Trace levels of peptidoglycan in serum underlie the NOD-dependent cytokine response to endoplasmic reticulum stress.

NOD1 and NOD2 are intracellular sensors of bacterial peptidoglycan that belong to the Nod-like receptor family of innate immune proteins. In addition to their role as direct bacterial sensors, it was proposed that the nucleotide-binding oligomerization domain (NOD) proteins could detect endoplasmic reticulum (ER) stress induced by thapsigargin, an inhibitor of the sarcoplasmic or endoplasmic reticulum calcium ATPase family that pumps Ca2+ into the ER, resulting in pro-inflammatory signaling. Here, we confirm that thapsigargin induces NOD-dependent pro-inflammatory signaling in epithelial cells. However, the effect was specific to thapsigargin, as tunicamycin and the subtilase cytotoxin SubAB from Shiga toxigenic Escherichia coli, which induce ER stress by other mechanisms, did not induce cytokine expression. The calcium ionophore A23187 also induced NOD-dependent signaling, and calcium chelators demonstrated a role for both intracellular and extracellular calcium in mediating thapsigargin-induced and NOD-dependent pro-inflammatory signaling, in part through the activation of plasma membrane-associated calcium release-activated channels. Moreover, our results demonstrate that both endocytosis and the addition of serum to the cell culture medium were required for thapsigargin-mediated NOD activation. Finally, we analyzed cell culture grade fetal calf serum as well as serum from laboratory mice using HPLC and MS identified the presence of various peptidoglycan fragments. We propose that cellular perturbations that affect intracellular Ca2+ can trigger internalization of peptidoglycan trace contaminants found in culture serum, thereby stimulating pro-inflammatory signaling. The presence of peptidoglycan in animal serum suggests that a homeostatic function of NOD signaling may have been previously overlooked.

Increased Endovascular Staphylococcus aureus Inoculum Is the Link Between Elevated Serum Interleukin 10 Concentrations and Mortality in Patients With Bacteremia.

BACKGROUND: Cell wall peptidoglycan stimulates interleukin 10 (IL-10) production in Staphylococcus aureus bacteremia (SaB) animal models, but clinical data are not available. This study evaluates the impact of intravascular bacterial cell numbers (ie, the level of bacteremia), in patients at the time of clinical presentation on IL-10 production and its association with S. aureus bacteremia (SaB) mortality. METHODS: Blood and isolates were collected in 133 consecutive SaB patients. Serum IL-10 was quantified by an electrochemoluminescence assay. Bacterial inoculum was measured in patient sera with elevated (n = 8) or low (n = 8) IL-10 using a magnetic bacterial capture assay. Staphylococcus aureus from these 2 groups were introduced into whole blood ex vivo to determine IL-10 production with variable inocula. RESULTS: IL-10 serum concentration was higher in SaB patient mortality (n = 27) vs survival (n = 106) (median, 36.0 pg/mL vs 10.4 pg/mL, respectively, P < .001). Patients with elevated IL-10 more often had endovascular SaB sources. The inoculum level of SaB was higher in patients with elevated serum IL-10 vs patients with low IL-10 (35.5 vs 0.5 median CFU/mL; P = .044). Ex vivo studies showed that 108 CFU/mL yielded greater IL-10 than did 103 CFU/mL (4.4 ± 1.8 vs 1.0 ± 0.6 pg/mL; P < .01). CONCLUSIONS: Elevated IL-10 serum concentrations at clinical presentation of SaB were highly associated with mortality. High intravascular peptidoglycan concentration, driven by a higher level of bacteremia, is a key mediator of IL-10 anti-inflammatory response that portends poor clinical outcome. Using IL-10 as an initial biomarker, clinicians may consider more aggressive antimicrobials for rapid bacterial load reduction in high-risk SaB patients.

Lysozyme is a component of the innate immune system linked to obesity associated-chronic low-grade inflammation and altered glucose tolerance.

