Assessing the immunopotency of Toll-like receptor agonists in an in vitro tissue-engineered immunological model.

SUMMARY: The in vitro Peripheral Tissue Equivalent (PTE) module is a three-dimensional tissue-engineered endothelial cell/collagen matrix culture system, which has been reported to reproduce in vivo physiological conditions and which generates dendritic cells (DC) autonomously. In the present study, we used the PTE module to investigate the immunopotency of Toll-like receptor (TLR) agonists, including polyinosine-polycytidylic acid, Gardiquimod, CpG 2006 and lipopolysaccharide. Application of TLR agonists in the PTE module induced a wide range of cytokines, including interleukins 1alpha/beta, 6, 8 and 10 and tumour necrosis factor-alpha. Compared with traditional peripheral blood mononuclear cell (PBMC) cultures, the PTE module produced twofold to 100-fold higher levels of cytokine secretion, indicating that it can be a highly sensitive assay system. This increased sensitivity is the result of the natural synergy between the leucocytes and the endothelium. Furthermore, the application of TLR agonists, such as lipopolysaccharide and Gardiquimod, to the PTE module enhanced DC differentiation and promoted DC maturation, as indicated by up-regulated expression of CD83, CD86 and CCR7(CD197). In addition, functional assays indicated PTE-derived DC treated with Gardiquimod, a TLR-7 agonist, significantly augmented anti-tetanus toxoid antibody production. Interestingly, replacing PBMC with purified myeloid cells (CD33(+)) significantly reduced the responsiveness of the PTE module to TLR stimulation. The reduced sensitivity was partly the result of the removal of plasmacytoid DC that participated in the response to TLR stimulation and sensitization of the PTE module. Overall, the in vitro PTE module clearly demonstrated the effects of TLR agonists on DC generation, maturation and antigen-presenting capacity, and may serve as a sensitive and predictive test bed for the evaluation of adjuvant candidates.

An immunologic model for rapid vaccine assessment -- a clinical trial in a test tube.

While the duration and size of human clinical trials may be difficult to reduce, there are several parameters in pre-clinical vaccine development that may be possible to further optimise. By increasing the accuracy of the models used for pre-clinical vaccine testing, it should be possible to increase the probability that any particular vaccine candidate will be successful in human trials. In addition, an improved model will allow the collection of increasingly more-informative data in pre-clinical tests, thus aiding the rational design and formulation of candidates entered into clinical evaluation. An acceleration and increase in sophistication of pre-clinical vaccine development will thus require the advent of more physiologically-accurate models of the human immune system, coupled with substantial advances in the mechanistic understanding of vaccine efficacy, achieved by using this model. We believe the best viable option available is to use human cells and/or tissues in a functional in vitro model of human physiology. Not only will this more accurately model human diseases, it will also eliminate any ethical, moral and scientific issues involved with use of live humans and animals. An in vitro model, termed "MIMIC" (Modular IMmune In vitro Construct), was designed and developed to reflect the human immune system in a well-based format. The MIMIC System is a laboratory-based methodology that replicates the human immune system response. It is highly automated, and can be used to simulate a clinical trial for a diverse population, without putting human subjects at risk. The MIMIC System uses the circulating immune cells of individual donors to recapitulate each individual human immune response by maintaining the autonomy of the donor. Thus, an in vitro test system has been created that is functionally equivalent to the donor's own immune system and is designed to respond in a similar manner to the in vivo response.

CCR8 expression identifies CD4 memory T cells enriched for FOXP3+ regulatory and Th2 effector lymphocytes.

CD4+ Th2 cells are important regulators of allergic inflammation. CCR8 is thought to play a role in Th2-mediated responses, however, expression of CCR8 in peripheral blood has not been fully characterized. Using a fluorescent form of the ligand selective for CCR8 (F-CCL1), we identified the leukocytes expressing CCR8 in human, monkey, and mouse peripheral blood. CCR8 expression is primarily restricted to a subset of human CD4 memory T lymphocytes (15%). Approximately 40% of CCR8+CD4+ T cells express Th2 cytokines IL-4 or IL-13 while 13% express the Th1 cytokine IFN-gamma. In fact, 50% of all Th2, but only 5% of Th1, cells express CCR8. Upon anti-CD3/anti-CD28 mAb-mediated activation, CCR8+CD4+ T cells secrete 3- to 7-fold higher levels of IL-4, IL-5, IL-9, and IL-13 and 10- to 20-fold lower levels of IFN-gamma or IL-17, compared with CCR8-CD4+ memory T cells. Two-thirds of CCR8+CD4 T cells express cutaneous lymphocyte-associated Ag while the majority lack gut-homing receptors. CCR8+CD4+ cells express CCR7 and CD62L and are present in spleen and lymph nodes of mice. Approximately 25% of CCR8+CD4 T cells express CD25high while 20% of CCR8+CD4+ express the T regulatory cell transcription factor FOXP3 accounting for 60% of all FOXP3-expressing CD4+ T cells. In conclusion, CCR8 marks a diverse subset of CD4 memory T cells enriched for T regulatory and Th2 cells which have the potential for recruitment into sites of allergic inflammation where they could participate in the induction and regulation of the allergic response.

Platelet expression profiling and clinical validation of myeloid-related protein-14 as a novel determinant of cardiovascular events.

BACKGROUND: Platelets participate in events that immediately precede acute myocardial infarction. Because platelets lack nuclear DNA but retain megakaryocyte-derived mRNAs, the platelet transcriptome provides a novel window on gene expression preceding acute coronary events. METHODS AND RESULTS: We profiled platelet mRNA from patients with acute ST-segment-elevation myocardial infarction (STEMI, n=16) or stable coronary artery disease (n=44). The platelet transcriptomes were analyzed and single-gene models constructed to identify candidate genes with differential expression. We validated 1 candidate gene product by performing a prospective, nested case-control study (n=255 case-control pairs) among apparently healthy women to assess the risk of future cardiovascular events (nonfatal myocardial infarction, nonfatal stroke, and cardiovascular death) associated with baseline plasma levels of the candidate protein. Platelets isolated from STEMI and coronary artery disease patients contained 54 differentially expressed transcripts. The strongest discriminators of STEMI in the microarrays were CD69 (odds ratio 6.2, P<0.001) and myeloid-related protein-14 (MRP-14; odds ratio 3.3, P=0.002). Plasma levels of MRP-8/14 heterodimer were higher in STEMI patients (17.0 versus 8.0 microg/mL, P<0.001). In the validation study, the risk of a first cardiovascular event increased with each increasing quartile of MRP-8/14 (Ptrend<0.001) such that women with the highest levels had a 3.8-fold increase in risk of any vascular event (P<0.001). Risks were independent of standard risk factors and C-reactive protein. CONCLUSIONS: The platelet transcriptome reveals quantitative differences between acute and stable coronary artery disease. MRP-14 expression increases before STEMI, and increasing plasma concentrations of MRP-8/14 among healthy individuals predict the risk of future cardiovascular events.