A short-term increase in dietary cholesterol and fat intake affects high-density lipoprotein composition in healthy subjects.

BACKGROUND AND AIMS: High-cholesterol and high-fat diets alter biochemical composition and anti-oxidant properties of high-density lipoproteins (HDL) in animals. Whether this occurs in humans is unknown. Therefore, we examined the effect of a short-term elevation in dietary cholesterol and fat intake on HDL composition in healthy subjects. METHODS AND RESULTS: In a randomized, crossover clinical trial, 14 healthy young volunteers followed a 14-day low-cholesterol/low-fat diet (LChF) and a 14-day isocaloric high-cholesterol/high-fat diet (HChF) in a random order. After each diet, we measured HDL concentrations of hydroxyeicosatetraenoic acids (HETE), hydroxyoctadecadienoic acids (HODE), and haptoglobin, as well as serum amyloid A (SAA) and paroxonase-1 activity (PON-1). HDL concentrations of 15-HETE (+254%, p = 0.002), 5-HETE (+116%, p = 0.004), 13-HODE (+102%, p = 0.049), and SAA levels (+75%, p = 0.007) were significantly higher after the HChF than after the LChF. Furthermore, haptoglobin was marginally increased (+32%, p = 0.091) while PON-1 activity was unaffected (-16%, p = 0.366) by the HChF. CONCLUSION: In healthy subjects, a short-term elevation in dietary cholesterol and fat intake increases HDL lipid hydroperoxide content (15-HETE, 5-HETE, 13-HODE) and SAA levels, which are key features of dysfunctional HDL. This is the first study showing that a physiologic manipulation of dietary cholesterol and fat intake affects HDL lipidome and proteome in healthy subjects independently of weight changes. CLINICAL TRIAL REGISTRATION: NCT02549144.

HDL lipid composition is profoundly altered in patients with type 2 diabetes and atherosclerotic vascular disease.

BACKGROUND AND AIMS: We have previously shown that the anti-inflammatory and anti-oxidant functions of HDL are impaired in T2D patients. In this study, we examined whether HDL from T2D patients contains elevated levels of oxidized fatty acids and whether those levels correlate with cardiovascular disease (CVD). METHODS AND RESULTS: HETEs and HODEs on HDL were determined by LC-MS/MS in 40 non-diabetic controls (ND), 40 T2D without CVD (D⁺CVD⁻) and 38 T2D with known history of CVD (D⁺CVD⁺). HDL oxidant index was evaluated by a cell-free assay using dichlorofluorescein. Twenty-six randomly selected subjects from the three groups underwent coronary calcium score evaluation (CAC). Major cardiovascular risk factors were similar among the groups. HETEs and HODEs content were significantly increased in HDL from D⁺CVD⁺ when compared to D⁺CVD⁻ and ND patients. HDL oxidant index was not different among the three groups; however, it was significantly higher in patients with CAC score >100 when compared to patients with CAC score <100. CONCLUSION: Patients with D⁺CVD⁻ and D⁺CVD⁺ are characterized by a severe, graded enrichment of oxidized fatty acids on HDL. In the present study, a loss of HDL function (as estimated by the HDL oxidant index) is observed only in patients with more advanced atherosclerosis.

ApoA-I mimetics favorably impact cyclooxygenase 2 and bioactive lipids that may contribute to cardiometabolic syndrome in chronic treated HIV.

OBJECTIVE: We investigated whether apolipoprotein A-I (apoA-I) mimetic peptides 4F and 6F can be a novel therapeutic strategy to reduce blood and gut bioactive lipids, proinflammatory effects of endotoxin (LPS) and aberrant activation of cyclooxygenase 2 (COX-2) as instigators of increased risk for cardiometabolic disease in chronic treated HIV. METHODS: We used two humanized murine models of chronic treated HIV infection (n = 109 mice) and gut explants from HIV infected (n = 10) persons to determine whether Tg6F and 4F attenuate in vivo and ex vivo increased blood and gut bioactive lipids (measured by mass spectrometry) and intestinal protein levels of COX-2 (measured by immunoassays) in chronic treated HIV. RESULTS: In these models of HIV, when compared to HIV-1 infected mice on antiretroviral therapy (ART) alone, oral Tg6F in combination with ART attenuated increases in plasma and gut bioactive lipids (and particularly COX lipids) and intestinal COX-2. 4F and Tg6F also reduced ex vivo production of COX-2 protein and associated secretion of bioactive lipids in gut explants from HIV-1 infected persons treated with LPS. CONCLUSION: ApoA-I mimetics favorably impact the proinflammatory effects of LPS, COX-2 and production of bioactive lipids that collectively drive gut and systemic inflammation in chronic treated HIV. Given prior experimental evidence that the proinflammatory effects of LPS, COX-2 and gut dysfunction contribute to cardiometabolic syndrome in chronic HIV, apoA-I mimetic peptides may be a novel therapy to treat cardiometabolic syndrome in chronic HIV.

