Background It is critical to develop new metrics to determine whether high density lipoprotein (HDL) is cardioprotective in humans. One promising approach is HDL particle concentration (HDL-P) – the size and concentration of HDL in plasma or serum. However, the two methods currently used to determine HDL-P yield concentrations that differ more than 5-fold. We therefore developed and validated an improved approach to quantify HDL-P, termed calibrated ion mobility analysis (calibrated IMA). Methods HDL was isolated from plasma by ultracentrifugation, introduced into the gas phase with electrospray ionization, separated by size, and quantified by particle counting. A calibration curve constructed with purified proteins was used to correct for the ionization efficiency of HDL particles. Results The concentrations of gold nanoparticles and reconstituted HDLs measured by calibrated IMA were indistinguishable from concentrations determined by orthogonal methods. In plasma of control (n=40) and cerebrovascular disease (n=40) subjects, three subspecies of HDL were reproducibility measured, with an estimated total HDL-P of 13.4±2.4 µM (mean±SD). HDL-C accounted for 48% of the variance in HDL-P. HDL-P was significantly lower in subjects with cerebrovascular disease, and this difference remained significant after adjustment for HDL cholesterol levels. Conclusions Calibrated IMA accurately and reproducibly determined the concentration of gold nanoparticles and synthetic HDL, strongly suggesting the method could accurately quantify HDL particle concentration. Importantly, the estimated stoichiometry of apoA-I determined by calibrated IMA was 3–4 per HDL particle, in excellent agreement with current structural models. Furthermore, HDL-P associated with cardiovascular disease status in a clinical population independently of HDL cholesterol.

Objective: HDL (high-density lipoprotein) infusion reduces atherosclerosis in animal models and is being evaluated as a treatment in humans. Studies have shown either anti- or proinflammatory effects of HDL in macrophages, and there is no consensus on the underlying mechanisms. Here, we interrogate the effects of HDL on inflammatory gene expression in macrophages. Approach and Results: We cultured bone marrow–derived macrophages, treated them with reconstituted HDL or HDL isolated from APOA1 Tg ;Ldlr −/− mice, and challenged them with lipopolysaccharide. Transcriptional profiling showed that HDL exerts a broad anti-inflammatory effect on lipopolysaccharide-induced genes and proinflammatory effect in a subset of genes enriched for chemokines. Cholesterol removal by POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) liposomes or β-methylcyclodextrin mimicked both pro- and anti-inflammatory effects of HDL, whereas cholesterol loading by POPC/cholesterol-liposomes or acetylated LDL (low-density lipoprotein) before HDL attenuated these effects, indicating that these responses are mediated by cholesterol efflux. While early anti-inflammatory effects reflect reduced TLR (Toll-like receptor) 4 levels, late anti-inflammatory effects are due to reduced IFN (interferon) receptor signaling. Proinflammatory effects occur late and represent a modified endoplasmic reticulum stress response, mediated by IRE1a (inositol-requiring enzyme 1a)/ASK1 (apoptosis signal-regulating kinase 1)/p38 MAPK (p38 mitogen-activated protein kinase) signaling, that occurs under conditions of extreme cholesterol depletion. To investigate the effects of HDL on inflammatory gene expression in myeloid cells in atherosclerotic lesions, we injected reconstituted HDL into Apoe −/− or Ldlr −/− mice fed a Western-type diet. Reconstituted HDL infusions produced anti-inflammatory effects in lesion macrophages without any evidence of proinflammatory effects. Conclusions: Reconstituted HDL infusions in hypercholesterolemic atherosclerotic mice produced anti-inflammatory effects in lesion macrophages suggesting a beneficial therapeutic effect of HDL in vivo.

Understanding the function of high-density lipoprotein (HDL) requires detailed knowledge of the structure of its primary protein, apolipoprotein A-I (APOA1). However, APOA1 flexibility and HDL heterogeneity have confounded decades of efforts to determine high-resolution structures and consistent models. Here, molecular dynamics simulations totaling 30 μs on two nascent HDLs, each with 2 APOA1 and either 160 phospholipids and 24 cholesterols or 200 phospholipids and 20 cholesterols, show that residues 1-21 of the N-terminal domains of APOA1 interact via strong salt bridges. Residues 26-43 of one APOA1 in the smaller particle form a hinge on the disc edge, which displaces the C-terminal domain of the other APOA1 to the phospholipid surface. The proposed structures are supported by chemical cross-linking, Rosetta modeling of the N-terminal domain, and analysis of the lipid-free ∆185APOA1 crystal structure. These structures provide a framework for understanding HDL maturation and revise all previous models of nascent HDL.

Previous studies indicate that overexpression of N-methylpurine DNA glycosylase (MPG) dramatically sensitizes cells to alkylating agent-induced cytotoxicity. We recently demonstrated that this sensitivity is preceded by an increased production of AP sites and strand breaks, confirming that overexpression of MPG disrupts normal base excision repair and causes cell death through overproduction of toxic repair intermediates. Here we establish through site-directed mutagenesis that MPG-induced sensitivity to alkylation is dependent on enzyme glycosylase activity. However, in contrast to the sensitivity seen to heterogeneous alkylating agents, MPG overexpression generates no cellular sensitivity to MeOSO2(CH2)2-lexitropsin, an alkylator which exclusively induces 3-meA lesions. Indeed, MPG overexpression has been shown to increase the toxicity of alkylating agents that produce 7-meG adducts, and here we demonstrate that MPG-overexpressing cells have dramatically increased removal of 7-meG from their DNA. These data suggest that the mechanism of MPG-induced cytotoxicity involves the conversion of non-toxic 7-meG lesions into highly toxic repair intermediates. This study establishes a mechanism by which a benign DNA modification can be made toxic through the overexpression of an otherwise well-tolerated gene product, and the application of this principle could lead to improved chemotherapeutic strategies that reduce the peripheral toxicity of alkylating agents.

