Human severe sepsis cytokine mixture increases ß2-integrin-dependent polymorphonuclear leukocyte adhesion to cerebral microvascular endothelial cells in vitro.

INTRODUCTION: Sepsis-associated encephalopathy (SAE) is a state of acute brain dysfunction in response to a systemic infection. We propose that systemic inflammation during sepsis causes increased adhesion of leukocytes to the brain microvasculature, resulting in blood-brain barrier dysfunction. Thus, our objectives were to measure inflammatory analytes in plasma of severe sepsis patients to create an experimental cytokine mixture (CM), and to use this CM to investigate the activation and interactions of polymorphonuclear leukocytes (PMN) and human cerebrovascular endothelial cells (hCMEC/D3) in vitro. METHODS: The concentrations of 41 inflammatory analytes were quantified in plasma obtained from 20 severe sepsis patients and 20 age- and sex-matched healthy controls employing an antibody microarray. Two CMs were prepared to mimic severe sepsis (SSCM) and control (CCM), and these CMs were then used for PMN and hCMEC/D3 stimulation in vitro. PMN adhesion to hCMEC/D3 was assessed under conditions of flow (shear stress 0.7 dyn/cm(2)). RESULTS: Eight inflammatory analytes elevated in plasma obtained from severe sepsis patients were used to prepare SSCM and CCM. Stimulation of PMN with SSCM led to a marked increase in PMN adhesion to hCMEC/D3, as compared to CCM. PMN adhesion was abolished with neutralizing antibodies to either ß2 (CD18), αL/ß2 (CD11α/CD18; LFA-1) or αM/ß2 (CD11ß/CD18; Mac-1) integrins. In addition, immune-neutralization of the endothelial (hCMEC/D3) cell adhesion molecule, ICAM-1 (CD54) also suppressed PMN adhesion. CONCLUSIONS: Human SSCM up-regulates PMN pro-adhesive phenotype and promotes PMN adhesion to cerebrovascular endothelial cells through a ß2-integrin-ICAM-1-dependent mechanism. PMN adhesion to the brain microvasculature may contribute to SAE.

Antibody microarray analysis of inflammatory mediator release by human leukemia T-cells and human non small cell lung cancer cells.

Cytokines and chemokines are responsible for regulating inflammation and the immune response. Cytokine and chemokine release is typically measured by quantitative enzyme-linked immunosorbant assay (ELISA) or Western blot analysis. To expedite the analysis of samples for multiple cytokines/chemokines, we have developed slide-based Thermo Scientific ExcelArray Antibody Sandwich Microarrays. Each slide consists of 16 subarrays (wells), each printed with 12 specific antibodies in triplicate and positive and negative control elements. This 16-well format allows for the analysis of 10 test samples using a six-point standard curve. The array architecture is based on the "sandwich" ELISA, in which an analyte protein is sandwiched between an immobilized capture antibody and a biotinylated detection antibody, using streptavidin-linked Thermo Scientific DyLight 649 Dye for quantitation. The observed sensitivity of this assay was <10 pg/mL. In our experiments, the Jurkat cell line was used as a model for human T-cell leukemia, and the A549 cell line was used as a model for human non-small cell lung cancer. To evoke a cytokine/chemokine response, cells were stimulated with tumor necrosis factor alpha (TNFalpha), phorbol-12-myristate-13-acetate (PMA, TPA), and phytohemagglutinin (PHA). Cell supernatants derived from both untreated and stimulated cells were analyzed on four different arrays (Inflammation I, Inflammation II, Angiogenesis, and Chemotaxis), enabling the quantitation of 41 unique analytes. Stimulated cells showed an increase in the expression level of many of the test analytes, including IL-8, TNF-alpha, and MIP-1alpha, compared to the non-treated controls. Our experiments clearly demonstrate the utility of antibody microarray analysis of cell-culture supernatants for the profiling of cellular inflammatory mediator release.

Robust microarray production of freshly expressed proteins in a human milieu.

PURPOSE: In vitro transcription/translation (IVTT) systems are widely used in proteomics. For clinical applications, mammalian systems are preferred for protein folding and activity; however, the level of protein obtained is low. A new system extracted from human cells (1-Step Human Coupled IVT (HCIVT)) has the potential to overcome this problem and deliver high yields of protein expressed in a human milieu. EXPERIMENTAL DESIGN: Western blots and self-assembled protein microarrays were used to test the efficiency of protein synthesis by HCIVT compared to rabbit reticulocyte lysate (RRL). The arrays were also used to measure the immune response obtained from serum of patients exposed to pathogens or vaccine. RESULTS: HCIVT performed better than RRL in all experiments. The yield of protein synthesized in HCIVT is more than ten times higher than RRL, in both Western blot and protein microarrays. Moreover, HCIVT showed a robust lot-to-lot reproducibility. In immune assays, the signals of many antigens were detected only in HCIVT-expressed arrays, mainly due to the reduction in the background signal and the increased levels of protein on the array. CONCLUSION AND CLINICAL RELEVANCE: HCIVT is a robust in vitro transcription and translation system that yields high levels of protein produced in a human milieu. It can be used in applications where protein expression in a mammalian system and high yields are needed. The increased immunogenic response of HCIVT-expressed proteins will be critical for biomarker discovery in many diseases, including cancer.

Elevated plasma matrix metalloproteinases and their tissue inhibitors in patients with severe sepsis.

PURPOSE: Matrix metalloproteinases (MMPs) are essential for tissue remodeling. Our objectives were to determine (1) the concentrations of MMPs and their tissue inhibitors (TIMPs) in plasma obtained from patients with severe sepsis, (2) to correlate changes in MMP and TIMP levels with disease severity, and (3) to investigate recombinant activated protein C (rAPC) actions on plasma MMP2, 9 activities from severe sepsis patients. MATERIALS AND METHODS: Matrix metalloproteinase and TIMP levels were quantified in plasma from patients with severe sepsis using antibody microarrays and gelatin zymography. RESULTS: Plasma MMPs (3, 7, 8, 9) and TIMPs (1, 2, 4) on microarray were increased in severe sepsis on intensive care unit (ICU) day 1, with more than 3-fold increases in MMP3, MMP7, MMP8, MMP9, and TIMP4. Latent forms of MMP2, 9 on zymography were increased in plasma from patients with severe sepsis, whereas only half of severe sepsis patients showed active MMP9. Elevated MMP7 and MMP9 on ICU days 1 and 3 negatively correlated with multiple organ dysfunctions. The temporal activity patterns of MMP2, 9 during 21 ICU days were not altered in patients treated with rAPC or by the addition of exogenous rAPC to plasma. CONCLUSION: Most plasma MMPs and TIMPS were elevated in patients with severe sepsis, but only a limited subset of MMPs (7, 9) negatively correlated with disease severity. Recombinant activated protein C does not appear to directly alter MMP2, 9 activities.