Endotoxin release and cytokine production in acute and chronic meningococcaemia

Chronic meningococcaemia is a relatively benign manifestation of meningococcal disease. Whether bacterial virulence factors are responsible for this benign course has not been studied. We compared the in vitro endotoxin-liberating ability and cytokine-inducing potential of 31 Neisseria meningitidis isolates obtained from children with acute septic shock with that of nine isolates obtained from patients with chronic meningococcaemia and 12 isolates obtained from carriers with respiratory symptoms. The median endotoxin level released in vitro after 3 h of incubation was significantly higher for isolates causing septic shock compared with isolates from the other two groups (P = 0.01 and 0.02, Mann–Whitney test). This was not explained by differences in bacterial growth rate in vitro. The median IL-6 levels in whole blood ex vivo after 4 h of incubation were also significantly lower for isolates causing chronic meningococcaemia (P = 0.04, Mann–Whitney test). The endotoxin and cytokine levels measured on admission in the 31 children with acute meningococcal septic shock showed a 1000-fold variation. No relationship was established between the amount of endotoxin released by the causative microorganisms in vitro and the endotoxin or cytokine levels in the corresponding 31 children. These results suggest a diminished bacterial virulence for isolates causing chronic meningococcaemia. However, other factors than the endotoxin-releasing potential of the microorganism involved are responsible for the wide variation in endotoxin and therefore cytokine levels in patients with acute meningococcal septic shock.     

Endotoxin from Haemophilus influenzae enhances IgE-mediated and non-immunological histamine release

Haemophilus influenza and its extracellular products (EP) did not release histamine from basophil leukocytes in cell suspensions from normal individuals, patients with chronic bronchitis or patients allergic to either house dust mite, grass pollen, cat dander or to their own bacteria. However, the EP was found to enhance their basophil histamine release. IgE-mediated histamine release was examined by stimulation of the cells with anti-IgE or the specific allergens, and non-immunological histamine release by stimulating the cells with the calcium ionophore A23187 or Staphylococcus aureus. In all the experiments EP caused a significant increase in the histamine release. When H. influenzae endotoxins were removed from the EP, the potentiating effect of EP was completely abolished, whereas heating (80 degrees C, 30 min) or treatment of EP with proteinase did not influence the potentiating effect. These results indicate that H. influenzae endotoxin potentiates histamine release caused by IgE-mediated reactions or by non-immunological mechanisms.     

Proteins adhering to Escherichia coli after exposure to guinea-pig serum

The constituents of guinea-pig serum that attach to the surface of a serum-sensitive strain of Escherichia coli have been determined. Antisera from rabbits immunized with bacteria exposed to serum (`sensitized' bacteria) were analysed by means of immunoelectrophoresis. It is concluded that at least seven constituents from the serum of the normal adult guinea-pig adhere to the E. coli during sensitization. These bind tightly enough to resist removal by at least six washings of the sensitized bacteria. Two of these constituents (IgG and IgM) are known to possess antibody activity. A third constituent, β_1C, is known to be the third component of the complement system of the guinea-pig. Another is probably the fourth component of this complement system. The other three serum components remain unidentified, although one of them is shown to possess esterase activity, and thus may be related to the first component of complement.

Evidence is presented suggesting that normal guinea-pigs may have little or no antibody against E. coli present in the IgG fraction of the serum.

If, before sensitization of the E. coli, the serum is heated to inactivate the activity of bactericidal complement, the serum does not kill this organism, but no change is detectable in the serum proteins adhering to these bacteria. Similarly, if, before sensitization of E. coli, the bactericidal antibody to the E. coli is absorbed from the serum, while bactericidal complement is left intact, there is no change in the serum proteins adhering to the bacteria. When the serum used for sensitizing E. coli is from guinea-pigs that have been actively immunized with the same strain of E. coli, rabbits immunized with these sensitized bacteria appear to form increased amounts of antibody directed against the 7S γ₂-globulin of the guinea-pig serum.