BACKGROUND & AIMS: Several proteins of the innate immune system are known to be deregulated with insulin resistance. We here aimed to investigate the relationship among circulating lysozyme (both plasma concentration and activity) and obesity-associated metabolic disturbances. METHODS: Plasma lysozyme concentration was determined cross-sectionally in a discovery (Cohort 1, n = 137) and in a replication cohort (Cohort 2, n = 181), in which plasma lysozyme activity was also analyzed. Plasma lysozyme was also evaluated longitudinally in participants from the replication cohort (n = 93). Leukocyte lysozyme expression (LYZ mRNA) were also investigated in an independent cohort (Cohort 3, n = 76), and adipose tissue (AT) LYZ mRNA (n = 25) and plasma peptidoglycan levels (n = 61) in subcohorts from discovery cohort. RESULTS: Translocation of peptidoglycan (as inferred from its increased circulating levels) was linked to plasma lysozyme, hyperinsulinemia and dyslipidemia in obese subjects. In both discovery and replication cohorts, plasma lysozyme levels and activity were significantly increased in obesity in direct association with obesity-associated metabolic disturbances and inflammatory parameters, being circulating lysozyme negatively correlated with fasting glucose, HbA1c and insulin resistance (HOMA-IR) in obese subjects. Of note, total cholesterol (p < 0.0001) and LDL cholesterol (p = 0.003) contributed independently to age-, gender- and BMI adjusted plasma lysozyme activity. Longitudinally, changes in HbA1c levels and serum LDL cholesterol were negatively associated with circulating lysozyme antimicrobial activity. On the contrary, the change in glucose infusion rate during the clamp (insulin sensitivity) was positively associated with lysozyme concentration. CONCLUSIONS: Increased plasma lysozyme levels and activity are found in obese subjects. The longitudinal findings suggest that plasma lysozyme might be protective on the development of obesity-associated metabolic disturbances.

Cleavage of proteoglycans, plasma proteins and the platelet-derived growth factor receptor in the hemorrhagic process induced by snake venom metalloproteinases.

Envenoming by viperid snakes results in a complex pattern of tissue damage, including hemorrhage, which in severe cases may lead to permanent sequelae. Snake venom metalloproteinases (SVMPs) are main players in this pathogenesis, acting synergistically upon different mammalian proteomes. Hemorrhagic Factor 3 (HF3), a P-III class SVMP from Bothrops jararaca, induces severe local hemorrhage at pmol doses in a murine model. Our hypothesis is that in a complex scenario of tissue damage, HF3 triggers proteolytic cascades by acting on a partially known substrate repertoire. Here, we focused on the hypothesis that different proteoglycans, plasma proteins, and the platelet derived growth factor receptor (PDGFR) could be involved in the HF3-induced hemorrhagic process. In surface plasmon resonance assays, various proteoglycans were demonstrated to interact with HF3, and their incubation with HF3 showed degradation or limited proteolysis. Likewise, Western blot analysis showed in vivo degradation of biglycan, decorin, glypican, lumican and syndecan in the HF3-induced hemorrhagic process. Moreover, antithrombin III, complement components C3 and C4, factor II and plasminogen were cleaved in vitro by HF3. Notably, HF3 cleaved PDGFR (alpha and beta) and PDGF in vitro, while both receptor forms were detected as cleaved in vivo in the hemorrhagic process induced by HF3. These findings outline the multifactorial character of SVMP-induced tissue damage, including the transient activation of tissue proteinases, and underscore for the first time that endothelial glycocalyx proteoglycans and PDGFR are targets of SVMPs in the disruption of microvasculature integrity and generation of hemorrhage.

Antibody neutralization of microbiota-derived circulating peptidoglycan dampens inflammation and ameliorates autoimmunity.

The human microbiota provides tonic signals that calibrate the host immune response1,2, but their identity is unknown. Bacterial peptidoglycan (PGN) subunits are likely candidates since they are well-known immunity-enhancing adjuvants, released by most bacteria during growth, and have been found in the blood of healthy people3-7. We developed a monoclonal antibody (mAb), 2E7, that targets muramyl-L-alanyl-D-isoglutamine (MDP), a conserved and minimal immunostimulatory structure of PGN. Using 2E7-based assays, we detected PGN ubiquitously in human blood at a broad range of concentrations that is relatively stable in each individual. We also detected PGN in the serum of several warm-blooded animals. However, PGN is barely detectable in the serum of germ-free mice, indicating that its origin is the host microbiota. Neutralization of circulating PGN via intraperitoneal administration of 2E7 suppressed the development of autoimmune arthritis and experimental autoimmune encephalomyelitis in mice. Arthritic NOD2-/- mice lacking the MDP sensor did not respond to 2E7, indicating that 2E7 dampens inflammation by blocking nucleotide-binding oligomerization domain-containing protein 2 (NOD2)-mediated pathways. We propose that circulating PGN acts as a natural immune potentiator that tunes the host immune response; altering its level is a promising therapeutic strategy for immune-mediated diseases.

