Oral administration of Shiga toxin-producing Escherichia coli induces intestinal and systemic specific immune response in mice

Hemolytic uremic syndrome (HUS) is the major complication of gastrointestinal infections with enterohemorrhagic Escherichia coli (EHEC) and is mediated by the production of Shiga toxins (Stx). Although it has been previously reported that not only HUS patients but healthy children have anti-Stx antibodies, very little is known about how these infections impact on mucosal immune system to generate a specific immune response. This work aimed to evaluate the immune responses elicited after a single oral dose of EHEC in a mouse model of HUS at weaning. We found sequential activation of T and B lymphocytes together with an increased percentage of IgA-bearing B cells in Peyer’s patches and mesenteric lymph nodes. We also found fecal anti-EHEC IgA and serum anti-Stx2 IgG in EHEC-inoculated mice. Besides, these mice were partially protected against an intravenous challenge with Stx2. These data demonstrate that one episode of EHEC infection is enough to induce activation in the gut-associated lymphoid tissue, especially the B cell compartment, and lead to the production of specific IgA in mucosal tissue and the generation of systemic protection against Stx2 in a percentage of intragastrically inoculated mice. These data also support the epidemiologic observation that a second episode of HUS is very rare.     

Properties of binding of Escherichia coli endotoxin to various matrices.

Binding of Escherichia coli O127:B8 endotoxin to a variety of resins and column materials was investigated by measuring the beta-hydroxy myristic acid content (a major component of the lipid A moiety) of endotoxin after hydrolysis by selected ion-monitoring gas chromatography-mass spectrometry. More than 80% of the endotoxin was bound to hydroxylapatite, polystyrene, Dowex 1-X2, and charcoal. The binding of endotoxin to these materials was markedly reduced by the addition of normal or delipidated serum. Phenyl- and octyl-Sepharose bound 56 and 50% of the endotoxin from saline solutions, respectively. Their percent binding was increased significantly in 1 M ammonium sulfate solutions, indicating hydrophobic interactions between endotoxin and phenyl- and octyl-Sepharose. Only 5% of the endotoxin was bound to plastic polymer PSI-HAP-100 beads, and no binding was observed with concanavalin A- and heparin-Sepharose. Study of the in vitro binding of endotoxin to the above material in the presence of serum suggests that the use of these materials in removing circulating endotoxin in vivo is limited.     

Airway immunopathology of asthma with exercise-induced bronchoconstriction

BACKGROUND: Exercise-induced bronchoconstriction (EIB) is a common cause of symptoms in a subgroup of asthmatic subjects. The pathobiology that makes this group of asthmatic subjects susceptible to bronchoconstriction after a brief period of exercise remains poorly understood. OBJECTIVE: We sought to determine whether there are differences in lower airway inflammation and production of cytokines and eicosanoids between asthmatic subjects with and without EIB. METHODS: Two distinct groups of asthmatic subjects based on a priori definitions were identified, one with moderate-to-severe EIB and the other without significant bronchoconstriction after exercise challenge. Both groups met the definition of asthma on the basis of bronchodilator response, bronchial hyperresponsiveness, or both. A comparative immunopathology study was conducted by using induced sputum to identify differences in lower airway inflammation and production of cytokines and eicosanoids. RESULTS: The groups had similar baseline lung function and bronchodilator response and did not have any asthma exacerbations within the prior year. The concentration of columnar epithelial cells was markedly higher in the group with EIB (1.4 x 10(5) vs 2.9 x 10(4) cells/mL, P=.01). The concentration of eosinophils was higher in the group with EIB (3.6 x 10(4) vs 4.9 x 10(3) cells/mL P=.04). Cysteinyl leukotrienes (CysLTs; 727.7 vs 151.9 pg/mL, P=.01) and the ratio of CysLTs to prostaglandin E(2) (1.85 vs 1.04, P=.002) in the airways were higher in the group with EIB. CONCLUSION: Injury to the airway epithelium, overexpression of CysLTs, relative under production of prostaglandin E(2), and greater airway eosinophilia are distinctive immunopathologic features of asthma with EIB.     