     

Effect ofE. coli endotoxin on erythrocyte morphology and blood iron level in rats

Laboratory of Histopathology and Electron Microscopy, Rostov Plague Research Institute. Department of Clinical Pharmacology, Rostov Medical Institute. (Presented by Academician of the Russian Academy of Medical Sciences N. K. Permyakov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 114, No. 10, pp. 439–442, October, 1992.     

Difference in exposure to airborne major rat allergen (Rat n 1) and to endotoxin in rat quarters according to tasks

Endotoxins found in occupational settings constitute a risk factor in the severity of respiratory allergic symptoms. OBJECTIVES: To assess the airborne concentrations of major rat allergen (Rat n 1) and endotoxin under various circumstances. METHODS: We took 483 airborne samples from 12 sites: 114 individual samples for endotoxin measurements and 113 for Rat n 1, from 38 workers (nine animal technicians, nine laboratory technicians, nine scientists and 11 students); and 256 static samples in rat rooms and experimental rooms, with or without disturbance, for simultaneous endotoxin and Rat n 1 measurements. Rat n 1 was measured with a two-site monoclonal ELISA and endotoxins with the Limulus method. RESULTS: Airborne Rat n 1 and endotoxin were significantly higher in rat rooms than in experimental rooms. Animal technicians had the greatest exposure to both Rat n 1 and endotoxin. Cage cleaning and rat feeding induced the highest exposure to Rat n 1 and endotoxin. Furthermore, we observed no significant difference in endotoxin exposure between researchers with or without rat contact during the sample period. There was no correlation between the number of rats present and airborne endotoxin concentrations. CONCLUSIONS: Exposure to airborne Rat n 1 and endotoxin is higher during cleaning and feeding tasks than during any other task, we feel that a major source of both is contaminated bedding that becomes airborne during disturbance.     

Renal blood flow distribution during E. coli endotoxin shock in dog

The effect of endotoxin on renal blood flow distribution was studied in anesthetized dogs. Renal blood flow was measured as hydrogen clearance by platinum electrodes placed in outer and in inner halves of cortex and by electromagnetic flowmeter. Intravenous injection of E. coli endotoxin, 3–5 mg/kg b. wt., promptly reduced arterial blood pressure (AP) and renal blood flow. After a transient increase for 45 min AP and renal blood flow declined to about 50% of the control 2½-3 h after injection. The reduction in outer cortical blood flow (OCF) was not significantly different from the reduction in inner cortical blood flow (ICF). The hematocrit (Hct) increased from 40.1±3.8% to 54.6±8%, but mean renal vascular resistance did not change. Total plasma protein concentration was not significantly elevated. A marked local flow variability was observed in some periods during the phase of shock with declining AP and total renal blood flow at high Hct. Thus renal blood flow showed phasic changes, but the OCF/ICF ratio was not changed during endotoxin shock. Local blood flow instability was observed periodically at high Hct.     

Exposure to house dust endotoxin and allergic sensitization in adults

BACKGROUND: It has been suggested that exposure to elevated levels of endotoxin decreases the risk of allergic sensitization. OBJECTIVE: To examine the associations between current exposure to bacterial endotoxin in house dust and allergic sensitization in adults. METHODS: In 1995-1996, we conducted a nested case-control study following a cross-sectional study performed within the European Community Respiratory Health Survey (ECRHS). Data of 350 adults aged 25-50 years was analysed. Allergic sensitization was assessed by measurement of specific immunoglobulin E (IgE) against several inhalant allergens. Living room floor dust samples were taken. The endotoxin content was quantified using a chromogenic kinetic Limulus amoebocyte lysate test. RESULTS: Multiple logistic regression analysis showed a negative association between exposure to house dust endotoxin and severe allergic sensitization. Odds ratios (95% CI) adjusted for place of residence, gender, age, and 'caseness' were 0.80 (0.64-1.00) for sensitization to >/=1 allergen and 0.72 (0.56, 0.92) for sensitization to >/=2 allergens using 3.5 kU/l as a cut-off value for sensitization. With regard to single allergens, the protective effect of endotoxin was strongest for pollen sensitization [aOR (95% CI) = 0.74 (0.58, 0.93)]. CONCLUSION: Our results indicate that current exposure to higher levels of house dust endotoxin might be associated with a decreased odds of allergic sensitization in adults.     