Detection of bacterial infection by a technetium-99m-labeled peptidoglycan aptamer.

Nuclear medicine clinicians are still waiting for the optimal scintigraphic imaging agents capable of distinguishing between infection and inflammation, and between fungal and bacterial infections. Aptamers have several properties that make them suitable for molecular imaging. In the present study, a peptidoglycan aptamer (Antibac1) was labeled with 99mTc and evaluated by biodistribution studies and scintigraphic imaging in infection-bearing mice. Labeling with 99mTc was performed by the direct method and the complex stability was evaluated in saline, plasma and in the molar excess of cysteine. The biodistribution and scintigraphic imaging studies with the 99mTc-Antibac1 were carried out in two different experimental infection models: Bacterial-infected mice (S. aureus) and fungal-infected mice (C. albicans). A 99mTc radiolabeled library, consisting of oligonucleotides with random sequences, was used as a control for both models. Radiolabeling yields were superior to 90% and 99mTc-Antibac1 was highly stable in presence of saline, plasma, and cysteine up to 6h. Scintigraphic images of S. aureus infected mice at 1.5 and 3.0h after 99mTc-Antibac1 injection showed target to non-target ratios of 4.7±0.9 and 4.6±0.1, respectively. These values were statistically higher than those achieved for the 99mTc-library at the same time frames (1.6±0.4 and 1.7±0.4, respectively). Noteworthy, 99mTc-Antibac1 and 99mTc-library showed similar low target to non-target ratios in the fungal-infected model: 2.0±0.3 and 2.0±0.6for 99mTc-Antibac1 and 2.1±0.3 and 1.9 ± 0.6 for 99mTc-library, at the same times. These findings suggest that the 99mTc-Antibac1 is a feasible imaging probe to identify a bacterial infection focus. In addition, this radiolabeled aptamer seems to be suitable in distinguishing between bacterial and fungal infection.

Silkworm larvae plasma (SLP) assay for detection of bacteria: False positives secondary to inflammation in vivo.

The silkworm larvae plasma (SLP) assay has been developed as a means to detect bacterial peptidoglycan as a surrogate for live bacteria. Here, we present results that indicate that generation of melanin by this assay is not fully reliable as a surrogate marker for bacterial count.

Mediator responses in surgical infections.

BACKGROUND: Surgical infections, and sepsis in particular, are characterized by extensive release of mediators. Our laboratories have been interested in understanding how these substances contribute to morbidity and mortality during various stages of surgical infections in order to develop new and more effective therapeutics and treatment strategies. METHODS: In a series of in vitro studies, human plasma was exposed to lipopolysaccharide (LPS), and whole blood was treated with peptidoglycan from Staphylococcus aureus. The activity of peptidoglycan also was studied in the rat, and LPS infusion was tested in dog and pig models. In a clinical study, the relation of serum LPS to multiple organ dysfunction and failure was studied in patients in the surgical intensive care unit. RESULTS: Exposure of plasma to LPS led to formation of bradykinin, activation of the plasma kallikrein-kinin system, and reduction of kallikrein inhibitor capacity. The coagulation, fibrinolysis, and complement cascades were activated. Peptidoglycan caused rapid release of tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 from macrophages and activation of the genes encoding pro-inflammatory and anti-inflammatory cytokines. In the rat, peptidoglycan induced cytokine release, caused liver and kidney dysfunction, and induced matrix metalloproteinase-9 (MMP-9) activity in the liver and lung. In the dog and pig, LPS caused substantial activation of plasma proteases. Clinically, a finding of LPS in the plasma was associated with multiple organ dysfunction and failure. These patients also revealed substantial activation of the plasma cascade systems, as well as systemic cytokine release. CONCLUSION: On the basis of these observations, we developed a monitoring system to recognize early signs of infection.

Bacterial translocation and plasma cytokines during transcatheter and open-heart aortic valve implantation.

