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Sepsis, among other pathologies, is an endotoxin tolerance (ET)-related disease. On admission, we classified 48 patients with sepsis into 3 subgroups according to the ex vivo response to lipopolysaccharide. This response correlates with the Acute Physiology and Chronic Health Evaluation (APACHE) II score and the ET degree. Moreover, the ET-related classification determines the outcome of these patients. Programmed cell death-ligand 1 (PD-L1) expression on septic monocytes is also linked with ET status. In addition to the regulation of cytokine production, one of the hallmarks of ET that significantly affects patients with sepsis is T-cell proliferation impairment or a poor switch to the adaptive response. PD-L1/programmed cell death-1 (PD-1) blocking and knockdown assays on tolerant monocytes from both patients with sepsis and the in vitro model reverted the impaired adaptive response. Mechanistically, the transcription factor hypoxia-inducible factor-1α (HIF1α) has been translocated into the nucleus and drives PD-L1 expression during ET in human monocytes. This fact, together with patient classification according to the ex vivo lipopolysaccharide response, opens an interesting field of study and potential personalized clinical applications, not only for sepsis but also for all ET-associated pathologies., (© The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Myeloid differentiation 2 (MD-2) recognizes endotoxin lipopolysaccharide (LPS), which is required for Toll-like receptor 4 (TLR4) activity. MD-2 represents a more attractive therapeutic target than TLR4 for intervention in severe inflammatory disorders due to microbial infection. Here, we suggest MD-2 as a molecular target of nonlipid chalcone in the inhibition of LPS-induced cellular inflammation. A chalcone derivative, 2',4-dihydroxy-6'-isopentyloxychalcone (JSH) competitively displaced LPS from MD-2, and was fitted into the ligand-binding site on the crystal structure of MD-2 under the most energetically favorable simulation. JSH nullified TLR4 activation mechanism and sequentially inhibited nuclear factor-κB (NF-κB) activation that involves the phosphorylation and degradation of inhibitory κBs and the nuclear import and transcriptional activity of NF-κB in LPS-activated macrophages. Moreover, JSH suppressed NF-κB-target inflammatory genes such as inducible nitric oxide synthase, cyclooxygenase-2, interleukin-1β (IL-1β) and IL-6. Taken together, this study assigns the chalcone structure as an LPS antagonist binding to MD-2 with therapeutic potential against inflammatory conditions.

Background: An experimental vaccine for sepsis, composed of detoxified Escherichia coli J5 lipopolysaccharide (LPS) complexed with the outer membrane protein (OMP) of Neisseria meningitidis group B, induces anti-core glycolipid antibody and has been tested in pilot studies in human volunteers., Methods: Mice were immunized with the LPS-J5/OMP vaccine with or without synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs as a vaccine adjuvant (CpG ODN). The efficacy of the vaccine-induced antibody response was tested in a cecal ligation and puncture model., Results: Immunization resulted in a >20-fold increase in anti-core glycolipid antibody levels, which were further increased 5-fold by the addition of CpG ODN, compared with the levels in mice in the control group. The vaccine provided a survival advantage after a cecal ligation and puncture was performed (P < .01) and significantly decreased the levels of bacteria in organs. Immunoglobulin G (IgG) anti-core glycolipid antibodies were decreased in mice to a significantly greater extent than were levels of total circulating IgG or IgG to the OMP part of the vaccine complex, suggesting specific epitope binding and clearance., Conclusions: These results indicate that the detoxified LPS-J5/OMP vaccine induces high levels of antibody against the core glycolipid of LPS and functions in vivo to promote clearance of gram-negative bacteria and improve the outcome of experimental polymicrobial intra-abdominal sepsis.