Prevalence of symptoms, sensitization to rats, and airborne exposure to major rat allergen (Rat n 1) and to endotoxin in rat-exposed workers: a cross-sectional study

OBJECTIVE: To analyse the relation between airborne exposure to major rat allergen and to endotoxins in exclusively rat-exposed workers and the prevalence of rat-related symptoms and sensitization. METHODS: A total of 113 workers answered a standardized questionnaire on their atopy status, occupational exposure to rats, and possible work-related symptoms. Specific IgE against rat urinary proteins (RUP) was measured for 73 subjects. Individual airborne exposure to Rat n 1 and endotoxin were determined with static (n = 256) samplings. Rat n 1 was measured with enzyme-linked immunosorbent assay (ELISA) and endotoxin by the Limulus method. RESULTS: Forty-four of 113 subjects (38.9%) reported at least one rat-related symptom: asthma (4.4%), rhinitis (34%) and conjunctivitis (16%). Twelve per cent were sensitized to RUP (specific IgE > 0.35 KU/L). But only 30.8% of all symptomatic subjects were sensitized to rat allergens. Airborne Rat n 1 levels were not related to symptoms in workers. Symptomatic patients not sensitized to rats were exposed to higher endotoxin levels, but airborne exposure to endotoxins did not significantly protect against or increase sensitization to RUP or rat-related symptoms. CONCLUSION: Most symptomatic workers were not sensitized to rat allergen; but no significant relation between rat-related symptoms and endotoxin levels was found. This suggests that more studies are needed to determine causes other than rat allergens or endotoxins that may be responsible for symptoms in rat-exposed workers.     

Resistance to second- and third-generation cephalosporins among Escherichia coli and Klebsiella species is rare in Finland

Objective: To investigate the antimicrobial resistance of Escherichia coli and Klebsiella spp. from pus, urine and respiratory specimens, with particular emphasis on the detection of third-generation cephalosporin resistance.
Methods: E. coli (698) and Klebsiella sp. (476) strains from pus, respiratory and urinary specimens from hospital patients were collected from 19 laboratories. Data about consumption of third-generation cephalosporins and cefuroxime were collected from 24 hospitals. Antimicrobial susceptibility was tested with disk diffusion in primary laboratories and by an agar dilution method. Extended-spectrum β-lactamase (ESBL) production was studied with a double disk synergy test and an ESBL Etest. The β-lactamase classes were characterized with polymerase chain reaction probes of the TEM and SHV β-lactamase families and isoelectric focusing.
Results: Only 0.6% of E. coli and 2.3% of Klebsiella spp. strains were resistant or intermediately resistant to cefotaxime, ceftriaxone and/or ceftazidime. The ESBL producers detected comprised one E. coli harboring TEM-like genes and five Klebsiella pneumoniae strains, two of which harbored SHV-like genes, two TEM-like genes and one both. Although consumption of cefuroxime has increased in the years 1990–1994, from 3.48 to 5.84 defined daily doses/100 bed-days, and the consumption of third-generation cephalosporins from 1.25 to 1.94 defined daily doses/100 bed-days, cefuroxime resistance of E. coli was only 3%.
Conclusion: Although the use of broad-spectrum cephalosporins has increased, resistance to second- and thirdgeneration cephalosporins is still rare in Finland.     

