Ansgar O. Aasen

Involvement of CD14 and Toll-Like Receptors in Activation of Human Monocytes by Aspergillus fumigatus Hyphae

ABSTRACT Invasive fungal infections represent an increasing problem associated with high mortality. The present study was undertaken to identify leukocyte subsets that are activated by hyphal fragments in a whole-human-blood model, as well as to examine the involvement of CD14 and Toll-like receptors (TLRs) in activation of monocytes by hyphae. Incubation of whole human blood with hyphal fragments fromAspergillus fumigatus and Scedosporium prolificans for 6 h caused induction of mRNAs for tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 in T cells, B cells, and monocytes, but not in granulocytes, as analyzed by reverse transcription-PCR with mRNA isolated from very pure populations of these leukocyte subsets. In primary adherent human monocytes, induction of TNF-α by hyphal fragments was dependent on plasma. Heat treatment of plasma at 56°C for 30 min strongly reduced the ability of plasma to prime for activation. Pretreatment of human monocytes with different concentrations (1, 3, and 10 μg/ml) of monoclonal antibody (MAb) HTA125 (anti-TLR4) or MAb 18D11 (anti-CD14) for 30 min inhibited the release of TNF-α induced by hyphal fragments in a dose-dependent manner. Maximal inhibitions of 35 and 70% were obtained with 10 μg of HTA125 and 18D11 per ml, respectively. In contrast, pretreatment with MAb TL2.1 (anti-TLR2) did not affect signaling induced by hyphae. Pretreatment with the lipid A antagonist B975 blocked lipopolysaccharide signaling but did not inhibit TNF-α production induced by hyphal fragments. Our results suggest that T cells, B cells, and monocytes are involved in the innate immune response to invasive fungal pathogens and that serum components are relevant for activation of monocytes by hyphae. CD14 and TLR4 may be involved in signaling of Aspergillus hyphae in monocytes, but further studies to elucidate this issue are warranted.

Peptidoglycan and Lipoteichoic Acid from Staphylococcus aureus Induce Tumor Necrosis Factor Alpha, Interleukin 6 (IL-6), and IL-10 Production in Both T Cells and Monocytes in a Human Whole Blood Model

ABSTRACT We have examined the ability of peptidoglycan (PepG) and lipoteichoic acid (LTA) isolated from Staphylococcus aureusto induce the release of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-10 in whole human blood and identified the cellular origins of these cytokines. Both PepG and LTA induced transient increases in TNF-α and IL-10 in plasma, with peak values at 6 and 12 h, respectively. IL-6 values increased throughout the experimental period (24 h). The TNF-α, IL-6, and IL-10 release induced by PepG and LTA was dose dependent. Only PepG was a potent inducer of TNF-α secretion. After stimulation of whole blood with PepG or LTA, very pure populations of monocytes (CD14 positive), T cells (CD2 positive), B cells (CD19 positive), and granulocytes (CD15 positive) were isolated by immunomagnetic separation and analyzed by reverse transcription-PCR for mRNA transcripts encoding TNF-α, IL-6, and IL-10. The TNF-α mRNA results were inconclusive. In contrast, PepG induced IL-6 and IL-10 mRNA accumulation in both T cells and monocytes. LTA, as well as lipopolysaccharide, induced IL-6 and IL-10 mRNA production in monocytes and possibly in T cells. Whether granulocytes and B cells produce cytokines in response to bacterial stimuli remains obscure. Blockade of the CD14 receptors with monoclonal antibodies (18D11) had no influence on the PepG-induced release of TNF-α but attenuated the LTA-induced release of the same cytokine. In conclusion, our data indicate that circulating T cells and monocytes contribute to cytokine production in sepsis caused by gram-positive bacteria.

Peptidoglycan and lipoteichoic acid in gram-positive bacterial sepsis: receptors, signal transduction, biological effects, and synergism.