OBJECTIVE: To determine whether the good safety profile of transarterial aortic valve implantation (TAVI) is related to lower levels of systemic bacterial translocation and systemic inflammation compared with open-heart surgery. BACKGROUND: Transcatheter aortic valve implantation via the transfemoral approach is increasingly used in very high-risk patients with aortic stenosis. The outcomes seem similar to those after open-heart aortic valve replacement (OHAVR). METHODS: Each of 26 consecutive high-risk patients (EuroSCORE >20% for risk of operative death) who underwent TAVI (cases) was matched to the first low-risk patient treated next in our department using elective OHAVR without coronary artery bypass (control subjects). We collected severity, outcome, and echocardiography indicators before and after surgery; complications; proinflammatory cytokine levels; and markers for microbial translocation. RESULTS: Despite greater illness severity, the TAVI patients had significantly lower vasopressor agent requirements, lower delirium rates, shorter hospital stays, and better hemodynamic findings compared with OHAVR patients. Vascular complications were more common after TAVI than after OHAVR (12, with seven requiring interventional therapy vs. 0, P = 0.006). Patients who underwent TAVI had lower blood transfusion requirements. Two TAVI patients died: one from iliac artery injury and the other from intracardiac prosthesis migration. Patients who underwent TAVI had lower plasma levels of endotoxin and bacterial peptidoglycan, as well as lower proinflammatory cytokine levels, suggesting less gastrointestinal bacterial translocation compared with OHAVR. CONCLUSIONS: Compared with OHAVR, TAVI was associated with decreases in bacterial translocation and inflammation. These differences may explain the lower delirium rate and better hemodynamic stability observed, despite the greater disease severity in TAVI patients.

Outer membrane protein A (OmpA), peptidoglycan-associated lipoprotein (PAL), and murein lipoprotein (MLP) are released in experimental Gram-negative sepsis.

We previously showed that Escherichia coli bacteria incubated in normal human serum release complexes that contain three conserved Gram-negative bacterial outer membrane proteins (OMPs) and LPS. We have identified the OMPs as outer membrane protein A (OmpA), peptidoglycan-associated lipoprotein (PAL), and murein lipoprotein (MLP). These OMPs are conserved among enteric Gram-negative bacteria and are bound by IgG in antisera raised to heat-killed rough bacteria such as E. coli J5 (J5 IgG). The present experiments were performed to further analyze the release of these OMPs in a rat wound infection model of sepsis. Plasma was collected from thermally injured rats with E. coli O18 sepsis and filtered. LPS was affinity-purified from plasma filtrates using monoclonal antibody specific for the O-polysaccharide side chain of E. coli O18 LPS. Plasma filtrates were also incubated with J5 IgG conjugated to magnetic beads. Affinity-purified samples were analyzed for the OMPs by immunoblotting. OmpA, PAL, and MLP were released into septic rat blood in complexes with LPS. PAL was consistently present in samples affinity-purified using J5 IgG. The results indicate that OmpA, PAL, and MLP are released and circulate in experimental Gram-negative sepsis and suggest that a proportion of released OMPs are tightly associated with LPS.

Detection of peptidoglycan in human plasma using the silkworm larvae plasma test.

Silkworm larvae plasma (SLP) reagent, which is prepared from the body fluid of the silkworm, reacts with peptidoglycan (PG), a fragment of both the Gram-positive and Gram-negative bacterial cell wall, as well as with beta-glucan, a component of fungi. We developed a quantitative method for the detection of PG in human plasma from cases with bacterial infection using the SLP reagent. Tested in this way, the SLP method showed 86.2% sensitivity, 90.6% specificity, 89.3% positive predictive value, and 88.5% efficiency. The SLP method provides a valuable tool for the diagnosis of systemic infection using patients' blood.

Bacterial translocation and peptidoglycan translocation by acute ethanol administration.

