Rattus Model Utilizing Selective Pulmonary Ischemia Induces Bronchiolitis Obliterans Organizing Pneumonia

Bronchiolitis obliterans organizing pneumonia (BOOP), a morbid condition when associated with lung transplant and chronic lung disease, is believed to be a complication of ischemia. Our goal was to develop a simple and reliable model of lung ischemia in the Sprague-Dawley rat that would produce BOOP. Unilateral ischemia without airway occlusion was produced by an occlusive slipknot placed around the left main pulmonary artery. Studies were performed 7 days later. Relative pulmonary and systemic flow to each lung was measured by injection of technetium Tc 99m macroaggregated albumin. Histological sections were examined for structure and necrosis and scored for BOOP. Apoptosis was detected by immunohistochemistry with an antibody against cleaved caspase 3. Pulmonary artery blood flow to left lungs was less than 0.1% of the cardiac output, and bronchial artery circulation was ~2% of aortic artery flow. Histological sections from ischemic left lungs consistently showed Masson bodies, inflammation, and young fibroblasts filling the distal airways and alveoli, consistent with BOOP. In quantitative evaluation of BOOP using epithelial changes, inflammation and fibrosis were higher in ischemic left lungs than right or sham-operated left lungs. Apoptosis was increased in areas exhibiting histological BOOP, but there was no histological evidence of necrosis. Toll-like receptor 4 expression was increased in ischemic left lungs over right. An occlusive slipknot around the main left pulmonary artery in rats produces BOOP, providing direct evidence that ischemia without immunomodulation or coinfection is sufficient to initiate this injury. It also affords an excellent model to study signaling and genetic mechanisms underlying BOOP

Myeloperoxidase inhibition ameliorates plaque psoriasis in mice

Plaque psoriasis is a common inflammatory condition of the skin characterized by red, flaking lesions. Current therapies for plaque psoriasis target many facets of the autoimmune response, but there is an incomplete understanding of how oxidative damage produced by enzymes such as myeloperoxidase contributes to skin pathology. In this study, we used the Aldara (Imiquimod) cream model of plaque psoriasis in mice to assess myeloperoxidase inhibition for treating psoriatic skin lesions. To assess skin inflammation severity, an innovative mouse psoriasis scoring system was developed. We found that myeloperoxidase inhibition ameliorated psoriasis severity when administered either systemically or topically. The findings of this study support the role of oxidative damage in plaque psoriasis pathology and present potential new therapeutic avenues for further exploration

Anion Exchange HPLC Isolation of High-Density Lipoprotein (HDL) and On-Line Estimation of Proinflammatory HDL

<div><p>Proinflammatory high-density lipoprotein (p-HDL) is a biomarker of cardiovascular disease. Sickle cell disease (SCD) is characterized by chronic states of oxidative stress that many consider to play a role in forming p-HDL. To measure p-HDL, apolipoprotein (apo) B containing lipoproteins are precipitated. Supernatant HDL is incubated with an oxidant/LDL or an oxidant alone and rates of HDL oxidation monitored with dichlorofluorescein (DCFH). Although apoB precipitation is convenient for isolating HDL, the resulting supernatant matrix likely influences HDL oxidation. To determine effects of supernatants on p-HDL measurements we purified HDL from plasma from SCD subjects by anion exchange (AE) chromatography, determined its rate of oxidation relative to supernatant HDL. SCD decreased total cholesterol but not triglycerides or HDL and increased cell-free (cf) hemoglobin (Hb) and xanthine oxidase (XO). HDL isolated by AE-HPLC had lower p-HDL levels than HDL in supernatants after apoB precipitation. XO+xanthine (X) and cf Hb accelerated purified HDL oxidation. Although the plate and AE-HPLC assays both showed p-HDL directly correlated with cf-Hb in SCD plasma, the plate assay yielded p-HDL data that was influenced more by cf-Hb than AE-HPLC generated p-HDL data. The AE-HPLC p-HDL assay reduces the influence of the supernatants and shows that SCD increases p-HDL.</p></div

Effects of XO, X, cf Hb alone and in combination on HDL conjugated diene formation.

<p>(A) XO, X, cf Hb alone and in combination were added to purified human HDL and initial rates of absorbance (A_{234 nm}) recorded over time. Absorbance at 234 nm was recorded and initial rates of absorbance calculated per test group. (A) Line graphs showing changes in A_{234 nm} with respect to time for the following test groups: a) HDL alone; b) HDL+XO+X; c) HDL+Hb (4 mg/dL) +XO+X; d) HDL+Hb (16 mg/dL) +XO+X; e) HDL+X; f) HDL+XO; and, g) XO+X. (B) The bar chart showed that XO/X increases A_{234 nm} at faster rates than HDL alone and that adding cf Hb increased initial rates of HDL oxidation greater than XO/X alone when cf Hb equals 16 mg/dL but, not 4 mg/dL. (**** = p<0.001, n = 5–12).</p

AE-HPLC separation of cf Hb and HDL from plasma.

<p>AE-HPLC of Hb alone revealed Hb elutes at 0–5 min. (A) Cf Hb (100 mg/dL) 30 µl was injected without DiI, which was measured by UV absorbance (A_{230 nm}). (B) Plasma treated with DiI (10 µg/mL) and then 30 µL was analyzed by fluorescent AE-HPLC (Ex 530 nm/Em 577 nm). (C) Plasma (30 µL) was injected, fractions collected, pooled, concentrated and examined by immunoblot analysis for apoA-I and Hb.</p

Separation of Xanthine Oxidase (XO) from HDL.

<p>DiI-treated plasma was injected (30 µL) into the AE-HPLC. Fractions were collected, concentrated and examined by immunoblot for XO. The chromatogram in A and immunoblot in B showed that the modified AE-HPLC protocol eluted HDL prior to XO.</p

P-HDL plotted as a function of cf Hb: Plate vs. HPLC Assay.

<p>For plate assay, control AA p-HDL data (* RFU/µg HDL_c⋅min) were plotted as open circles and SCD p-HDL data were plotted as open squares. For HPLC assay, controls (AA, meaning homozygous for hemoglobin AA) p-HDL data (* RFU/apoA-I (µg/mL)) were plotted as closed circles and SCD p-HDL data were plotted as closed squares. (y = ax+b; Plate assay: y = 0.03004x+3.554; HPLC assay: y = 0.01688x+0.84140). These data showed that p-HDL isolated by apo B precipitation was more sensitive to the effects of cf Hb and was more variable than p-HDL determined by the AE-HPLC-PCR assay.</p

AE-HPLC-PCR Quantification of p-HDL in Control (AA) and SCD Plasma.

<p>(A) DCF chromatogram showing relative levels of DCF fluorescence (index of oxidizability) for p-HDL in control and SCD plasma. At 30–44 minutes, DCF fluorescence intensity in separated HDL in SCD plasma (black line) is greater than in control plasma (gray line). (B) Dividing DCF fluorescence intensity under these peaks by the subject’s plasma apoA-I concentration (µg/mL) yields relative fluorescence units (RFU) per apoA-I (µg/mL). P-HDL are increased in SCD subjects compared to control subjects (** = p<0.01, n = 6).</p