In sepsis and multiple organ dysfunction syndrome (MODS) caused by gram-negative bacteria, lipopolysaccharide (LPS) initiates the early signaling events leading to the deleterious inflammatory response. However, it has become clear that LPS can not reproduce all of the clinical features of sepsis, which emphasize the roles of other contributing factors. Gram-positive bacteria, which lack LPS, are today responsible for a substantial part of the incidents of sepsis with MODS. The major wall components of gram-positive bacteria, peptidoglycan and lipoteichoic acid, are thought to contribute to the development of sepsis and MODS. In this review, the literature underlying our current understanding of how peptidoglycan and lipoteichoic acid activate inflammatory responses will be presented, with a focus on recent advances in this field.

ABANDON THE MOUSE RESEARCH SHIP? NOT JUST YET!

ABSTRACT Many preclinical studies in critical care medicine and related disciplines rely on hypothesis-driven research in mice. The underlying premise posits that mice sufficiently emulate numerous pathophysiologic alterations produced by trauma/sepsis and can serve as an experimental platform for answering clinically relevant questions. Recently, the lay press severely criticized the translational relevance of mouse models in critical care medicine. A series of provocative editorials were elicited by a highly publicized research report in the Proceedings of the National Academy of Sciences (PNAS; February 2013), which identified an unrecognized gene expression profile mismatch between human and murine leukocytes following burn/trauma/endotoxemia. Based on their data, the authors concluded that mouse models of trauma/inflammation are unsuitable for studying corresponding human conditions. We believe this conclusion was not justified. In conjunction with resulting negative commentary in the popular press, it can seriously jeopardize future basic research in critical care medicine. We will address some limitations of that PNAS report to provide a framework for discussing its conclusions and attempt to present a balanced summary of strengths/weaknesses of use of mouse models. While many investigators agree that animal research is a central component for improved patient outcomes, it is important to acknowledge known limitations in clinical translation from mouse to man. The scientific community is responsible to discuss valid limitations without overinterpretation. Hopefully, a balanced view of the strengths/weaknesses of using animals for trauma/endotoxemia/critical care research will not result in hasty discount of the clear need for using animals to advance treatment of critically ill patients.

Human monocytes stimulation by particles of hydroxyapatite, silicon carbide and diamond: in vitro studies of new prosthesis coatings

Aseptic loosening due to wear and debris formation constitutes the major problem in longevity of joint replacements. Diamond coated onto the prosthesis surface may reduce wear, owing to its excellent tribological properties. A thin diamond coating may be brittle, and we plan eventually to reinforce it with silicon carbide whiskers (SiC). In the present study we compared particles of diamond, SiC and hydroxyapatite (HA) in serum-free cultures of human monocytes. All particles were found to be phagocytozed, and monocyte morphology changed except after the ingestion of diamond. Interleukin-1 beta production was increased on average 30-fold and 38-fold in cultures exposed to HA and SiC, respectively, compared to control and diamond cultures (n = 6). Addition of the phagocytosis inhibitor cytochalasin B inhibited the morphological changes of the monocytes and reduced interleukin-1 beta production. In some experiments particles of polymethylmethacrylate were also included, and the interleukin-1 beta stimulation was in the same range as after HA and SiC stimulation. The results show that diamond particles in serum-free monocyte culture are inert, while SiC and HA have a stimulatory effect comparable to polymethylmethacrylate. With its excellent tribological and biocompatible properties, future studies with diamond coating are warranted.

Heparin-coated cardiopulmonary bypass equipment. II. Mechanisms for reduced complement activation in vivo