Nuclear factor (NF)-kappaB is involved in regulating the transcription of many of the immunomodulatory mediators involved in the development of sepsis-induced organ failure. Kinase pathways involving p38 and Akt and initiated by engagement of Toll-like receptors modulate transcriptional activity of NF-kappaB, but apparently through different mechanisms. Increased activation of NF-kappaB occurs with sepsis, and greater levels of nuclear accumulation of NF-kappaB are associated with higher rates of mortality and worse clinical outcome. The percentage of apoptotic neutrophils is reduced in sepsis, and inhibition of nuclear translocation of NF-kappaB restores neutrophil apoptosis to baseline levels. In models of sepsis, suppression of NF-kappaB activation decreases acute inflammatory processes and organ dysfunction. Because NF-kappaB occupies a central role in signaling pathways important in sepsis, modulation of NF-kappaB activity may be an appropriate therapeutic target in patients with sepsis.

Anticoagulants have gained increasing attention in the treatment of sepsis. This study used danaparoid to investigate the role of factor Xa in endotoxin-induced coagulation and inflammation and its effectiveness when coagulation activation has already occurred. Thirty healthy volunteers were enrolled in the randomized, placebo-controlled trial. Subjects received 2 ng/kg endotoxin and danaparoid 10 min or 3 h thereafter or placebo. Endotoxin increased prothrombin fragment 1+2 (F(1+2)) levels from 0.5 to 7.0 nmol/L at 5 h in the placebo group. Early danaparoid infusion inhibited endotoxin-induced thrombin formation: maximum F(1+2) levels reached only 1.8 nmol/L (P<.01, vs. baseline or placebo). Delayed danaparoid infusion effectively blocked further thrombin formation. However, danaparoid did not alter endotoxin-induced changes in the fibrinolytic system, cytokine levels, activation of leukocytes, or tissue factor expression on monocytes. Danaparoid therefore selectively attenuates endotoxin-induced coagulopathy, even with delayed administration when coagulation activation is well under way.

Inflammatory responses to lipo-oligosaccharide (LOS) contribute to the severity of meningococcal disease. Strains that express the L(3,7,9) LOS immunotypes are isolated from the majority of patients, but other immunotypes are isolated predominantly from carriers. Inflammatory responses elicited from a human monocytic cell line (THP-1) that had been pretreated with vitamin D3 (VD3) were compared after stimulation with purified LOSs from standard immunotype strains. The neutralizing effects of normal human serum and serum from mice immunized with strain B:2a:P1.5,2:L3 were compared. LOSs of immunotypes L3, L7, L8, and L9 induced significantly higher levels of tumor necrosis factor-alpha and interleukin-6, compared with other immunotypes. Normal human serum neutralized the proinflammatory responses to LOSs of all immunotypes tested. Immune mouse serum neutralized inflammatory responses against LOSs from immunotypes with epitopes cross-reactive with L(3,7,9) moieties. Antibodies found in normal human serum and immune mouse serum to the oligosaccharide, core, and lipid A moieties of meningococcal endotoxin contribute to neutralizing activity.

The protective effect of volume support, imipenem, and the anti-endotoxin antibody E5 given as mono- or combination therapy was studied in fulminant intraabdominal sepsis in rats. Mortality, blood levels of bacteria, endotoxin, tumor necrosis factor (TNF), and lactate, and packed cell volume were measured. All monotherapy regimens improved survival, and protection decreased with delayed intervention. Volume support prevented dehydration and lactate accumulation but had no effect on levels of bacteria, endotoxin, or TNF. Imipenem killed bacteria and released endotoxin, and lactacidosis was reduced. E5 reduced endotoxin, TNF, and lactate but not bacteremia. The imipenem-induced increase in plasma endotoxin was abolished by E5. When E5 was added to a regimen of volume support plus imipenem 6 h after induction, it gave an extra improvement of survival, but this synergism disappeared when intervention was delayed 4 h more. The primary effect of E5 was a reduction in plasma endotoxin level.