BACKGROUND: We examined bacterial translocation (BT) by acute ethanol administration, using a peptidoglycan detecting system. METHODS: Rats were given 20% (v/w) ethanol (10 ml/kg body weight), and heparinized samples of portal blood were collected at specific time points after administration. Plasma peptidoglycan, beta-glucan, and endotoxin concentrations of portal blood were measured. The rats were divided into three groups: a 20% ethanol group (20ET group), a 5% ethanol group (5ET group), and a control group. The groups were given 10 ml/kg body weight of either 20% (v/w) ethanol (20ET group), 5% (v/w) ethanol (5ET group), or distilled water (control group). Femoral arterial blood, portal blood, mesenteric lymph nodes (MLNs), spleen, and liver were cultured to assess BT. Plasma peptidoglycan, beta-glucan, and endotoxin concentrations of femoral arterial blood and portal blood were measured. RESULTS: The plasma peptidoglycan concentration of portal blood was significantly increased 24 h after the administration of 20% ethanol. There was no significant difference in the incidence and magnitude of viable BT to the MLNs, spleen, and liver among any of the groups at this time point. The rate of plasma peptidoglycan positivity and the plasma peptidoglycan concentration were increased significantly in the portal blood of the 20ET group 24 h after administration. CONCLUSIONS: Peptidoglycan was translocated into the portal blood by acute administration of 20% ethanol. Our findings suggest that viable bacterial flora may translocate from the intestine under the influence of ethanol, and BT may be one of the causes of chronic alcoholic liver disease.

Plasma exchange therapy for the treatment of Escherichia coli O-157 associated hemolytic uremic syndrome.

Plasma exchange (PE) therapy was administered to three patients with Escherichia coli O-157 associated hemolytic uremic syndrome (HUS) in early phase. Following several PE treatments, all cases completely recovered without any apparent complications. The usefulness of PE therapy in removing microbial fragments and inflammatory cytokines was evaluated. The peptidoglycan (PG) level, interleukin-1 receptor antagonist (IL-1Ra) were higher in HUS patients starting PE therapy than in patients who had received several sessions of PE therapy. PE therapy was an effective early phase treatment for Escherichia coli O-157 associated HUS.

Bacterial translocation secondary to small intestinal mucosal ischemia during cardiopulmonary bypass. Measurement by diamine oxidase and peptidoglycan.

OBJECTIVES: To demonstrate that small intestinal mucosal ischemia occurs during cardiopulmonary bypass by measuring serum diamine oxidase activity, an index of small intestinal mucosal ischemia, in perioerative patients undergoing cardiovascular surgery with and without cardiopulmonary bypass. METHODS: Twelve successive patients who underwent coronary artery bypass grafting with cardiopulmonary bypass (Group I) were compared to 10 patients who underwent off-pump coronary artery bypass grafting (Group II). Serum diamine oxidase activity, blood lactate concentration, and serum peptidoglycan concentration were measured perioperatively. RESULTS: Serum diamine oxidase activity rose after the start of cardiopulmonary bypass and continued to rise throughout cardiopulmonary bypass in Group I, while activity was unchanged in Group II. The serum lactate concentration mirrored the change in the diamine oxidase activity in both groups. The peptidoglycan concentration in Group I rose after the start of cardiopulmonary bypass and returned to near normal concentrations after surgery. CONCLUSIONS: The parallel rise in diamine oxidase activity and the serum lactate concentration in Group I implies that ischemic injury to the mucosa of the small intestine occurs during cardiopulmonary bypass, and the rise in the serum peptidoglycan concentration indicates that bacteremia did occur. Thus, cardiopulmonary bypass causes hypoperfusion of small intestinal mucosa and consequently bacterial translocation.

Role of inflammatory pathways, blood mononuclear cells, and gut-derived bacterial products in alcohol dependence.

