Effect of anticoagulants on 162 circulating immune related proteins in healthy subjects.

Diagnosis of complex disease and response to treatment is often associated with multiple indicators, both clinical and laboratorial. With the use of biomarkers, various mechanisms have been unraveled which can lead to better and faster diagnosis, predicting and monitoring of response to treatment and new drug development. With the introduction of multiplex technology for immunoassays and the growing awareness of the role of immune-monitoring during new therapeutic interventions it is now possible to test large numbers of soluble mediators in small sample volumes. However, standardization of sample collection and laboratory assessments remains suboptimal. We developed a multiplex immunoassay for detection of 162 immune related proteins in human serum and plasma. The assay was split in panels depending on natural occurring concentrations with a maximum of 60 proteins. The aim of this study was to evaluate precision, accuracy, reproducibility and stability of proteins when repeated freeze-thaw cycles are performed of this in-house developed panel, as well as assessing the protein signature in plasma and serum using various anticoagulants. Intra-assay variance of each mediator was <10%. Inter-assay variance ranged between 1.6 and 37% with an average of 12.2%. Recoveries were similar for all mediators (mean 99.8 ± 2.6%) with a range between 89-107%. Next we measured all mediators in serum, EDTA plasma and sodium heparin plasma of 43 healthy control donors. Of these markers only 19 showed similar expression profiles in the 3 different matrixes. Only 5 mediators were effected by multiple freeze-thawing cycles. Principal component analysis revealed different coagulants cluster separately and that sodium heparin shows the most consistent profile.

A multiplex immunoassay for human adipokine profiling.

BACKGROUND: Adipose tissue secretory proteins, called adipokines, play pivotal roles in the pathophysiology of obesity and its associated disorders such as metabolic syndrome, type 2 diabetes, and cardiovascular disease. Because methods for comprehensive adipokine profiling in patient plasma and other biological samples are currently limited, we developed a multiplex immunoassay for rapid and high-throughput measurement of 25 adipokines in only 50 microL of sample. METHODS: (Pre)adipocyte and ex vivo cultured adipose tissue supernatants were generated and together with plasma from 5 morbidly obese patients and 5 healthy and normal weight controls used to develop the adipokine multiplex immunoassay and test its usefulness in biological samples. We assessed adipokine dynamic ranges, lower limits of detection and quantification, cross-reactivity, intra- and interassay variation, and correlation with adipokine ELISAs. RESULTS: The limits of quantification and broad dynamic ranges enabled measurement of all 25 adipokines in supernatants and patient plasmas, with the exception of TNF-alpha in plasma samples. Intraassay variation was <10% for all adipokines; interassay variation was < 15%. The multiplex immunoassay results correlated significantly with ELISA measurements. Plasma adipokine profiling showed significantly higher concentrations of the novel adipokines cathepsin S (5.1 x 10(4) vs 4.3 x 10(4) ng/L, P = 0.003) and chemerin (4.1 x 10(5) vs 2.7 x 10(5) ng/L, P = 0.0008) in morbidly obese patients than normal weight controls, besides the established differences in adiponectin and leptin concentrations. CONCLUSIONS: Our findings underscore the relevance of the novel adipokines cathepsin S and chemerin, but foremost the potential of this novel method for both comprehensive adipokine profiling in large patient cohorts and for biological discovery.

CD8+ T cell responses in bronchoalveolar lavage fluid and peripheral blood mononuclear cells of infants with severe primary respiratory syncytial virus infections.

A protective role for CD8+ T cells during viral infections is generally accepted, but little is known about how CD8+ T cell responses develop during primary infections in infants, their efficacy, and how memory is established after viral clearance. We studied CD8+ T cell responses in bronchoalveolar lavage (BAL) samples and blood of infants with a severe primary respiratory syncytial virus (RSV) infection. RSV-specific CD8+ T cells with an activated effector cell phenotype: CD27+CD28+CD45RO+CCR7-CD38+HLA-DR+Granzyme B+CD127- could be identified in BAL and blood. A high proportion of these CD8+ T cells proliferated and functionally responded upon in vitro stimulation with RSV Ag. Thus, despite the very young age of the patients, a robust systemic virus-specific CD8+ T cell response was elicited against a localized respiratory infection. RSV-specific T cell numbers as well as the total number of activated effector type CD8+ T cells peaked in blood around day 9-12 after the onset of primary symptoms, i.e., at the time of recovery. The lack of a correlation between RSV-specific T cell numbers and parameters of disease severity make a prominent role in immune pathology unlikely, in contrast the T cells might be involved in the recovery process.

Characterization of the CD8+ T cell responses directed against respiratory syncytial virus during primary and secondary infection in C57BL/6 mice.