OBJECTIVE Our objective was to study mechanisms for reduced complement activation by heparin coating of cardiopulmonary bypass equipment in clinical heart surgery. METHODS Adults undergoing elective coronary artery bypass grafting were randomized to cardiopulmonary bypass with Duraflo II heparin-coated (n = 15) or uncoated (n = 14) sets (Duraflo coating surface; Baxter International, Inc, Deerfield, Ill). Blood samples were analyzed with the use of enzyme immunoassays for C1rs-C1 inhibitor complexes and the activation products Bb, C4bc, C3bc, C5a-desArg, and the terminal complement complex. Data were compared by repeated-measures analysis of variance. RESULTS C1 was activated during bypass, and increases in C1rs-C1 inhibitor complexes were larger with heparin coating (P =.03). C4bc increased after administration of protamine, without intergroup differences (P =.69). Bb (P =.22) and C5a-desArg (P =.13) tended to increase less with heparin coating. Formation of C3bc (P =.03) and the terminal complement complex (P <.01) was significantly reduced with heparin coating. C5a-desArg increased 2-fold during bypass, whereas the terminal complement complex increased 10- to 20-fold. Maximal terminal complement complex concentrations were significantly correlated to maximal Bb and C3bc (R = 0.6, P <.001), but not to C1rs-C1 inhibitor complexes or C4bc (R < 0.05, P >.8). CONCLUSIONS C1 activation during bypass was increased by heparin coating, but further classical pathway activation was held in check until administration of protamine. Heparin coating significantly inhibited C3bc and terminal complement complex formation. Terminal complement complex concentrations were related to alternative pathway activation and may be useful for evaluation of differences in bypass circuitry. Increases and intergroup differences in terminal complement complex concentrations were much larger than those in C5a-desArg.

Peptidoglycan primes for LPS-induced release of proinflammatory cytokines in whole human blood

The pathophysiological mechanisms involved in mixed bacterial infections caused by gram-positive and gram-negative bacteria are largely unknown. The present study examines the potential interaction between lipopolysaccharide (LPS) and peptidoglycan (PepG) in the induction of the sepsis-associated cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-10 in whole human blood. Plasma values of these cytokines were measured by enzyme immunoassays and a TNF bioassay. Co-administration of PepG (10 microg/mL) or muramyl dipeptide (MDP, 1 microg/mL) with LPS (10 ng/mL) caused significantly elevated values of TNF-alpha and IL-6 in the blood that could not be obtained by the sum of the values obtained by each stimulant alone, or by 3-fold higher doses of either bacterial component alone. This phenomenon was observed 1 h after stimulation, throughout the experimental period (24 h), and with different doses of LPS and PepG. In contrast, the release of IL-10 was not influenced by the co-administration of PepG or MDP with LPS. The TNF-alpha release induced by co-administration of LPS and PepG was abrogated after pretreatment with a monoclonal antibody against CD14 (18D11). Addition of PepG or MDP to whole blood caused a 2-fold increase in the surface expression of CD14 on monocytes, as measured by flow cytometry. In contrast, LPS caused decreased expression of this receptor. Our data suggest that PepG and MDP primes human whole blood leukocytes for LPS-induced release of proinflammatory cytokines. We speculate that synergy between PepG and LPS may contribute to the pathogenesis in sepsis caused by mixed bacterial infections.

The importance of anal endosonography in the evaluation of idiopathic fecal incontinence

PURPOSE: The aim of the study was to evaluate the use of anal endosonography in idiopathic incontinence. METHODS: In 29 patients and 26 normal controls, the relationship between sonography images and physiologic parameters was studied. RESULTS: External anal sphincter function, measured as fiber density by single-fiber electromyography (P=0.0001) and pudendal nerve terminal motor latency (P=0.04), was significantly impaired in patients with idiopathic incontinence compared with controls. Both the external and internal anal sphincter could be identified by anal endosonography, and the thickness directly measured. The thickness of the external anal sphincter was significantly negatively correlated to muscle fiber density (r=−0.65,P=0.0002) and to pudendal nerve distal conduction velocity (r=−0.74,P=0.008). The thickness of the internal anal sphincter was significantly correlated to resting pressure (r=−0.67,P=0.0001). CONCLUSION: The ratio between the thickness of the external and internal sphincter muscles measured on the sonography screen was significantly reduced in patients with neurogenic incontinence compared with controls (P<0.01).