Vacuum ultraviolet laser photodissociation (UVPD) of peptide ions leads to unusual dissociation channels involving backbone C-C bond breaking. However, the molecular basis for the observed behavior is not clearly understood. We now report theoretical investigations using ab initio/density functional theory (DFT) techniques on neutral and protonated dipeptides undergoing vacuum ultraviolet (VUV) induced fragmentation via a Rydberg excitation (and/or electron detachment) and subsequent C-C bond cleavage. New experimental results on VUV photodissociation of dipeptides (protonated Ala_Arg and Arg_Ala) provide strong support for our proposed model. Our mechanism also provides a natural explanation for the presence of immonium ions that are sometimes observed in such experiments.

Rationale: High-density lipoprotein (HDL) may be cardioprotective because it accepts cholesterol from macrophages via the cholesterol transport proteins ABCA1 and ABCG1. The ABCA1-specific cellular cholesterol efflux capacity (ABCA1 CEC) of HDL strongly and negatively associates with cardiovascular disease (CVD) risk, but how diabetes impacts that step is unclear. Objective: To test the hypothesis that HDL's cholesterol efflux capacity is impaired in subjects with type 2 diabetes. Methods and Results: We performed a case-control study with 19 subjects with type 2 diabetes and 20 control subjects. Three sizes of HDL particles, small-HDL, medium-HDL and large-HDL, were isolated by high-resolution size exclusion chromatography from study subjects. Then we assessed the ABCA1 CEC of equimolar concentrations of particles. Small-HDL accounted for almost all of ABCA1 CEC activity of HDL. ABCA1 CECbut not ABCG1 CECof small-HDL was lower in the subjects with type 2 diabetes than the control subjects. Isotope dilution tandem mass spectrometry demonstrated that the concentration of serpin family A member 1 (SERPINA1) in small-HDL was also lower in subjects with diabetes. Enriching small-HDL with SERPINA1 enhanced ABCA1 CEC. Structural analysis of SERPINA1 identified 3 amphipathic alpha-helices clustered in the N-terminal domain of the protein; biochemical analyses demonstrated that SERPINA1 binds phospholipid vesicles. Conclusions: The ABCA1 CEC of small-HDL is selectively impaired in type 2 diabetes, likely because of lower levels of SERPINA1. SERPINA1 contains a cluster of amphipathic α-helices that enable apolipoproteins to bind phospholipid and promote ABCA1 activity. Thus, impaired ABCA1 activity of small HDL particles deficient in SERPINA1 could increase CVD risk in subjects with diabetes.

Objective. This study aimed to explore the laboratory markers associated with perforation in children with acute appendicitis. Methods. This retrospective study reviewed 1895 children (3–18 years old) with confirmed acute appendicitis from 2007 to 2017. Clinical (demographic characteristics, symptoms, and signs) and laboratory data (white blood cell count, C-reactive protein (CRP), procalcitonin, D-lactate, platelet count, bilirubin, aspartate aminotransferase, and alanine aminotransferase) were collected and compared between perforated and nonperforated groups. The logistic regression analysis was performed to identify independent risk factors. Results. Of all patients, 613 children were perforated. Children with perforation had significantly longer duration of symptoms, higher white blood cell count, CRP level, and neutrophils percentage, and lower serum sodium level. Elevated white blood cell count with CRP level and elevated neutrophils percentage with CRP level were found to be associated with risk of perforation. Conclusions. White blood cell count with C-reactive protein and neutrophils percentage with CRP are important markers in distinguishing perforated appendicitis from nonperforated appendicitis in pediatric subjects.

Reversal of immunodeficiency in the lung by gene therapy is limited in part by the difficulty of transfecting lung cells in vivo.Many options exist for successfully transfecting cells in vitro, but they are not easily adapted to the in vivo condition. To overcome this limitation, we transduced macrophages in vitro with the murine IFN-␥ (mIFN-␥) gene and intratracheally delivered the macrophages to express mIFN-␥ in vivo. A recombinant retroviral vector pSF91 system was modified to encode mIFN-␥ and enhanced green fluorescent protein (EGFP). A murine macrophage cell line J774A.1 transduced with the retroviral supernatant increased secretion from undetectable levels to 131.6 ؎ 4.2 g/ml mIFN-␥ at 24 h in vitro. The mIFN-␥-producing macrophages were intratracheally instilled into mechanically ventilated scid mice. mIFN-␥ levels in the bronchoalveolar lavage increased from undetectable levels at baseline to 158.8 ؎ 5.1 pg/ml at 48 h (P < 0.001). Analysis of the lavaged cells for EGFP expression revealed that EGFP expression was directly proportional to the number of transduced macrophages instilled into the lung. Immune function was partially restored in the alveolar spaces of scid mice with evidence of enhanced MHC class II antigen expression and increased phagocytosis (P < 0.05). Tumor necrosis factor ␣ was increased from undetectable at baseline to 103.5 ؎ 11.4 pg/ml. In contrast, i.p. administration of the engineered macrophages did not enhance IFN-␥ levels in the lung. Our study suggests airway delivery of genetically engineered macrophages expressing mIFN-␥ gene can partially restore significant immune activity in the lungs of immunodeficient mice.