. Due to low frequencies in peripheral blood, knowledge of the breadth of ex vivo human NKT cell functions has been limited. To bridge this gap, we studied highly purified NKT cells from PBMC of healthy donors and assessed the production of 27 effector functions using sensitive Elispot and multiplex bead assays. We found the ex vivo human NKT cell response is predominantly comprised of the chemokines MIP1-α, and MIP1-β as well as the Th1 cytokines IFN-γ and TNF-α. Although lower in magnitude, there was also significant production of IL-2, IL-4, and perforin after mitogen stimulation. Surprisingly, little/no IL-5, IL-6, IL-10, or IL-13 was detected, and no subjects' NKT cells produced IL-17. Comparison of the NKT functional profiles between age-matched male and female subjects revealed similar IL-4 responses, but higher frequencies of cells producing IFN-γ and MIP1-α, from males. There were no gender differences in the circulating NKT subset distribution. These findings implicate chemokines as a major mechanism by which NKT cells control responses in humans. In addition, the panoply of Th2 and Th17 cytokine secretion by NKT cells from healthy donors may not be as pronounced as previously believed. NKT cells may therefore contribute to the gender bias found in many diseases.

Current treatment strategies for asthma in teenagers derive primarily from information on chronic disease in adults. More detailed understanding of risk factors related to teenage asthma might aid in the development of improved preventive and treatment strategies for this age group.
We sought to identify biomarkers associated with asthma phenotypes in teenagers, particularly atopic asthma, and to identify markers that aid in discriminating between atopic subjects at high versus low risk of asthma.
We studied 1380 unselected 14-year-olds and collected data on clinical history, allergic sensitization, and respiratory and immunoinflammatory function. The latter comprised measurements of circulating inflammatory markers and in vitro innate and adaptive immune functions, including house dust mite T-cell responses. We integrated the data into regression models to identify variables most strongly associated with asthma risk and severity among atopic subjects.
Eight hundred twenty-seven subjects were atopic, 140 subjects were asthmatic, and 81% of asthmatic subjects were also atopic. We identified asthma risk variables related to atopy intensity, including specific IgE and eosinophil levels, plus an additional series external to the T(H)2 cascade but that modified risk only in atopic subjects, including IFN-gamma, IL-10, and IL-12 responses and neutrophil numbers in blood. Moreover, bronchial hyperresponsiveness was associated strongly with atopic but not nonatopic asthma, and the bronchial hyperresponsiveness risk profile was itself dominated by atopy-associated variables.
Asthma in teenagers is predominantly driven by atopy acting in concert with a second tier of T(H)2-independent immunoinflammatory mechanisms, which contribute to pathogenesis only against the background of pre-existing inhalant allergy.

A decrease in the frequency and activation state of dendritic cells in the sentinel lymph node (SLN) has been observed in early stages of melanoma development. This may hinder the generation of effective antitumor T-cell responses and increase the likelihood of metastatic spread. Immunopotentiation of the melanoma SLN may therefore be a valuable adjuvant treatment option. One way to achieve this is through the use of bacterially derived unmethylated cytosine-phosphate-guanine (CpG) DNA sequences that bind Toll-like receptor 9 and activate plasmacytoid dendritic cells (PDC). CpG-activated PDC, in turn, release IFN alpha and may thus boost T-cell and natural killer cell responses as well as activate conventional myeloid dendritic cells (MDC).
We studied the effects of preoperative local administration of the CpG B-type oligodeoxynucleotide (ODN) PF-3512676 (formerly known as CPG 7909) on dendritic cell and T-cell subsets in the SLN of 23 stage I to III melanoma patients, randomized to receive intradermal injections of either PF-3512676 or saline (NaCl 0.9%).
PF-3512676 administration resulted in bulkier SLN, higher yields of isolated SLN leukocytes, and activation of BDCA-2(+)CD123(+) PDC as well as of CD1a(+) MDC. In addition, PF-3512676 administration was associated with the presence of a newly identified CD11c(hi)CD123(+)CD83(+)TRAIL(+) mature SLN-MDC subset, an increased release of a variety of inflammatory cytokines, and lower frequencies of CD4(+)CD25(hi)CTLA-4(+)FoxP3(+) regulatory T cells in the SLN.
These findings point to the possible utility of the conditioning of SLN by PF-3512676 as an adjuvant immunotherapeutic modality for early-stage melanoma.