ACTIVATION OF THE LIVER X RECEPTOR PROTECTS AGAINST HEPATIC INJURY IN ENDOTOXEMIA BY SUPPRESSING KUPFFER CELL ACTIVATION

ABSTRACT Recent reports have demonstrated that liver X receptors (LXRs) of the nuclear receptor family have anti-inflammatory effects on macrophages. Here we examine whether activation of LXR by the synthetic agonist GW3965 can ameliorate the liver injury/dysfunction caused by endotoxins in the rat. Male Wistar rats received GW3965 (0.3 mg/kg) or vehicle (50% dimethyl sulfoxide) 30 min before coadministration of lipopolysaccharide (LPS, 5 mg/kg i.v.) and peptidoglycan (1 mg/kg i.v.). Treatment with GW3965 attenuated the increase in the plasma levels of alanine aminotransferase and bilirubin (markers of liver injury/dysfunction) as well as the focal hepatocyte necrosis (histology) caused by coadministration of LPS and peptidoglycan. This protective effect of GW3965 treatment was associated with reduced infiltration of mast cells in the liver (histopathology) and reduced gene expression of the chemokines eotaxins 1 and 2, whereas MIP-2 mRNA levels were not affected. Plasma levels of tumor necrosis factor &agr; and prostaglandin E2 were significantly attenuated by GW3965, whereas plasma interleukins 6 and 10 were not altered. High expression of LXR&agr; mRNA was observed in Kupffer cell cultures, suggesting that Kupffer cells are targets of GW3965. Subsequent in vitro studies in Kupffer cells demonstrated that exposure to GW3965 attenuated the LPS-induced release of tumor necrosis factor &agr; and prostaglandin E2 in a dose-dependent manner. In conclusion, this study demonstrates that activation of LXR by GW3965 protects against liver injury and dysfunction in a rat model of endotoxemia, in part by exerting an anti-inflammatory effect on Kupffer cells.

Peptidoglycan of Staphylococcus aureus causes inflammation and organ injury in the rat.

ObjectivePrevious studies have implicated a role of peptidoglycan in the pathophysiology of organ injury in sepsis. However, the systemic response to, and organ injury caused by, peptidoglycan have been scarcely studied in vivo. DesignProspective, randomized study. SettingUniversity-based research laboratory. SubjectsFifty-seven anesthetized, male Wistar rats. InterventionsAfter surgical preparation, anaesthetized rats were administered 3 mg/kg Staphylococcus aureus peptidoglycan (n = 9), 10 mg/kg S. aureus peptidoglycan (n = 14), or an equal volume of saline (sham, n = 12) in the jugular vein over a 10-min period. Measurements and Main ResultsInjection of low-dose peptidoglycan (3 mg/kg) had no measurable effects on the rats. In contrast, high-dose peptidoglycan (10 mg/kg) caused increased serum values of aspartate aminotransferase (p ≤ .005), alanine aminotransferase (p ≤ .001), &ggr;-glutamyltransferase, and bilirubin (p ≤ .05) (indicators of liver injury/dysfunction) as well as a moderate, but significant, increase in serum creatinine and urea (p ≤ .05) (indicators of renal dysfunction). Plasma analyses showed a substantial increase in plasma values of tumor necrosis factor-&agr;, interleukin-6, and interleukin-10 (p ≤ .05 for all vs. sham) at 1 and 3 hrs (enzyme-linked immunosorbent assay). This was accompanied by accumulation of messenger RNAs for tumor necrosis factor-&agr;, interleukin-6, and interleukin-10 in both the liver and the lung (p ≤ .05 for all cytokines vs. sham) (real-time polymerase chain reaction). Peptidoglycan also caused increased DNA binding of nuclear factor-&kgr;B (band-shift assays) and phosphorylation of c-Jun and Jun N-terminal kinase (Western blots). In the kidney, interleukin-6 messenger RNA was increased, whereas Toll-like receptor 4 messenger RNA was significantly decreased. ConclusionsThese results demonstrate that injection of peptidoglycan alone causes organ injury/dysfunction, organ inflammation, and systemic inflammation in the rat, involving nuclear factor-&kgr;B and possibly activator protein 1. These data support the contention that peptidoglycan is a contributing factor in the pathophysiology of organ injury in sepsis.