BACKGROUND: Inflammation might play a role in the development of several psychiatric diseases. However, the origins of processes that mediate inflammation are unknown. We previously reported increased intestinal permeability, elevated blood lipopolysaccharide levels, and low-grade systemic inflammation associated with psychological symptoms of alcohol dependence in alcohol-dependent subjects. In this study, we tested inflammatory responses of peripheral blood mononuclear cells (PBMCs) to gut-derived bacterial products during detoxification and the relationship to alcohol craving. METHODS: In 63 actively drinking noncirrhotic alcohol-dependent subjects, testing was performed at the beginning (day 2) and end (day 18) of alcohol detoxification and compared with testing in 14 healthy subjects. Activation of various intracellular signaling pathways by gut-derived bacterial products was analyzed by quantitative polymerase chain reaction, Western blotting, and DNA binding assays (for transcription factors). Toll-like receptor activation was assessed by cell cultures. RESULTS: In addition to lipopolysaccharides, we showed that peptidoglycans may also cross the gut barrier to reach the systemic circulation. Both activate their respective Toll-like receptors in peripheral blood mononuclear cells. Chronic alcohol consumption inhibited the nuclear factor kappa B proinflammatory cytokine pathway but activated the mitogen-activated protein kinase/activator protein 1 pathway, together with the inflammasome complex. This activity resulted in increased messenger RNA and plasma levels of interleukin (IL)-8, IL-1ß, and IL-18. Activated proinflammatory pathways, in particular, IL-8 and IL-1ß, were positively correlated with alcohol consumption and alcohol-craving scores. Short-term alcohol withdrawal was associated with the recovery of lipopolysaccharide-dependent receptors but not peptidoglycan-dependent receptors. CONCLUSIONS: Lipopolysaccharides and peptidoglycans from the gut microbiota stimulate specific inflammatory pathways in peripheral blood mononuclear cells that are correlated with alcohol craving.

Expression of bovine granulocyte chemotactic protein-2 (GCP-2) in neutrophils and a mammary epithelial cell line (MAC-T) in response to various bacterial cell wall components.

CXC chemokines are potential attractants for polymorphonuclear leucocytes (PMNs) and play an important role in resistance to infectious diseases, such as bovine mastitis. In this study, a bovine mammary epithelial cell line (MAC-T) and blood PMNs were stimulated with bacterial cell wall components of gram negative and gram positive bacteria, including lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). The expression of two CXC chemokines, interleukin (IL)-8 and granulocyte chemotactic protein (GCP)-2 was analysed by real-time PCR. High concentrations (1 or 10 µg/mL) of LPS and LTA, but not PGN, significantly increased the expression of GCP-2 and IL-8 in both MAC-T and PMNs. Biopsies from mammary glands of cattle with clinical Escherichia coli mastitis also had increased expression of GCP-2. Using an in vitro transepithelial migration assay, recombinant human GCP-2 (rhGCP-2) showed weak chemoattractant effects on bovine blood PMNs. It was concluded that PMNs and MAC-T cells can express GCP-2 in response to certain bacterial cell components during the course of mastitis.

Thymocytes, pre-B cells, and organ changes in a mouse model of chronic ethanol ingestion--absence of subset-specific glucocorticoid-induced immune cell loss.

BACKGROUND: The well-known immune deficiency of the chronic alcoholic dictates the need for a long-term rodent ethanol administration model to evaluate the baseline immunologic effects of chronic ethanol abuse, and investigate the genetic determinants of those effects. Much published work with rodents has shown clearly that acute ethanol administration and short-term ethanol-containing liquid diets both cause elevated corticosterone and can cause significant thymocyte, pre-B cell and peripheral lymphocyte losses. Such losses may mask more subtle alterations in immune homeostasis, and in any case are generally short-lived compared with the span of chronic ethanol abuse. Thus, it is important to have a model in which long-term immune alterations can be studied free of corticosteroid-induced cell losses. METHODS: We have utilized chronic 20% (w/v) ethanol in water administration to several mouse strains for prolonged periods of time and evaluated serum corticosterone, immunologic stress parameters, and other organ changes by standard methods. RESULTS: We now confirm earlier reports that chronic ethanol in water administration to mice does not produce net elevations of corticosterone, although diurnal variation is altered. Importantly, there is neither selective loss of immune cell populations known to be corticosteroid sensitive, CD4+CD8+ thymocytes and pre-B cells, nor are changes observed in the histologic appearance of the thymus. Nonetheless, there are significant chronic ethanol effects in other tissues, including reduced heart weight, mild hepatic steatosis, alterations of gut flora, increased serum peptidoglycan, and as published elsewhere, immune system abnormalities. CONCLUSIONS: This model of ethanol administration is convenient, sustainable for up to 1 year, demonstrably feasible in several mouse strains, permits good weight gains in most strains, and results in significant changes in a number of organs. The administration method also will permit modeling of long-term steady abuse punctuated by major binges, and is suitable for supplementation studies using water soluble additives. Overall, the method is useful for a wide range of studies requiring a chronic low-stress method of ethanol administration.

