Variables to be controlled in the assessment of blood innate immune responses to Toll-like receptor stimulation.

Variability in TLR function influences susceptibility to infectious as well as immune-mediated diseases. Given the outbred nature of humans, identifying functional Toll-like receptor variability and its role in clinical disease requires such analysis to be conducted in large, often multi-centered cohorts. Yet the technically complex measurements involved in innate immune analysis benefit from centralized processing of samples.

Ontogeny of Toll-like receptor mediated cytokine responses of human blood mononuclear cells.

Newborns and young infants suffer increased infectious morbidity and mortality as compared to older children and adults. Morbidity and mortality due to infection are highest during the first weeks of life, decreasing over several years. Furthermore, most vaccines are not administered around birth, but over the first few years of life.

A single immunization near birth elicits immediate and lifelong protective immunity.

Most existing vaccines do not induce protective immunity immediately following birth, nor do they retain protective efficacy in the latter years of life without booster doses. Using a mouse model, we present evidence that a live-replicating vaccine administered only once shortly after birth was able to induce both immediate and lifelong protection. Newborn mice immunized with a safe, highly attenuated strain of Listeria monocytogenes (Lm) were already protected by day 7 post-vaccination when challenged with a virulent strain of Lm.

Correlation analysis of intracellular and secreted cytokines via the generalized integrated mean fluorescence intensity.

The immune response in humans is usually assessed using immunogenicity assays to provide biomarkers as correlates of protection (CoP). Flow cytometry is the assay of choice to measure intracellular cytokine staining (ICS) of cell-mediated immune (CMI) biomarkers. For CMI analysis, the integrated mean fluorescence intensity (iMFI) was introduced as a metric to represent the total functional CMI response as a CoP.

Identification of B cells through negative gating-An example of the MIFlowCyt standard applied.

Polychromatic flow cytometric analysis takes advantage of the increasing number of available fluorophores to positively identify and simultaneously assess multiple parameters in the same cell (1). Additional parameters may be analyzed through negative identification (i.e.

Neonatal innate TLR-mediated responses are distinct from those of adults.

The human neonate and infant are unduly susceptible to infection with a wide variety of microbes. This susceptibility is thought to reflect differences from adults in innate and adaptive immunity, but the nature of these differences is incompletely characterized. The innate immune response directs the subsequent adaptive immune response after integrating information from TLRs and other environmental sensors.

Systems biology evaluation of immune responses induced by human host defence peptide LL-37 in mononuclear cells.

The immune system is very complex, it involves the integrated regulation and expression of hundreds of proteins. To understand in greater detail how the human host defence immunomodulatory peptide LL-37 interacts with innate immunity, a systems approach was pursued. Polychromatic flow cytometry was employed to demonstrate that within human peripheral blood mononuclear cells, CD14+ monocytes, myeloid and plasmocytoid dendritic cells and T- and B-lymphocytes, all responded to LL-37, with the differential production of intracellular cytokines.

Polychromatic flow cytometric high-throughput assay to analyze the innate immune response to Toll-like receptor stimulation.

Polychromatic flow cytometry allows the capture of multidimensional data, providing the technical tool to assess complex immune responses. Interrogation of the adaptive T cell response to infection or vaccination already has benefited greatly from standardized protocols for polychromatic flow cytometric analysis. The innate immune system plays an important role in health and disease, and presents potentially important therapeutic and diagnostic modalities.

Induction of protective immunity to Listeria monocytogenes in neonates.

Neonates suffer unduly from infections and also respond suboptimally to most commonly used vaccines. However, a CD8 T cell response can be elicited in neonates if the Ag is introduced into the cytoplasm of APCs. Listeria monocytogenes (Lm) targets the cytoplasm of APC and is a strong CD8 and CD4 Th1-promoting vaccine vehicle in adult mice.