A recombinant endotoxin-neutralizing protein (ENP) from Limulus polyphemus and a monoclonal IgM anti-lipid A antibody (HA-1A) were compared in a rat model of Escherichia coli sepsis. One hour after intraperitoneal challenge with 10(6) cfu of E. coli O18ac K1, animals were sensitized to endotoxin with lead acetate and treated with ENP, HA-1A, or saline, followed by ceftriaxone and gentamicin. Before treatment, 95% of rats had high-grade bacteremia and high serum endotoxin concentrations, which were similar in all treatment groups (P > .60). One hour after treatment, there was no bacterial growth in any blood sample, and endotoxin concentrations were significantly lower in the ENP group than in the HA-1A and saline groups (P < .01). At 24 h after challenge, survival in the ENP group was significantly higher than in the HA-1A saline group (P < .001). ENP improved survival in a rat model of E. coli sepsis with high mortality despite effective antibiotic therapy.

Monoclonal antibodies (MAbs) specific for O polysaccharide or core oligosaccharide/lipid A of Escherichia coli O111:B4 lipopolysaccharide (LPS) were compared in canine septic shock. Animals received O-specific, core-specific, or control murine IgG2a MAbs (or saline) before intraperitoneal implantation of an E. coli O111:B4-infected clot. Animals were further randomized to ceftriaxone or saline. O-specific MAb significantly reduced bacteremia and endotoxemia but not serum tumor necrosis factor. Core-specific MAb significantly increased mean arterial pressure from day 4 to 28 (P = .02). In dogs not receiving ceftriaxone, survival was enhanced by O-specific MAb (4/5) compared with core-specific MAb (0/5) and control (1/8) (P = .03). Survival rates were similar (P = .22) but survival was prolonged in antibiotic-treated animals also receiving O-specific MAb (P = .02 vs. core-specific MAb and controls) or core-specific MAb (P = .08 vs. controls). These data support the complex role of LPS in sepsis and the discrete functional effects of MAbs specific for different elements of LPS.

Pathogen-free rats were rendered neutropenic, given oral feedings of Pseudomonas aeruginosa 12.4.4, then monitored for fever. At the onset of fever, rats were given intravenous treatment with either anti-endotoxin monoclonal antibody (MAb) E5 or control MAb B55. Survival was significantly greater in E5- than in B55-treated animals (P < .01). Serum levels of both lipopolysaccharide and tumor necrosis factor-alpha were significantly reduced in E5- versus B55-treated rats 24 h after treatment (P < .01 and < .05, respectively). Rats were also treated with E5 or B55 in combination with a suboptimal dose of ciprofloxacin at fever onset and again 24 h later. Survival was significantly greater in ciprofloxacin-treated animals given E5 than in animals given B55 (P < .005). Posttreatment endotoxin levels were decreased in animals receiving E5 in combination with ciprofloxacin (P < .001) compared with B55-treated animals. These results indicate that therapy with anti-endotoxin MAb E5 alone or in combination with antimicrobial therapy improves survival in this bacteremic infection model of Pseudomonas sepsis.

Gram-negative sepsis is caused by endotoxin-induced release of tumor necrosis factor (TNF) and other cytokines. HA-1A is a human monoclonal antibody that binds specifically to endotoxin. HA-1A should prevent death in endotoxemic patients and reduce serum levels of TNF and interleukin-6 (IL-6). This hypothesis was tested in 82 septic patients who were randomly allocated to receive a single intravenous 100-mg dose of HA-1A or placebo. Pretreatment endotoxemia was detected in 27 patients (33%). Death occurred within 28 days of treatment in 8 (73%) of 11 placebo recipients and in 5 (31%) of 16 HA-1A recipients (P = .02). The median decrease in serum TNF level 24 h after treatment was 12 ng/L in patients given HA-1A and 0 ng/L in placebo recipients (n = 65; P = .04). For IL-6, this was 204 ng/L in patients given HA-1A and 44 ng/L in placebo recipients (n = 67; P = .4). Thus, HA-1A reduces mortality in septic patients with endotoxemia and lowers serum TNF levels.