Hand-off education and evaluation: piloting the observed simulated hand-off experience (OSHE).

AIM: The Observed Simulated Hand-off Experience (OSHE) was created to evaluate medical students' sign-out skills using a real-time assessment tool, the Hand-off CEX. SETTING: Thirty-two 4th year medical students participated as part of an elective course. PROGRAM DESCRIPTION: One week following an interactive workshop where students learned effective hand-off strategies, students participated in an experience in which they performed a hand-off of a mock patient using simulated history and physical examination data and a brief video. PROGRAM EVALUATION: Internal medicine residents served as standardized hand-off receivers and were trained on expectations. Students were provided feedback using a newly developed Hand-off CEX, based on the "Mini-CEX," which rates overall hand-off performance and its components on a 9-point Likert-type scale. Outcomes included performance ratings and pre- and post-student self-assessments of hand-off preparedness. Data were analyzed using Wilcoxon signed-rank tests and descriptive statistics. Resident receivers rated overall student performance with a mean score of 6.75 (range 4-9, maximum 9). Statistically significant improvement was observed in self-perceived preparedness for performing an effective hand-off (67% post- vs. 27% pre-reporting 'well-prepared,' p<0.009). DISCUSSION: This brief, standardized hand-off training exercise improved students' confidence and was rated highly by trained observers. Future work focuses on formal validation of the Hand-off CEX instrument. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11606-009-1170-y) contains supplementary material, which is available to authorized users.

Remodeling of the HDL proteome with treatment response to abatacept or adalimumab in the AMPLE trial of patients with rheumatoid arthritis.

BACKGROUND AND AIMS: To evaluate changes in the high-density lipoprotein (HDL) proteome and HDL function in active rheumatoid arthritis (RA) patients initiating therapy with abatacept or adalimumab in the Abatacept Versus Adalimumab Comparison in Biologic-Naïve RA Subjects with Background Methotrexate (AMPLE) study. METHODS: Ultra high-pressure liquid chromatography (UHPLC) coupled with ion mobility mass spectrometry (LC-IM-MS) was used to analyze proteins associated with immunoaffinity-captured HDL from plasma of 30 patients with RA randomized to either abatacept (n = 15) or adalimumab (n = 15) therapy. Paraoxonase 1 (PON1) activity, HDL anti-oxidant capacity, cholesterol profiles, and homocysteine levels were also measured at baseline and following treatment. Repeated-measures analyses were performed using mixed-effect linear models to model the within-subject covariance over time. RESULTS: In models controlling for age, sex and treatment group, improvement in inflammation measured by decreases in CRP was associated with improvement in HDL function and changes in several HDL-associated proteins including significant decreases in lipopolysaccharide-binding protein, serum amyloid A-I (SAA-I) and inter-alpha-trypsin inhibitor heavy chain H4 (p values < 0.05). Improvement in disease activity was also associated with changes in multiple HDL-associated proteins. Adalimumab was associated with higher PON1 activity, HDL-associated serotransferrin, and HDL-associated immunoglobulin J chain, and lower HDL-associated SAA-I over time compared with abatacept. CONCLUSIONS: Improvement in inflammation associated with treatment of RA, using either abatacept or adalimumab in the AMPLE study, was associated with improvement in HDL function and significant alterations in the HDL proteome, including proteins involved in the immune response, proteinase inhibition, and lipid metabolism.

Increased transcription of IL-8 in endothelial cells is differentially regulated by TNF-alpha and oxidized phospholipids.

