Eric W. Olle

Harmful and protective roles of neutrophils in sepsis.

The current studies demonstrate protective and harmful effects of neutrophils (PMN) during experimental sepsis after cecal ligation and puncture (CLP). It is known that CLP induces signaling defects in blood PMN. When PMN were depleted 12 h after CLP, there were dramatic reductions in levels of bacteremia, evidence for reduced liver and renal dysfunction, sharp reductions in serum levels of cytokines (IL-1β, IL-6, IL-10, TNF-α, and IL-2), and improved survival. In contrast, PMN depletion before CLP resulted in substantial increases in bacteremia and no evidence for attenuation of liver and renal failure dysfunction. These data suggest that at the onset of sepsis, PMN are important in regulating the levels of bacteremia, whereas after the onset of sepsis, as they lose innate immune functions, their presence is associated with higher levels of bacteremia and intensified organ dysfunction.

Matrix metalloproteinase‐9 is an important factor in hepatic regeneration after partial hepatectomy in mice

Partial hepatectomy triggers hepatocyte proliferation, hepatic matrix remodeling, and hepatocyte apoptosis, all of which are important processes in the regenerating liver. Previous studies have shown an increase in the levels of matrix metalloproteinases gelatinase A (MMP‐2) and gelatinase B (MMP‐9) after partial hepatectomy. The goal of this study was to investigate the role of MMP‐9 in liver regeneration after partial hepatectomy. A 70% hepatectomy or sham laparotomy was performed in wild‐type or MMP‐9–deficient (MMP‐9−/−) mice. Hepatic regeneration was determined by liver weight/total body weight ratios and BrdU staining, which was used to a calculate mitotic index at several times postoperatively. Cytokine and growth factor expression was evaluated by Luminex™ bead–based ELISA and Western blots. Finally, the effect of MMP‐9 on apoptosis was measured using TUNEL and caspase expression. The MMP‐9−/− animals had a delayed hepatic regenerative response when compared with wild‐type controls. The MMP‐9–deficient animals expressed significantly less VEGF, HGF, and TNF‐α between days 2 and 3 post‐hepatectomy. Apoptosis, as measured by TUNEL staining and caspase expression, was decreased in the MMP‐9−/−. In conclusion, MMP‐9 plays an important role in liver regeneration after partial hepatectomy by affecting matrix remodeling, as well as cytokine, growth factor, and caspase expression. (HEPATOLOGY 2006;44:540–549.)

Development of an Internally Controlled Antibody Microarray

Antibody microarrays are a high throughput technology used to concurrently screen for protein expression. Most antibody arrays currently used are based on the ELISA sandwich approach that uses two antibodies to screen for the expression of a limited number of proteins. Also because antigen-antibody interactions are concentration-dependent, antibody microarrays need to normalize the amount of antibody that is used. In response to the limitations with the currently existing technology we have developed a single antibody-based microarray where the quantity of antibody spotted is used to standardize the antigen concentration. In addition, this new array utilizes an internally controlled system where one color represents the amount of antibody spotted, and the other color represents the amount of the antigen that is used to quantify the level of protein expression. When compared with median fluorescence intensity alone, normalization for antibody spot intensity decreased variability and lowered the limits of detection. This new antibody array was tested using standard cytokine proteins and also cell lysates obtained from mouse macrophages stimulated in vitro and evaluated for the expression of the cytokine proteins interleukin (IL)-1β, IL-5, IL-6, and macrophage inflammatory proteins 1α and 1β. The levels of protein expression seen with the antibody microarray was compared with that obtained with Western blot analysis, and the magnitude of protein expression observed was similar with both technologies with the antibody array actually showing a greater degree of sensitivity. In summary, we have developed a new type of antibody microarray to screen for protein expression that utilizes a single antibody and controls for the amount of antibody spotted. This type of array appears at least as sensitive as Western blot analysis, and the technology can be scaled up for high throughput screening for hundreds of proteins in complex biofluids such as blood.

Influence of matrix application timing on spectral reproducibility and quality in SELDI-TOF-MS

Timing is Everything Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a robust platform for protein profiling and differential gene expression analysi...

Role of interleukin-6 in immune complex induced models of vascular injury

Previous studies have suggested that Interleukin-6 (IL-6) acts as a marker of vasculitis. To determine the role of IL-6 in vasculitis we utilized two models of immune complex induced vascular injury (dermal Arthus and acute pulmonary alveolitis) in IL-6 deficient (IL-6−/−) and IL-6 sufficient (IL-6+/+) mice. Plasma and bronchoalveolar lavage (BAL) levels of IL-6 were elevated in the injured IL-6+/+ mice with acute alveolitis and in the plasma of IL-6+/+ mice with dermal Arthus vasculitis. While, IL-6 levels in IL-6−/− mice were near or below the levels of detection. Histological examination of the intensity of vascular injury response demonstrated no significant differences between IL-6−/− and IL6+/+ mice. More specifically, lung permeability (total protein in the BAL) in the lung injury model in IL-6−/− mice was the same as injured IL-6+/+ mice. As a corollary, assessment of vascular permeability in both models was the same in the IL-6−/− as the IL-6+/+ mice. Quantification of leukocyte influx into the injured tissues in both models also revealed no differences between the IL-6−/− and IL-6+/+ mice. These data demonstrate that while IL-6 is upregulated in acute vascular injury it does not appear to be critical in the development of the vascular inflammatory response.

Screening of serum samples from Wegener's granulomatosis patients using antibody microarrays

Wegeners Granulomatosis (WG) is an idiopathic granulomatosis autoimmune vasculitis that primarily affects small vessels and is associated with glomerulonephritis and pulmonary granulomatous vasculitis. Anti‐neutrophil cytoplasmic auto‐antibodies (cANCA) against proteinase‐3 are used to identify WG, but ANCA titers are not present in some patients with the localized disease. The objective of this study was to develop an antibody array to help identify protein expression patterns in serum from patients with WG as compared to normals. The arrays were tested for limits of detection, background, and cross reactivity using standard proteins. The arrays were hybridized with either normal patient serum (n = 30) or with serum samples from a population of WG patients (n = 26) that were age and sex matched. Data analysis and curve fitting of the standard dilution series calculated r2 values and determined a sensitivity of <50 pg/mL for the majority of proteins. A total of 24 proteins were assessed. Several statistically significant increases (p<0.05) were seen in the expression of: angiotensin converting enzyme‐I, IFN‐γ, IL‐8, s‐ICAM‐1 and s‐VCAM in WG patients as compared to controls. Utilizing the antibody microarray technology has led to the identification of potential biomarkers of vascular injury in the serum of WG patients.