Fever in rats after intravenousE. coli endotoxin administration

Summary In conscious unrestrained rats, at an ambient temperature of 22°C, oesophageal temperature was measured and temperature effect of single and repeated intravenous injection ofE. coli endotoxin was examined. The first injection of endotoxin in a dose of 10.0 g/rat did not change the rat body temperature. The second injection of this dose in the same animals repeated after 48 h produced fever. With following injections the fevers observed were less pronounced. The absence of fever after a single injection of endotoxin was accompanied by the rapid loss of pyretic activity of the rat plasma samples (bioassayed in rabbits). When fever was observed (48 h interval between endotoxin injections) the pyretic activity of the rat plasma remained unchanged for 90 min following endotoxin injection. It was concluded that after a single injection endotoxin is rapidly detoxified in the rat circulation while this process does not take place after the second endotoxin injection (48 h interval). The process of endotoxin detoxification can be depressed by the pretreatment with nitrogen mustard. Analysis of changes of skin temperature following endotoxin injections and the influence of aspirin on endotoxin-induced fever suggest that the fever observed was of central origin.     

Nonspecific resistance to Escherichia coli in mice.

Nonspecific cell-mediated immunity to a relatively virulent strain of Escherichia coli was studied in mice infected with Staphylococcus aureus and elicited with specific antigens. The infected and elicited mice were protected against as intraperitoneal challenge by E. coli for an observation period of 7 days, whereas normal mice, given the same number of bacteria, died within 18 to 24 h. However, the amount of time elapsing between elicitation and challenge greatly affected the rate of protection. Little or no protection was observed in mice injected with S. aureus but not elicited or in mice injected with staphylococcal antigens but not infected with staphylococci.     

Uropathogenic Escherichia coli induces chronic pelvic pain

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a debilitating syndrome of unknown etiology often postulated, but not proven, to be associated with microbial infection of the prostate gland. We hypothesized that infection of the prostate by clinically relevant uropathogenic Escherichia coli (UPEC) can initiate and establish chronic pain. We utilized an E. coli strain newly isolated from a patient with CP/CPPS (strain CP1) and examined its molecular pathogenesis in cell culture and in a murine model of bacterial prostatitis. We found that CP1 is an atypical isolate distinct from most UPEC in its phylotype and virulence factor profile. CP1 adhered to, invaded, and proliferated within prostate epithelia and colonized the prostate and bladder of NOD and C57BL/6J mice. Using behavioral measures of pelvic pain, we showed that CP1 induced and sustained chronic pelvic pain in NOD mice, an attribute not exhibited by a clinical cystitis strain. Furthermore, pain was observed to persist even after bacterial clearance from genitourinary tissues. CP1 induced pelvic pain behavior exclusively in NOD mice and not in C57BL/6J mice, despite comparable levels of colonization and inflammation. Microbial infections can thus serve as initiating agents for chronic pelvic pain through mechanisms that are dependent on both the virulence of the bacterial strain and the genetic background of the host.     

Effect of Escherichia coli endotoxin on dog lung fluid balance

Endotoxin may cause an increase in pulmonary capillary permeability and thus promote edema formation. We used a gravimetric technique to estimate the pulmonary capillary filtration coefficient (KF) and the maximum capillary pressure at which the lung could maintain a constant weight (Pccritical) in dogs after intravenous administration of Escherichia coli (E. coli) endotoxin. KF should be increased and Pccritical should be decreased by an increase in permeability. Four groups of three to four dogs were given 1, 10, 1,000, or 3,000 micrograms/kg of endotoxin. A fifth group of five dogs, which served as controls, was given no endotoxin. KF was significantly (P less than 0.05) greater than control [0.049 +/- 0.031 (SD) ml . min-1 . mmHg-1] in only the 1-micrograms/kg group (0.100 +/- 0.027), indicating a possible increase in permeability. However, changes in capillary surface area may have affected KF. Pccritical was not significantly different from control (20.7 +/- 2.4 mmHg) in any of the E. coli groups. We conclude from these results that E. coli endotoxin may have caused a slight increase in permeability; however, the lung retained its ability to resist edema formation.     