Limulus antilipopolysaccharide factor (LALF), an 11.8-kDa peptide isolated from amebocytes of Limulus polyphemus, neutralizes meningococcal lipooligosaccharide (LOS)-induced gelation of limulus amebocyte lysate. Rabbits challenged with an LD90 of LOS (10 micrograms/kg) premixed with LALF in vitro (n = 10) had significantly higher mean arterial pressure, arterial pH, serum bicarbonate concentrations, and survival (90% vs. 8%, P = .005) than did rabbits challenged with LOS alone. Relative to untreated controls, rabbits pretreated with LALF intravenously (iv) at 1.2 mg/kg (n = 21) also had significant improvements in physiologic measurements and higher survival (52% vs. 8%, P = .003). Even when LALF (1.2 mg/kg iv) was given 1/2 h after LOS challenge, animals showed significant improvements in physiologic measurements and survival (33% vs. 8% in untreated controls P = .028). LALF-treated animals also had significantly lower circulating endotoxin activity and tumor necrosis factor concentrations. Thus, LALF attenuates the toxic effects of meningococcal LOS in rabbits even when administered after LOS challenge and deserves further evaluation as a potential therapeutic agent for treating gram-negative septic shock.

In vitro and in vivo antibacterial and protective properties of murine monoclonal antibodies (MAbs) to Escherichia coli O111:B4 lipopolysaccharide (LPS) were evaluated in relation to antibody isotype and complement-fixing activity. Six O side chain-specific MAbs, including two IgMs and one of each IgG subclass, were analyzed for quantitative binding and C3 deposition on intact bacteria, complement-mediated bactericidal and opsonophagocytic activity, and protection against intraperitoneal infections in mice. Although C3 was deposited on bacteria in the presence of normal human serum (NHS) alone, LPS-specific MAbs increased C3 attachment in a dose-dependent manner. Bacterial killing occurred only in the presence of both antibody and complement NHS and required an intact alternative pathway. The efficiency of bacterial killing varied by antibody isotype (IgM greater than IgG2a greater than other IgG subclasses) and correlated with C3-fixing capacity. Opsonophagocytic activity of MAbs exhibited a similar isotype-related rank order. Likewise, IgM was more active than IgG, and IgG2a was superior to other IgG subclasses, in MAb-mediated protection against intraperitoneal infection. These data document the interdependent antibacterial and complement-fixing properties of LPS-reactive MAbs and the degree to which both activities are determined by antibody class and isotype.

To study the role of cytokines as mediators of endotoxin-induced shock, the serum levels of tumor necrosis factor (TNF) and interleukin-6 (IL-6) were compared in mice receiving either a monoclonal antibody to endotoxin core (clone 20), an irrelevant monoclonal antibody (A1), or culture media (DMEM/FCS) alone before lethal challenge with live Escherichia coli O111:B4. Clone 20 given 1.5 h before the bacterial challenge protected mice from death (mortality at 48 h 3% vs. 87%, P less than .001). The pattern of IL-6 release was indistinguishable in clone 20 recipients and controls: The area under the curve (AUC) for 5 h was 1.22 +/- 0.07 x 10(6), 1.03 +/- 0.17 x 10(6), and 1.22 +/- 0.07 x 10(6) units/ml for clone 20, A1, and DMEM/FCS, respectively. Similarly, the timing and extent of TNF release in the serum was virtually identical in clone 20 recipients that survived and control animals that died. AUC for 5 h was 30.8 +/- 4.0 x 10(3), 28.1 +/- 1.1 x 10(3), and 30.4 +/- 4.7 x 10(3) ng/ml in clone 20, A1, and DMEM/FCS recipients, respectively. Thus, TNF and IL-6 appear insufficient to cause death in this model of experimental gram-negative shock.

