Biochemical and functional characterization of charge-defined subfractions of high-density lipoprotein from normal adults.

High-density lipoprotein (HDL) is regarded as atheroprotective because it provides antioxidant and anti-inflammatory benefits and plays an important role in reverse cholesterol transport. In this paper, we outline a novel methodology for studying the heterogeneity of HDL. Using anion-exchange chromatography, we separated HDL from 6 healthy individuals into five subfractions (H1 through H5) with increasing charge and evaluated the composition and biologic activities of each subfraction.

Porphyromonas gingivalis mediates inflammasome repression in polymicrobial cultures through a novel mechanism involving reduced endocytosis.

The interleukin (IL)-1β-processing inflammasome has recently been identified as a target for pathogenic evasion of the inflammatory response by a number of bacteria and viruses. We postulated that the periodontal pathogen, Porphyromonas gingivalis may suppress the inflammasome as a mechanism for its low immunogenicity and pathogenic synergy with other, more highly immunogenic periodontal bacteria. Our results show that P.

NLRP1-dependent pyroptosis leads to acute lung injury and morbidity in mice.

Acute inflammation in response to both exogenous and endogenous danger signals can lead to the assembly of cytoplasmic inflammasomes that stimulate the activation of caspase-1. Subsequently, caspase-1 facilitates the maturation and release of cytokines and also, under some circumstances, the induction of cell death by pyroptosis. Using a mouse line lacking expression of NLRP1, we show that assembly of this inflammasome in cells is triggered by a toxin from anthrax and that it initiates caspase-1 activation and release of IL-1β.

The NLR adaptor ASC/PYCARD regulates DUSP10, mitogen-activated protein kinase (MAPK), and chemokine induction independent of the inflammasome.

ASC/PYCARD is a common adaptor for a diverse set of inflammasomes that activate caspase-1, most prominently the NLR-based inflammasome. Mounting evidence indicates that ASC and these NLRs also elicit non-overlapping functions, but the molecular basis for this difference is unclear. To address this, we performed microarray and network analysis of ASC shRNA knockdown cells.

Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling.

High-fat diet (HFD) and inflammation are key contributors to insulin resistance and type 2 diabetes (T2D). Interleukin (IL)-1β plays a role in insulin resistance, yet how IL-1β is induced by the fatty acids in an HFD, and how this alters insulin signaling, is unclear. We show that the saturated fatty acid palmitate, but not unsaturated oleate, induces the activation of the NLRP3-ASC inflammasome, causing caspase-1, IL-1β and IL-18 production.

Cutting edge: NLRC5-dependent activation of the inflammasome.

The nucleotide-binding domain leucine-rich repeat-containing proteins, NLRs, are intracellular sensors of pathogen-associated molecular patterns and damage-associated molecular patterns. A subgroup of NLRs can form inflammasome complexes, which facilitate the maturation of procaspase 1 to caspase 1, leading to IL-1β and IL-18 cleavage and secretion. NLRC5 is predominantly expressed in hematopoietic cells and has not been studied for inflammasome function.

Deletion of ripA alleviates suppression of the inflammasome and MAPK by Francisella tularensis.

Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Evasion of the immune response contributes to the extraordinary virulence of this organism although the mechanism is unclear. Whereas wild-type strains induced low levels of cytokines, an F.

Effects of the putative transcriptional regulator IclR on Francisella tularensis pathogenesis.

Francisella tularensis is a highly virulent Gram-negative bacterium and is the etiological agent of the disease tularemia. IclR, a presumed transcriptional regulator, is required for full virulence of the animal pathogen, F. tularensis subspecies novicida U112 (53).

Inflammasome inhibition as a pathogenic stealth mechanism.

The activation of inflammasomes containing NBD-LRR (NLRs) or non-NLRs is critical for effective host defense against microbial pathogens. Recent discoveries have uncovered a plethora of pathogenic strategies to inhibit inflammasome-mediated processing of IL-1beta and IL-18. We review recent evidence for viral and bacterial manipulation of the inflammasome, ranging from perturbation of caspase-1 activation to targeting of specific inflammasome components.

Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown.

Shortly after the cellular mechanism of RNA interference (RNAi) was first described, scientists began using this powerful technique to study gene function. This included designing better methods for the successful delivery of small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) into mammalian cells. While the simplest method for RNAi is the cytosolic delivery of siRNA oligonucleotides, this technique is limited to cells capable of transfection and is primarily utilized during transient in vitro studies.

NLRP3 (NALP3, Cryopyrin) facilitates in vivo caspase-1 activation, necrosis, and HMGB1 release via inflammasome-dependent and -independent pathways.

Bacterial infection elicits a range of beneficial as well as detrimental host inflammatory responses. Key among these responses are macrophage/monocyte necrosis, release of the proinflammatory factor high-mobility group box 1 protein (HMGB1), and induction of the cytokine IL-1. Although the control of IL-1beta has been well studied, processes that control macrophage cell death and HMGB1 release in animals are poorly understood.

Neisseria gonorrhoeae activates the proteinase cathepsin B to mediate the signaling activities of the NLRP3 and ASC-containing inflammasome.

Neisseria gonorrhoeae is a common sexually transmitted pathogen that significantly impacts female fertility, neonatal health, and transmission of HIV worldwide. N. gonorrhoeae usually causes localized inflammation of the urethra and cervix by inducing production of IL-1beta and other inflammatory cytokines.

Critical role of apoptotic speck protein containing a caspase recruitment domain (ASC) and NLRP3 in causing necrosis and ASC speck formation induced by Porphyromonas gingivalis in human cells.

Periodontal disease is a chronic inflammatory disorder that leads to the destruction of tooth-supporting tissue and affects 10-20 million people in the U.S. alone.

Pro-apoptotic low-density lipoprotein subfractions in type II diabetes.

Objective: To test the hypothesis that differences in subfractions of circulating lipoproteins between diabetic and non-diabetic subjects exist and might contribute to the increased risk for atherosclerosis in type II diabetics.

Methods And Results: LDL isolated from diabetic (D) and control subjects (N) were separated by FPLC into five subfractions (L1-L5). The fractional distributions of N- and D-LDL were not different, but the most strongly retained subfractions of D-LDL (D-L5) were markedly more pro-apoptotic to bovine aortic endothelial cells in vitro than were the other subfractions in D- or N-LDL.

Modulation of angiogenic processes in cultured endothelial cells by low density lipoproteins subfractions from patients with familial hypercholesterolemia.

Objective: Electronegative low density lipoprotein (LDL) subfractions are cytotoxic to endothelial cells. To continue our study of homozygotic familial hypercholesterolemic (FH)-LDL, we report the effects of FH-LDL subfractions (FH-L1 to FH-L5) on the angiogenic processes in cultured endothelial cells.

Methods And Results: Subconfluent bovine aortic endothelial cells (BAEC) were treated with LDL subfractions (20 microg/ml), and the effects on angiogenic functions, including cell proliferation, migration, apoptosis, tube formation, secretion of matrix metalloproteinases (MMPs), and vascular endothelial growth factor (VEGF) were determined.