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) upregulates a spectrum of inflammatory cytokines and adhesion molecules different from those induced by classic inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide. Interestingly, Ox-PAPC also induces the expression of a set of proteins similar to those induced by TNF-alpha or lipopolysaccharide, which include the chemokines monocyte chemotactic protein-1 (MCP-1) and interleukin (IL)-8. To elucidate the molecular mechanisms of Ox-PAPC-induced gene expression and to determine whether Ox-PAPC and other inflammatory mediators such as TNF-alpha utilize common signaling pathways, we examined the transcriptional regulation of IL-8 by Ox-PAPC and TNF-alpha in human aortic endothelial cells. Both Ox-PAPC and TNF-alpha induced the expression of IL-8 mRNA in a dose-dependent fashion; however, the kinetics of IL-8 mRNA accumulation between the 2 ligands differed. Ox-PAPC-induced IL-8 mRNA was seen as early as 30 minutes, peaked between 4 and 8 hours, and decreased substantially by 24 hours. In contrast, TNF-alpha-induced IL-8 mRNA synthesis was elevated at 30 minutes, peaked at 2 hours, and reached basal/undetectable levels by 6 hours. Actinomycin D experiments suggested that both Ox-PAPC and TNF-alpha regulate the expression of IL-8 at the transcriptional level. Furthermore, the half-life of IL-8 mRNA for both ligands was similar (<30 minutes), suggesting that mRNA stability was not responsible for the differences in the kinetics of IL-8 accumulation between the 2 ligands. Transient transfection studies with reporter constructs containing 1.48 kb of the IL-8 promoter identified an Ox-PAPC-specific response region between -133 and -1481 bp of the IL-8 promoter. In contrast, TNF-alpha activation of the IL-8 promoter was mediated almost entirely through the nuclear factor-kappaB and activation protein-1 response elements present between -70 and -133 bp of the IL-8 promoter. Thus, although Ox-PAPC and TNF-alpha both induced IL-8 synthesis, our data suggest that the 2 ligands utilize different mechanisms in the regulation of IL-8 transcription.

Human paraoxonase-3 is an HDL-associated enzyme with biological activity similar to paraoxonase-1 protein but is not regulated by oxidized lipids.

Paraoxonase-1 (PON1) is a secreted protein associated primarily with high density lipoprotein (HDL) and participates in the prevention of low density lipoprotein (LDL) oxidation. Two other paraoxonase (PON) family members, namely, PON2 and PON3, have been identified. In this study, we report the cloning and characterization of the human PON3 gene from HepG2 cells. Tissue Northern analysis identifies an approximately 1.3-kb transcript for PON3 primarily in the liver. PON3-specific peptide antibodies detect an approximately 40-kDa protein associated with HDL and absent from LDL. Pretreatment of cultured human aortic endothelial cells with supernatants from HeLa Tet On cell lines overexpressing PON3 prevents the formation of mildly oxidized LDL and inactivates preformed mildly oxidized LDL. In contrast to PON1, PON3 is not active against the synthetic substrates paraoxon and phenylacetate. Furthermore, PON3 expression is not regulated in HepG2 cells by oxidized phospholipids and is not regulated in the livers of mice fed a high-fat atherogenic diet.

TGF-beta 1 inhibits both endotoxin-induced prostaglandin synthesis and expression of the TIS10/prostaglandin synthase 2 gene in murine macrophages.

Activated macrophages produce substantial quantities of paracrine mediators, including cytokines, nitric oxide, and prostaglandins. Transforming growth factor beta 1 (TGF-beta) is a potent modulator of immune function. TGF-beta inhibits the cytotoxic activity of endotoxin/lipopolysaccharide (LPS)-activated macrophage cell lines and primary macrophage cultures, reducing their expression of cytokines and nitric oxide. In this report we demonstrate that TGF-beta also attenuates the LPS-induced synthesis and secretion of prostaglandin E2 in murine RAW 264.7 macrophage cells. Macrophage activation also induces accumulation of the recently described ligand-responsive prostaglandin synthase (PGS) TIS10/PGS-2. While TGF-beta alone has no effect on expression from the TIS10/PGS-2 gene, this cytokine inhibits LPS-induced TIS10/PGS-2 protein accumulation and synthesis, as well as LPS-induced TIS10/PGS-2 message accumulation in RAW 264.7 cells. TGF-beta concentrations in the range of 0.1-1.0 ng/ml (4-40 pM) maximally inhibit LPS-induced TIS10/PGS-2 message accumulation. In contrast, neither LPS nor TGF-beta has any effect on the level of PGS-1 (EC 1.14.99.1) message. TGF-beta also attenuates LPS-induced accumulation of unspliced TIS10/PGS-2 transcripts in RAW 264.7 cells, suggesting that this cytokine exerts its effects on TIS10/PGS-2 expression at the transcriptional level. TGF-beta inhibits the LPS-induced accumulation of TIS10/PGS-2 protein and message in cultured murine peritoneal macrophages, as well as in macrophage cell lines.

Deep sequencing in library selection projects: what insight does it bring?

High throughput sequencing is poised to change all aspects of the way antibodies and other binders are discovered and engineered. Millions of available sequence reads provide an unprecedented sampling depth able to guide the design and construction of effective, high quality naïve libraries containing tens of billions of unique molecules. Furthermore, during selections, high throughput sequencing enables quantitative tracing of enriched clones and position-specific guidance to amino acid variation under positive selection during antibody engineering. Successful application of the technologies relies on specific PCR reagent design, correct sequencing platform selection, and effective use of computational tools and statistical measures to remove error, identify antibodies, estimate diversity, and extract signatures of selection from the clone down to individual structural positions. Here we review these considerations and discuss some of the remaining challenges to the widespread adoption of the technology.