Airway administration of Escherichia coli endotoxin to mice induces glucocorticosteroid-resistant bronchoconstriction and vasopermeation

The effects of the administration of Escherichia coli endotoxin (lipopolysaccharide, LPS) into the airways of C57Bl/6 mice were studied. Neutrophil sequestration in the lungs and their enrichment, together with tumor necrosis factor (TNF)-alpha, in bronchoalveolar lavage fluid (BALF) were associated with bronchoconstriction and bronchopulmonary hyperreactivity (BHR) to methacholine and alveolocapillary dysfunction. Granulocyte depletion by the myelotoxic drug vinblastine failed to modify TNF-alpha production and prevented LPS-induced neutrophil recruitment to lungs and BALF, bronchoconstriction, and BHR. Neutrophils were again sequestered in the lungs when LPS was administered 4 to 5 d after vinblastine, whereas inhibition of their passage to BALF persisted. Under those conditions, bronchoconstriction and BHR by LPS also recovered, showing that these functional effects are independent from BALF neutrophil enrichment but require lung sequestration. Administration of granulocyte colony-stimulating factor after vinblastine counteracted its effects and allowed the recovery of lung neutrophil sequestration by LPS and a partial recovery of bronchoconstriction under conditions where neutrophils still failed to migrate to BALF. Dexamethasone (the phosphate salt and its free base) suppressed LPS-induced TNF-alpha generation in BALF and its neutrophil enrichment, whereas neutrophil lung sequestration, bronchoconstriction, BHR, and alveolocapillary dysfunction were marginally reduced and only so at low doses of dexamethasone, higher doses being inactive or aggravating. In situ neutrophil activation could account for LPS-induced bronchoconstriction and BHR, both of which are refractory to steroids and appear to be mediated by unrelated mechanisms, which may be relevant for acute respiratory distress syndrome, a condition for which LPS administration is used as a model.     

Evaluation of absorption with Limulus amebocyte lysate to remove contaminating endotoxin from interferon and lymphokine preparations

Alpha and beta human interferon (IFN) preparations and lymphokines (supernatants of PHA-stimulated blood lymphocytes) were deliberately contaminated with endotoxin (20 ng/ml) and subsequently rendered endotoxin-free by absorption with Limulus amebocyte lysate (LAL). Absorption with LAL did not appreciably affect the antiviral activity of IFN and lymphokines in 8 experiments and caused a 30-50% reduction in two. The capacity of these agents to stimulate natural killer cell activity and monocyte cytotoxicity was not consistently modified by absorption on LAL. When the chemotactic activity of lymphokine for monocytes was measured, the maximal number of monocytes induced to migrate and the maximal active lymphokine concentration were not affected by absorption with LAL. LAL-treated lymphokines, however, showed a prozone phenomenon, presumably related to the release of chemotaxis inhibitor(s) from the LAL gel.     

大肠杆菌内毒素诱导的SD大鼠眼内炎

目的 观察细菌内毒素脂多糖(LPS)诱导的大鼠眼内炎性反应的临床、组织病理学和血眼屏障的特点.方法 SD大鼠玻璃体腔内注人大肠杆菌LPS(1μg)建立LPS诱导的眼内炎动物模型,对照组注入无菌生理盐水.在LPS注入后6h至7d的不同时点,分别对眼部炎性反应评分、浸润白细胞计数、前房水蛋白质浓度和玻璃体内LPS水平进行测定和评估.结果 在LPS注射后6～72 h眼内可见严重的炎性反应,至术后7d炎性反应基本消退.眼内白细胞的浸润数量在术后24h达到高峰[(1182.63±191.15)细胞/眼],至术后3 d浸润细胞数量迅速下降[(331.25±57.9)细胞/眼].在各观察时点,均可观察到眼内炎组房水蛋白质浓度较对照组显著增高(P<0.01). LPS注入眼内后前3 d,玻璃体腔内LPS含量迅速下降[前3d分别为(327.02±51.54)、(176.0±53.68)和(54.91±13.26)ng],在7d后玻璃体腔内LPS基本被清除.结论 大肠杆菌内毒素在SD大鼠可诱导出严重的实验性眼内炎,大量白细胞眼内浸润、血眼屏障破坏和玻璃体腔自发性细菌成分清除是本实验性眼内炎模型的主要病理特点.     

Endotoxin neutralization with rabbit antisera to Escherichia coli J5 and other gram-negative bacteria.

