The novel Group A Streptococcus antigen SpnA combined with bead-based immunoassay technology improves streptococcal serology for the diagnosis of acute rheumatic fever.

OBJECTIVES: Streptococcal serology provides evidence of prior Group A Streptococcus (GAS) exposure, crucial to the diagnosis of acute rheumatic fever (ARF) and post-streptococcal glomerulonephritis. However, current tests, which measure anti-streptolysin-O and anti-DNaseB antibodies, are limited by false positives in GAS endemic settings, and incompatible methodology requiring the two tests to be run in parallel. The objective was to improve streptococcal serology by combining the novel GAS antigen, SpnA, with streptolysin-O and DNaseB in a contemporary, bead-based immunoassay. METHODS: Recombinant streptolysin-O, DNAseB and SpnA were conjugated to polystyrene beads with unique fluorescence positions so antibody binding to all three antigens could be detected simultaneously by cytometric bead array. Multiplex assays were run on sera collected in three groups: ARF; ethnically matched healthy children; and healthy adults. RESULTS: The ability of the antigens to detect a previous GAS exposure in ARF was assessed using the 80th centile of the healthy children group as cut-off (upper limit of normal). SpnA had the highest sensitivity at 88%, compared with 75% for streptolysin-O and 56% for DNaseB. CONCLUSIONS: SpnA has favorable immunokinetics for streptococcal serology, and can be combined with anti-streptolysin-O and anti-DNaseB in a multiplex format to improve efficiency and accuracy.

MicroRNA regulation in human CD8+ T cell subsets--cytokine exposure alone drives miR-146a expression.

BACKGROUND: microRNAs (miRNAs) are emerging as key regulators of the immune system, but their role in CD8+ T cell differentiation is not well explored. Some evidence suggests that signals from cell surface receptors influence the expression of miRNAs in CD8+ T cells, and may have consequent effects on cell phenotype and function. We set out to investigate whether common gamma chain cytokines modulated human CD8+ T cell expression of miR-146a, which previous studies have associated with different stages of CD8+ differentiation. We also investigated how changes in miR-146a related to other miRNAs that alter with CD8+ differentiation status. METHODS: We treated human CD8+ T cells with the cytokines IL-2, IL-7 or IL-15 either at rest or after stimulation with anti-CD3 and anti-CD28. For some experiments we also purified human CD8+ T cell subsets ex vivo. Flow cytometry was used in parallel to assess cell surface memory marker expression. Total RNA from these cells was subjected to microarray analysis and real-time PCR for miRNA expression. Nucleofection studies were performed to assess potential mRNA targets of miR-146a. RESULTS: We find that miR-146a is up-regulated in naïve CD8+ T cells exposed to IL-2 or IL-15, even in the absence of an activating T cell receptor stimulus, but not when IL-7 is also present. miR-146a expression correlates with a memory phenotype in both ex vivo and in vitro cultured cells although in our hands overexpression of miR-146a was not sufficient alone to drive a full memory phenotype. In ex vivo analysis, miR-146a was one of a small number of miRNAs that was differentially expressed between naïve and memory CD8+ T cells. CONCLUSIONS: miR-146a is emerging as a critical regulator of immune system. Our data shows that miR-146a expression is strongly influenced by the cytokine milieu even in the absence of a T cell receptor stimulus. Our results have implications for studies designed to assess the function of miR-146a, help to define a fingerprint of miRNA expression in CD8+ T cell subsets and may be useful when designing optimal protocols for T cell expansion as efficacy of T cell immunotherapy is correlated with an 'early' memory phenotype.

Direct peptide lipidation through thiol-ene coupling enables rapid synthesis and evaluation of self-adjuvanting vaccine candidates.

A radical lipidation: Application of a novel thiol-ene lipidation enables the one-step synthesis of self-adjuvanting antigenic peptides as vaccine candidates. The resultant monoacyl lipopeptides are shown to activate monocytes in a robust manner.

Rotavirus NSP4 is secreted from infected cells as an oligomeric lipoprotein and binds to glycosaminoglycans on the surface of non-infected cells.

BACKGROUND: Nonstructural glycoprotein 4 (NSP4) encoded by rotavirus is the only viral protein currently believed to function as an enterotoxin. NSP4 is synthesized as an intracellular transmembrane glycoprotein and as such is essential for virus assembly. Infection of polarized Caco-2 cells with rotavirus also results in the secretion of glycosylated NSP4 apparently in a soluble form despite retention of its transmembrane domain. We have examined the structure, solubility and cell-binding properties of this secreted form of NSP4 to further understand the biochemical basis for its enterotoxic function. We show here that NSP4 is secreted as discrete detergent-sensitive oligomers in a complex with phospholipids and demonstrate that this secreted form of NSP4 can bind to glycosaminoglycans present on the surface of a range of different cell types. METHODS: NSP4 was purified from the medium of infected cells after ultracentrifugation and ultrafiltration by successive lectin-affinity and ion exchange chromatography. Oligomerisation of NSP4 was examined by density gradient centrifugation and chemical crosslinking and the lipid content was assessed by analytical thin layer chromatography and flame ionization detection. Binding of NSP4 to various cell lines was measured using a flow cytometric-based assay. RESULTS: Secreted NSP4 formed oligomers that contained phospholipid but dissociated to a dimeric species in the presence of non-ionic detergent. The purified glycoprotein binds to the surface of various non-infected cells of distinct lineage. Binding of NSP4 to HT-29, a cell line of intestinal origin, is saturable and independent of divalent cations. Complementary biochemical approaches reveal that NSP4 binds to sulfated glycosaminoglycans on the plasma membrane. CONCLUSION: Our study is the first to analyze an authentic (i.e. non-recombinant) form of NSP4 that is secreted from virus-infected cells. Despite retention of the transmembrane domain, secreted NSP4 remains soluble in an aqueous environment as an oligomeric lipoprotein that can bind to various cell types via an interaction with glycosaminoglycans. This broad cellular tropism exhibited by NSP4 may have implications for the pathophysiology of rotavirus disease.