Lipopolysaccharide from strains of Haemophilus influenzae was extracted and isolated by the hot phenol-water procedure. The preparations were relatively insoluble in water but could be solubilized with surface-active agents. The preparations contained carbohydrate (30%), fatty acid (29%), and phosphate (4.7%); protein content was less than 1%. Thin-layer chromatography, gas-liquid chromatography, and colorimetric assays detected glucose, galactose, glucosamine, heptose, and a 2-keto-3-deoxy-octonate-like molecule (less than 1%). Neither methylpentose nor dideoxyhexose was detected. The lipid portion was composed of fatty acids common to lipopolysaccharide of Salmonella. The preparations provoked positive dermal Shwartzman reactions and biphasic febrile responses in rabbits, responses typical of endotoxic activity. The 50% lethal dose for mice was decreased from 16.5 microgram/g to 0.015 microgram/g by concomitant administration of actinomycin D. The preparations were shown to be polyclonal activators of bone marrow-derived (B) cells. Limulus lysate gelation was seen with 8.0 ng of lipopolysaccharide. Preliminary hemagglutination data suggested at least three different antigenic factors associated with the lipopolysaccharide of H. influenzae type b. The H. influenzae lipopolysaccharide appeared biologically similar to that of enterobacteria but chemically different.

The pyrogenic efect of the synthetic adjuvant N-acetylmuramyl-L-alanine-D-isoglutamine, also known as muramyl dipeptide (MDP), was studied in rabbits. MDP induced biphasic fevers in rabbits, but two structural analogues, N-acetylmuramyl-L-alanine-D-glutamic acid (MDPA) and the dimethylester of MDPA, were 10 times less pyrogenic. This finding was supported by studies in which MDP and its analogues released leukocytic pyrogen (LP) from rabbit phagocytic cells in vitro. In addition, MDP released LP from human phagocytes. Human phagocytes, however, required a 10-fold greater concentration of MDP than did rabbit cells. The structural analogues were similarly less effective than the parent molecule in releasing LP from human cells. All preparations of MDP were negative in the limulus amebocyte lysate test and failed to show pyrogenic cross-tolerance with bacterial endotoxin. Thus MDP, which is a pyrogenic molecule, is also able to release LP from rabbit phagocytes and to a lesser degree from human phagocytes, but does not cause gelation of limulus amebocyte lysate.

The protection by antisera to O antigens and antigens (lipid A, the Re 595 mutant of Salmonella minnesota, and the J5 mutant of Escherichia coli) of the core portion of endotoxin against lethal challenge with lipopolysaccharide (LPS) was compared. Rabbits immunized with the Re mutant developed antibody that protected mice against challenge with S. minnesota or Salmonella typhosa LPS. Antisera to heterologous O antigen and lipid A were not protective, whereas homologous antisera and antiserum to the Re or J5 mutant protected against lethal challenge. On a volumetric basis, O-specific antiserum was consistently somewhat more protective than antiserum to the Re mutant, which was slightly more protective than antiserum to the J5 mutant. When comparisons were made using passive transfer to similar quantities of hemagglutinating or precipitating antibody, antiserum to the Re mutant was considerably more protective than O-specific antiserum. This greater protection is hypothesized to result from more effective masking of the toxic lipid A moiety by antibody to the rough mutants than by O-specific antibody.

Antiserum to rough gram-negative mutants such as Escherichia coli J5 and Salmonella minnesota Re595 is thought to neutralize the toxic effects of lipopolysaccharides (LPSs). To verify that such antisera are capable of binding heterologous endotoxins, we examined IgG and IgM class antibodies induced in rabbits to a variety of LPSs. Immunization with rough mutants or lipid A induced high IgG antibody responses to the homologous purified LPS and relatively low but significant responses to heterologous LPSs. Increases in IgM antibodies were also primarily to homologous LPS. Immunization with smooth organisms induced little or no antibody to heterologous LPSs. Soluble LPS, outer membrane vesicles, and whole bacteria produced strong homologous inhibition but little or no heterologous inhibition in enzyme-linked immunosorbent assays. Cross-adsorption of antisera to rough mutants suggested that the IgG and IgM antibodies induced to heterologous LPS were adsorbed by the heterologous LPS and not by the core LPS used to immunize the animals. Rabbit antibody directed to J5 or Re595 LPS fails to bind to any substantial degree to heterologous LPS. Immunization with whole bacterial vaccines, particularly the rough mutants and lipid A, does increase antibody to a wide variety of antigens. The possibilities that the protective effects of antisera to rough mutants are due to a polyclonal antibody response or to the induction of as yet unidentified factor(s) deserve further investigation.