Identification and purification of the 69-kDa intracellular protease involved in the proteolytic processing of the crystal delta-endotoxin of Bacillus thuringiensis subsp. tenebrionis.

The dynamics of appearance of intracellular proteases in relation to the synthesis of crystal delta-endotoxin was studied to identify the native intracellular protease(s) involved in the proteolytic processing of the 73-kDa protoxin of Bacillus thuringiensis subsp. tenebrionis. In vitro proteolytic activation of the 73-kDa protoxin indicated the possible role of 69-kDa protease in the proteolytic processing of 73-kDa protoxin. The purified 69-kDa protease was able to cause the proteolytic activation of the 73-kDa protoxin to 68-kDa toxin and this conversion was inhibited by ethylenediamine tetraacetic acid and 1,10-phenanthroline.

Calcium- and phospholipid-dependent protein kinase C and phosphatidylinositol kinase: two major phosphorylation systems in the cornea.

The signaling messengers for the regulation of intracellular calcium-dependent functions involve a phospholipid-sensitive protein kinase (protein kinase C) modulated by diacylglycerol, which is a product of phosphoinositide degradation. We found that protein kinase C activity is two times higher in the epithelium than in the stroma-endothelial layers of the rabbit cornea. 12-0-tetradecanoylphorbol-13-acetate (TPA) and phosphatidylserine stimulate protein kinase C at low Ca2+ concentrations. The TPA-phosphatidylserine stimulated activity of corneal epithelium was recovered in the soluble fraction; in the membrane-bound fraction, a very active phosphatidylinositol kinase accounted for half the basal phosphorylation of the corneal epithelium.

Hypoparathyroidism, intracranial calcification, and seizures 61 years after thyroid surgery.

Though hypocalcemic symptoms from hypoparathyroidism following thyroidectomy most often occur in the immediate postoperative period, hypoparathyroidism can present itself many years after the thyroid surgery with nonspecific symptoms. We present herein the case of a 74-year-old woman with previously undiagnosed hypoparathyroidism who had tonic-clonic seizures and intracranial calcification 61 years after her thyroidectomy. This case is unusual because of the long latency between thyroidectomy and clinical presentation.

PON3 is upregulated in cancer tissues and protects against mitochondrial superoxide-mediated cell death.

To achieve malignancy, cancer cells convert numerous signaling pathways, with evasion from cell death being a characteristic hallmark. The cell death machinery represents an anti-cancer target demanding constant identification of tumor-specific signaling molecules. Control of mitochondrial radical formation, particularly superoxide interconnects cell death signals with appropriate mechanistic execution. Superoxide is potentially damaging, but also triggers mitochondrial cytochrome c release. While paraoxonase (PON) enzymes are known to protect against cardiovascular diseases, recent data revealed that PON2 attenuated mitochondrial radical formation and execution of cell death. Another family member, PON3, is poorly investigated. Using various cell culture systems and knockout mice, here we addressed its potential role in cancer. PON3 is found overexpressed in various human tumors and diminishes mitochondrial superoxide formation. It directly interacts with coenzyme Q10 and presumably acts by sequestering ubisemiquinone, leading to enhanced cell death resistance. Localized to the endoplasmic reticulum (ER) and mitochondria, PON3 abrogates apoptosis in response to DNA damage or intrinsic but not extrinsic stimulation. Moreover, PON3 impaired ER stress-induced apoptotic MAPK signaling and CHOP induction. Therefore, our study reveals the mechanism underlying PON3's anti-oxidative effect and demonstrates a previously unanticipated function in tumor cell development. We suggest PONs represent a novel class of enzymes crucially controlling mitochondrial radical generation and cell death.

DNA immunization using a non-viral promoter.

Most DNA vaccines rely on strong viral promoters to optimize levels of transgene expression. Some studies have demonstrated that the potency of viral promoters does not necessarily correlate with DNA vaccine efficacy in vivo. This has partly been attributed to downregulation of these promoters by cytokines such as interferon gamma induced by the CpG motives of these vaccines. In an attempt to avoid downregulation of viral promoters by IFN-gamma, we tested vaccine vectors driven by the MHC class II promoter. To enhance the activity of this promoter, another plasmid expressing the human MHC class II transactivator driven by a viral promoter, the native IFN-gamma inducible CIITA type IV promoter (PIV) or a synthetic promoter containing IFN-gamma inducible elements was co-inoculated. Our data show that a non-viral promoter such as the MHC class II promoter tested in this study can indeed be used in DNA vaccines.