The peptidoglycan-degrading property of lysozyme is not required for bactericidal activity in vivo.

Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in pulmonary host defense. Increased concentration of lysozyme in the airspaces of transgenic mice enhanced bacterial killing whereas lysozyme deficiency resulted in increased bacterial burden and morbidity. Lysozyme degrades peptidoglycan in the bacterial cell wall leading to rapid killing of Gram-positive organisms; however, this mechanism cannot account for the protective effect of lysozyme against Gram-negative bacteria. The current study was therefore designed to test the hypothesis that the catalytic activity (muramidase activity) of lysozyme is not required for bacterial killing in vivo. Substitution of serine for aspartic acid at position 53 (D53S) in mouse lysozyme M completely ablated muramidase activity. Muramidase-deficient recombinant lysozyme (LysM(D53S)) killed both Gram-positive and Gram-negative bacteria in vitro. Targeted expression of LysM(D53S) in the respiratory epithelium of wild-type (LysM(+/+)/LysM(D53S)) or lysozyme M(null) mice (LysM(-/-)/LysM(D53S)) resulted in significantly elevated lysozyme protein in the airspaces without any increase in muramidase activity. Intratracheal challenge of transgenic mice with Gram-positive or Gram-negative bacteria resulted in a significant increase in bacterial burden in LysM(-/-) mice that was completely reversed by targeted expression of LysM(D53S). These results indicate that the muramidase activity of lysozyme is not required for bacterial killing in vitro or in vivo.

Diagnostic and predictive value of the silkworm larvae plasma test for postoperative infection following gastrointestinal surgery.

OBJECTIVE: To determine whether the silkworm larvae plasma (SLP) test is a reliable diagnostic marker of infection in patients with infectious complications following gastrointestinal surgery. DESIGN: Prospective study. SETTING: Department of Surgery, University Hospital, Shiga University of Medical Science. PATIENTS: One hundred and twelve adult patients undergoing gastrointestinal surgery INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Thirty-nine of 112 patients developed infectious complications (infected group). Seven patients with sepsis (severely infected group) and 32 patients without sepsis (minor infected group) were observed. The operation time, blood loss, and age were significantly greater in both infected groups than in the noninfected group. The systemic inflammatory response syndrome score on postoperative day (POD) 1 and POD7 was highest in the severely infected group. The increase in C-reactive protein on POD3 and POD7 was significantly higher in both infected groups than in the noninfected group. White blood cell counts on POD7 were elevated significantly higher in the severely infected group than in the other groups. Immediately after surgery, SLP activity significantly increased compared with presurgery in all groups and was significantly higher in the minor and severely infected groups than in the noninfected group. The increased SLP activity returned to preoperative levels in the minor and noninfected groups; however, SLP activity in the severely infected groups remained high throughout the observational period. The most significant factor and time point that predicted infectious complications were the SLP test on POD1; sensitivity 66.7%, specificity 90.4%, positive and negative predictive values 78.8% and 83.5%. The area under the receiver operating characteristic curve for the SLP test was 0.813 +/- 0.046. CONCLUSIONS: The SLP test appears to be a useful marker of diagnosis and prediction of infectious complications following gastrointestinal surgery. Moreover, the SLP test may be able to evaluate not only the existence but also the severity of infection in surgical patients.

Bacterial peptidoglycan-associated lipoprotein: a naturally occurring toll-like receptor 2 agonist that is shed into serum and has synergy with lipopolysaccharide.

Sepsis is initiated by interactions between microbial products and host inflammatory cells. Toll-like receptors (TLRs) are central innate immune mediators of sepsis that recognize different components of microorganisms. Peptidoglycan-associated lipoprotein (PAL) is a ubiquitous gram-negative bacterial outer-membrane protein that is shed by bacteria into the circulation of septic animals. We explored the inflammatory effects of purified PAL and of a naturally occurring form of PAL that is shed into serum. PAL is released into human serum by Escherichia coli bacteria in a form that induces cytokine production by macrophages and is tightly associated with lipopolysaccharide (LPS). PAL activates inflammation through TLR2. PAL and LPS synergistically activate macrophages. These data suggest that PAL may play an important role in the pathogenesis of sepsis and imply that physiologically relevant PAL and LPS are shed into serum and act in concert to initiate inflammation in sepsis.