An enzyme-linked immunosorbent assay for antibody to the lipopolysaccharides (LPSs) of a rough strain of Escherichia coli (J5) and Neisseria gonorrhoeae was developed. When standard methods of this assay were applied to these LPSs, no antibody was measured. By radiolabeling smooth and rough endotoxins with 51Cr, it was discovered that the rough LPS was not adhering to the polystyrene tubes. When Mg++ was added, however, all endotoxins absorbed to the solid phase. With this assay antiserum to core glycolipid had higher titers against its smooth parent strain than did homologous glycolipid had higher titers against its smooth parent strain that did homologous antiserum, and antisera to gonococci had high homologous but low heterologous titers. Thus, these data demonstrate that bulky O side chains do not hinder the penetration of antibody to core glycolipid and that LPSs from gonococci are heterogeneous but do cross-react.

Three mouse strains in the C3H lineage--C3H/HeJ, C3HeB/FeJ, and C3H/HeNCr1BR--were tested for their ability to be protected against infection with Salmonella typhimurium by a panel of nonviable vaccines and by passive transfer of hyperimmune serum. These strains differ in their innate susceptibilities to infection with S. typhimurium, but all are histocompatible. The same vaccines showed a widely different ability to protect different mouse strains. Ability to protect was not closely related to the capacity of the mice to make either agglutinating or anti-O antibody (as shown by ELISA) in response to a particular vaccine. Passive transfer of antibody was shown to protect inherently resistant mice but not inherently susceptible strains. These observations suggest that reported discrepancies in vaccine efficacy among laboratories may be attributable to differences in the mouse strains used and raise the question as to what might be an appropriate mouse model for human infections with Salmonella species.

For study of the kinetics of endotoxin release from bacterial cells during therapy for gram-negative bacterial sepsis, serial blood samples were obtained from rabbits with Escherichia coli sepsis that were treated with either antibiotic or placebo. The concentrations of viable bacteria, free endotoxin, and total endotoxin in each blood sample were quantitated. In animals treated with placebo the concentration of free endotoxin was proportional to the level of bacteremia. In contrast, in animals treated with antibiotic the plasma levels of free endotoxin increased 10- to 2,000-fold, in spite of decreasing levels of bacteremia. Free endotoxin that was present in the plasma following antibiotic treatment appeared to be derived in part from the breakdown of circulating bacteria and in part from the disintegration of bacteria in tissues other than the blood. The results of this study demonstrate that significant amounts of endotoxin are released from bacterial cells following administration of antibiotics in vivo.

The pharmacological factors involved in lipopolysaccharide (LPS)-induced host resistance against infection were investigated in relation to the problem of endotoxin contamination of preparations of monoclonal antibody to the common structure of endotoxin. When administered prophylactically, purified LPS (as low as 4 ng/kg of mouse body weight) or antibody preparations contaminated with endotoxin (assayed by Limulus amoebocyte lysate test) were protective against lethal challenge with a clinical isolate of Escherichia coli (P less than .01). Antibodies that were nonreactive to LPS were similarly protective (P less than .001) when spiked with low doses of LPS (100 ng/mg of protein) but, as with LPS, were without effect when administered after infection. Endotoxin contamination of core-reactive antibody to LPS is mostly a problem associated with the large-scale production, purification, and concentration of the monoclonal antibody. The efficacy (as reported in studies in animal models of gram-negative bacterial sepsis) of antibody to LPS core is controversial. We suggest that endotoxin contamination is a likely factor in this controversy.