Greg Kilmer

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.

Robust microarray production of freshly expressed proteins in a human milieu

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.

Human severe sepsis cytokine mixture increases β2-integrin-dependent polymorphonuclear leukocyte adhesion to cerebral microvascular endothelial cells in vitro

IntroductionSepsis-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.MethodsThe 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/cm2).ResultsEight 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 β2 (CD18), αL/β2 (CD11α/CD18; LFA-1) or αM/β2 (CD11β/CD18; Mac-1) integrins. In addition, immune-neutralization of the endothelial (hCMEC/D3) cell adhesion molecule, ICAM-1 (CD54) also suppressed PMN adhesion.ConclusionsHuman SSCM up-regulates PMN pro-adhesive phenotype and promotes PMN adhesion to cerebrovascular endothelial cells through a β2-integrin-ICAM-1-dependent mechanism. PMN adhesion to the brain microvasculature may contribute to SAE.

Abstract 4958: Analysis of the AKT signaling pathway in prostate cancer using a streamlined Western blot workflow

Relative protein levels in the Akt pathway were analyzed in metastasized and normal prostate cancer cell lines using a new streamlined Western blot workflow and compared to a traditional Western blot method. Protein expression levels in the Akt pathway are commonly studied using Western blotting; however, current Western blot techniques involve multiple time-consuming steps taking more than 5 hours or even days. We used a streamlined Western blot workflow that combines a rapid 10-minute protein transfer and a one-hour immunoblotting protocol. When using an overnight primary antibody incubation, the protocol requires less than 30 minutes. Importantly, this method does not require the use of additional equipment or consumables and does not limit throughput. These data illustrate that current Western blot techniques involving multiple time-consuming steps can be streamlined to yield comparable results for phospho-specific and total protein targets. Results obtained in the analysis of the Akt pathway yielded comparable sensitivity to traditional Western blotting methods for both low- and high-abundance target proteins. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4958.

Abstract LB-119: Production of functionally active glycoproteins and phosphoproteins based on a novel human in vitro expression system

One of the key features of the human proteome is the abundance of post-translation modification (PTM), with more than 50% of proteins being glycosylated and more than 30% of all the proteins regulated by kinase activities. The alteration of protein glycosylation and other PTMs such as phosphorylation in cancer cells is closely related to the onset and progression of cancer and other disease states. In collaboration with RIKEN, we have developed a novel human in vitro translation (also known as cell-free expression) system, that can produce biologically active glycoproteins such as human chorionogonadotrophic hormone as well as active protein kinases such as Akt, MAPK and Aurora A kinase. A highly efficient PCR based approach will also be discussed that eliminates the need for cloning of genes into the expression vector and provides an easier expression of genes present in any other vector. In addition we demonstrate that our in vitro human expression, unlike the only other commercially existing mammalian expression system based on rabbit reticulocytes, can sustain expression of proteins for over 6 hours without the need to remove of inhibitory by products and is compatible with fluorimetric and colorimetric assays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-119.