The BALB/c mouse model for human respiratory syncytial virus infection has contributed significantly to our understanding of the relative role for CD4+ and CD8+ T cells to immune protection and pathogenic immune responses. To enable comparison of RSV-specific T cell responses in different mouse strains and allow dissection of immune mechanisms by using transgenic and knockout mice that are mostly available on a C57BL/6 background, we characterized the specificity, level and functional capabilities of CD8+ T cells during primary and secondary responses in lung parenchyma, airways and spleens of C57BL/6 mice. During the primary response, epitopes were recognized originating from the matrix, fusion, nucleo- and attachment proteins, whereas the secondary response focused predominantly on the matrix epitope. C57BL/6 mice are less permissive for hRSV infection than BALB/c mice, yet we found CD8+ T cell responses in the lungs and bronchoalveolar lavage, comparable to the responses described for BALB/c mice.

Respiratory syncytial virus infection of monocyte-derived dendritic cells decreases their capacity to activate CD4 T cells.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in children, the elderly, and immune-compromised individuals. CD4 and CD8 T cells play a crucial role in the elimination of RSV from the infected lung, but T cell memory is not sufficient to completely prevent reinfections. The nature of the adaptive immune response depends on innate immune reactions initiated after interaction of invading pathogens with host APCs. For respiratory pathogens myeloid dendritic cell (DC) precursors that are located underneath the epithelial cell layer lining the airways may play a crucial role in primary activation of T cells and regulating their functional potential. In this study, we investigated the role of human monocyte-derived DC in RSV infection. We showed that monocyte-derived DC can be productively infected, which results in maturation of the DC judged by the up-regulation of CD80, CD83, CD86, and HLA class II molecules. However, RSV infection of DC caused impaired CD4 T cell activation characterized by a lower T cell proliferation and ablation of cytokine production in activated T cells. The suppressive effect was caused by an as yet unidentified soluble factor produced by RSV-infected DC.

HLA-DP4 presents an immunodominant peptide from the RSV G protein to CD4 T cells.

CD4 T cells play a crucial role during virus infections by producing antiviral cytokines and by regulating humoral and cellular immune responses. Unfortunately however, exaggerated CD4 T cell responses can cause significant immune-mediated disease as was observed during RSV infections in children previously vaccinated with a formalin-inactivated virus in the 1960s. It has been observed that vaccination with the G protein of RSV tends to prime mice for a similar Th2-mediated enhanced disease. Whether the G protein may play a role in enhanced disease in man is unclear. In the present study, we identified an immunodominant epitope in the conserved region of the G protein encompassing amino acid residues 162-175. This epitope is presented in the context of HLA-DPB1*0401 and DPB1*0402, the most prevalent HLA class II alleles. Importantly, in some patients, a mixed Th1/Th2 response against this epitope was found in bronchoalveolar lavage samples during primary RSV infections.

Matrix metalloproteinases as targets for the immune system during experimental arthritis.

Novel therapies for rheumatoid arthritis aiming at intervention in the inflammatory process by manipulation of autoreactive T and B lymphocytes receive major interest. However, the development of such therapies is largely hampered by the lack of knowledge of self-Ags recognized during the disease process. Recently, we predicted putative T cell self-epitopes based on a computer search profile. In the present study, the predicted self-epitopes were tested for T cell recognition in two experimental arthritis models, and their arthritogenic capacity was analyzed. Fourteen of n = 51 predicted self-epitopes were recognized during experimental arthritis of which six were able to actively induce arthritis. Interestingly, three of these six peptides were derived from matrix metalloproteinases (MMP), and only T cells responsive to MMP-derived epitopes were able to passively transfer arthritis to naive rats. Moreover, we demonstrate the presence of Abs to MMP-3 during the course of adjuvant arthritis. Together these data indicate that MMPs play a pivotal role as target for T and B cells during the development of inflammatory arthritis. This finding sheds new light on the pathophysiological role of MMPs during arthritis and opens novel possibilities for Ag-specific immunotherapy.

Successful immunotherapy with matrix metalloproteinase-derived peptides in adjuvant arthritis depends on the timing of peptide administration.

We have recently found that matrix metalloproteinases (MMPs) are targets for T-cell and B-cell reactivity in experimental arthritis. In the present article, we investigate whether modulation of MMP-specific T-cell responses could influence the course of adjuvant arthritis (AA). Lewis rats were treated nasally with MMP peptides prior to or after AA induction. Administration of the MMP-10 or the MMP-16 peptide prior to AA induction reduced the arthritic symptoms. In contrast, administration of the MMP-10 peptide after AA induction aggravated the arthritic symptoms. The present study shows the possible usefulness of MMP peptides for immunotherapy. However, a clear understanding of proper timing of peptide administration is crucial for the development of such therapies.