To study the mechanisms of protection against endotoxin challenge offered by antisera to smooth and rough gram-negative organisms, we have developed an assay to quantitate endotoxin neutralization based on inhibition of the Limulus amoebocyte lysate test. Dilutions of different bacterial lipopolysaccharides (LPSs) were incubated with hyperimmune rabbit sera against Escherichia coli O113, E. coli O18, and rough mutants E. coli J5 and Salmonella minnesota Re595 and were then combined with limulus lysate. The gelation reaction induced by LPS in the lysate was monitored spectrophotometrically, and the concentration of LPS resulting in a 50% lysate response was determined and correlated with antibody titers measured by enzyme-linked immunosorbent assay. Antisera to smooth organisms neutralized homologous LPS markedly and heterologous LPSs only minimally relative to neutralization by preimmune serum. Neutralization of homologous LPS occurred immediately without preincubation of serum and LPS. Antisera to rough mutants neutralized more heterologous LPS than did antisera to smooth organisms. However, this heterologous neutralization required preincubation of serum and LPS and did not appear to be correlated with antibody concentrations. We conclude that antisera to LPS rapidly neutralize the biological activity of the homologous LPS, as detected by limulus lysate, and that neutralization is at least in part antibody mediated. Antisera to rough-mutant organisms slowly neutralized the activity of heterologous LPSs, but this effect appeared not to be correlated with concentrations of antibody to the LPS of the rough mutant, as measured by enzyme-linked immunosorbent assay.     

Low-dose endotoxin in allergic asthmatics: effect on bronchial and inflammatory response to cat allergen

BACKGROUND: Endotoxin was proposed to increase the severity of asthma. Endotoxin levels greatly differ according to settings. In domestic environments, airborne concentrations may be dramatically low compared with levels reported in occupational settings. OBJECTIVE: Our first objective was therefore to assess the effect of inhalation of low-level lipopolysaccharide (LPS) on the immediate and late-phase asthmatic bronchial response. Our second objective was to evaluate the effect of exposure to LPS on the local and systemic inflammatory response. METHODS: Nineteen asthmatics sensitized to cat underwent on two separate occasions a bronchial challenge test to cat allergen (cat BCT) preceded randomly by a pre-exposure to either saline or LPS (2 microg). Methacholine challenge test was performed 24 h before exposure to LPS or saline. The Borg scale for dyspnoea and lung function were recorded before and after exposure to LPS or saline, and before and after cat BCT. Induced sputum and blood samples were collected before and after cat BCT, and analysed for cell counts and eosinophil cationic protein (ECP) levels. RESULTS: Inhalation of 2 microg LPS did not induce any changes in forced expiratory volume in 1 s (FEV1), peak expiratory flow (PEF), FEF 25-75 and Borg scale of dyspnoea. It neither modified Fel d 1 PD20 (45.03 ng as compared with 87.03; P=0.42). As well, there was no significant difference in late-phase reaction. Pre-exposure to LPS did not influence eosinophil counts or ECP levels in blood and sputum. CONCLUSION: Our study demonstrated that pre-exposure to LPS at low levels, which may be encountered in domestic environment, had no significant effect on the immediate and late-phase bronchial response to cat allergen. It neither modified local and systemic eosinophilic inflammation.     

Endotoxin Tolerance Induced by Lipopolysaccharides Derived from Porphyromonas gingivalis and Escherichia coli: Alternations in Toll-Like Receptor 2 and 4 Signaling Pathway