Uropathogenic Escherichia coli induces extrinsic and intrinsic cascades to initiate urothelial apoptosis

A murine model of urinary tract infection identified urothelial apoptosis as a key event in the pathogenesis mediated by uropathogenic Escherichia coli (UPEC), yet the mechanism of this important host response is not well characterized. We employed a culture model of UPEC-urothelium interactions to examine the biochemical events associated with urothelial apoptosis induced by the UPEC strain NU14. NU14 induced DNA cleavage within 5 h that was inhibited by the broad caspase inhibitor ZVAD, and urothelial caspase 3 activity was induced within 3 h of exposure to type 1 piliated NU14 and was dependent upon interactions mediated by the type 1 pilus adhesin FimH. Flow cytometry experiments using chloromethyl-X-rosamine and Indo-1 revealed FimH-dependent mitochondrial membrane depolarization and elevated [Ca(2+)](in), respectively, indicating activation of the intrinsic apoptotic pathway. Consistent with this possibility, overexpression of Bcl(XL) inhibited NU14 activation of caspase 3. Immunoblotting, caspase inhibitors, and caspase activity assays implicated both caspase 2 and caspase 8 in apoptosis, suggesting the involvement of the intrinsic and extrinsic apoptotic cascades. To reconcile the apparent activation of both extrinsic and intrinsic pathways, we examined Bid-green fluorescent protein localization and observed translocation from the cytosol to mitochondria in response to either NU14 or purified FimH. These data suggest that FimH acts as a tethered toxin of UPEC that activates caspase-dependent urothelial apoptosis via direct induction of the extrinsic pathway and that the intrinsic pathway is activated indirectly as a result of coupling by caspase 8-mediated Bid cleavage.     

Antibody response to Escherichia coli O antigens and influence of the protein moiety of the endotoxin

Exposure of mice to different serotypes of E. coli bacteria, either O4 or O6, resulted in an enhanced indirect IgG-PFC response to the alternate bacteria. This effect seemed to be mediated by a protein connected with the endotoxin structure. This protein moiety had some weak adjuvant activities and increased the antibody response against sheep red blood cell about two-fold. This effect was not likely to be due to any contamination with the B-cell mitogen lipid A, a constituent of the endotoxin from which the protein was isolated.

In addition, experiments were performed in which irradiated spleen cells (0–400 R) from mice injected with E. coli O4 bacteria were transferred to irradiated (800 R) recipients together with E. coli O6 bacteria. Decreasing numbers of antibody forming cells with increasing irradiation dose were found. The parallel experiment employing E. coli O6 bacteria for both primary and secondary antigen injections revealed an increased immune response for an irradiation dose of 50 R, showing that suppressor cells are more irradiation sensitive than the other cells involved in this immune response, but that the effect of such cells is possibly overcome by the influence of the protein residue isolated from endotoxin.

A secondary response to E. coli O6 bacteria was also noted in agreement with previous results. It was found that this immune response could be reduced drastically by injecting primed thymocytes, simultaneously with the second injection.

     

Regulation of antibody synthesis against Escherichia coli endotoxin: III. Induction of immunological paralysis in non-immune and pre-immunized mice

Normal mice injected with a paralysing dose of endotoxin are unresponsive to an immediate subsequent injection of an immunogenic dose of the corresponding bacteria.

In pre-sensitized mice the injection of a paralysing dose of endotoxin suppresses the immune response after a lag period of 80–90 hours during which the responding cells appear normally. The suggested explanation to this was that antigen-sensitive cells once triggered by the antigen divide and produce antibodies for a certain time period and thereafter disappear. During this period they are unaffected by the paralysing dose of antigen. It is suggested that the cells amenable to suppression are those from which the antibody producing cells are recruited, e.g. the antigen sensitive cells.

The kinetics of suppression of an active immune response was the same whether antiserum or a paralysing dose of antigen was used as suppressive agent. This finding further supported the conclusion that actively antibody producing cells are antigen independent.

     

Effects of Escherichia coli endotoxin on structure and permeability of myocardial capillaries.

A study was carried out to investigate changes in myocardial capillaries induced by endotoxin, in order to clarify the pathogenesis of myocardial damage in endotoxemia. Wistar rats were injected intraperitoneally with 100 mg/kg Escherichia coli lipopolysaccharide and then sacrificed at 1, 2, 3,4, 5, 6, 8, and 24 h after injection. The myocardium was observed by electron microscopy with histochemistry using horseradish peroxidase and immunocytochemistry for Na+, K(+)-ATPase/TPase. The earliest evident endothelial alterations were swelling, increased numbers of pinocytotic vesicles, and formation of cytoplasmic projections. Interstitial edema and focal detachment of the endothelial cells from the basement membrane occurred with time. Vascular permeability was increased after endotoxin injection. Activity of Na+, K(+)-ATPase was reduced on the plasma membrane of the endothelial cells. It is concluded that endotoxin induces structural and enzymatic changes in the myocardial capillary endothelium and an increase of capillary permeability.