Periodontitis is a chronic inflammatory disease induced by bacteria. Exposure of the host to periodontal pathogens and their virulence factors induces a hyporesponsive state to subsequent challenge, which is termed endotoxin tolerance. In this experiment, we studied the cytokine production in THP-1 cells upon single or repeated Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) or Escherichia coli (E. coli) LPS stimulation by ELISA. In addition, the protein expression profiles of Toll-like receptor 2 (TLR2), TLR4, IL-1 receptor-associated kinase 4 (IRAK4) and IRAK-M and the gene expression changes of Toll-interacting protein (Tollip) and suppressor of cytokine-signaling-1 (SOCS1) were explored to identify possible mechanisms for changes in cytokine secretion. After repeated stimulation with P. gingivalis LPS or E. coli LPS, secretions of TNF-α and IL-1β were decreased significantly compared with those following single challenge, while the levels of IL-10 were increased (p?<?0.05). Only comparable levels of IL-8 were confirmed in P. gingivalis LPS-tolerized cells (p?>?0.05). In addition, severe downregulation of TLR2 was detected in THP-1 cells retreated with P. gingivalis LPS, and the reduction of TLR4 expression was observed in cells restimulated with E. coli LPS (p?<?0.05). Precondition with P. gingivalis LPS or E. coli LPS also led to an enhancement of IRAK-M and SOCS1, while maintaining the expressions of IRAK4 and Tollip. This pattern of cytokine production indicates the different effects of endotoxin tolerance triggered by P. gingivalis LPS and E. coli LPS, which might contribute to limiting inflammatory damage. Moreover, TLR2, TLR4, IRAK-M, and SOCS1 might play important roles in developing tolerance.     

Endotoxin contamination contributes to the pulmonary inflammatory and functional response to Aspergillus fumigatus extract inhalation in heaves horses

BACKGROUND: As part of a primary prevention of asthma study, we measured the effect of environmental control measures on Der p 1, Fel d 1 and Can f 1 over a 3.5-year period. METHODS: High-risk infants (both parents atopic) without pets, were randomized to the Active group (n = 142, vinyl flooring in child's room, allergen-impermeable cot mattress, hot-washable toy, mite allergen-impermeable encasings to parental bed and to child's bed when older, high filtration vacuum cleaner, hot-washing of bedding) or the Control group (n = 136, no intervention), in early pregnancy. Dust samples from the parental mattress, living room floor, child's mattress and floor at baseline (pregnancy), birth and at 3 years were analysed for Der p 1, Fel d 1 and Can f 1. RESULTS: A total of 278 families completed the baseline visit, 259 the birth visit and 239 the 3-year visit. In the Active group at 3 years, 58% remained compliant with all measures likely to reduce the child's exposure to allergen and 77% of parents still used encasings on their bed. Levels of Der p 1, Fel d 1 and Can f 1 were significantly lower in the Active group in the child's floor and the child's mattress at 3 years compared to the Control group (P < 0.001). For the parental mattress, the levels of Der p 1 and Fel d 1 were lower in the Active group (P < 0.001) and there was a strong trend towards a lower level for Can f 1. There was no difference in the levels of any of the allergens between the groups in the living room floor. Childrens' bedrooms with no detectable mite, cat or dog allergen were significantly more common in the Active than the Control group (25 vs. 2, P < 0.001). CONCLUSIONS: Environmental control measures are effective in substantially reducing levels of Der p 1, Fel d 1 and Can f 1 in homes without pets in the long term and are acceptable to families. The effect of this environmental manipulation on the development of sensitization and allergic disease remains to be seen.     

Bacteria and endotoxin enhance basophil histamine release and potentiation is abolished by carbohydrates

Histamine release caused by anti-IgE, specific antigens and calcium ionophore A23187 was examined in leukocyte suspensions from healthy individuals and patients allergic to house dust mite and birch pollen. Staphylococcus aureus and LPS from Salmonella typhimurium were found to cause a synergistic enhancement of the release. The potentiation of mediator release by the bacteria and the endotoxin depends on a binding to the basophilocyte, followed by a non-transient event, since the potentiating effect persists after preincubation of the cells with the LPS followed by washout and leaving the cells for 30 min at 37 degrees C before stimulation with anti-IgE. The potentiation was abolished or reduced by galactose (10(-7) and 10(-6) M) and N-acetylglucosamine (10(-6) and 10(-5) M), acting by a binding to the basophil cell membrane, demonstrated by the persistence of effect after preincubation and washout of unbound sugar.