Interleukin 17A evoked mucosal damage is attenuated by cannabidiol and anandamide in a human colonic explant model.

Interleukin 17A (IL-17A) is a cytokine linked to inflammatory bowel disease. We investigated IL-17A expression in human colonic mucosa, whether IL-17A can elicit colonic mucosal damage in a human explant model and modulate gastrointestinal epithelial permeability in cell culture. We also tested if select cannabinoid ligands, shown to be protective in colitis models could attenuate damage caused by IL-17A. In addition, the ability of pro-inflammatory cytokines TNF-α and IL-1ß to modulate levels of IL-17A in the explant colitis model was also explored. IL-17A incubation caused significant mucosal epithelial and crypt damage which were attenuated following hydrocortisone treatment, and also reduced following anandamide or cannabidiol incubation. IL-17A-evoked mucosal damage was also associated with an increase in matrix metalloprotease activity. However, IL-17A did not induce any significant changes in epithelial permeability in confluent Caco-2 cell monolayers over a 48h incubation period. IL-17A was located predominantly in human mucosal epithelium together with IL-17C, but both IL-17A and IL-17C were also expressed in the lamina propria and submucosa. Incubation of human colonic mucosal tissue or Caco-2 cells with pro-inflammatory cytokines TNF-α and IL-1ß however did not alter IL-17A expression. These results indicate IL-17A has a widespread distribution in the human colon and the capacity to elicit mucosal damage which can be attenuated by cannabinoid ligands.

Cannabinoid CB2 receptor activation attenuates cytokine-evoked mucosal damage in a human colonic explant model without changing epithelial permeability.

Cannabinoid receptor activation is protective in animal colitis models. We sought to investigate if cannabinoids attenuated colitis-like tissue damage in human colonic specimens, with the hypothesis that cannabinoids would be protective in a cytokine-driven model of human colonic mucosal damage. Healthy human colonic mucosa was incubated with pro-inflammatory cytokines TNF-α and IL-1ß to elicit colitis-like tissue damage. The cytokine-driven increase in scored crypt and mucosal damage and lymphocyte density was attenuated with concomitant hydrocortisone pretreatment. The cannabinoid receptor 2 (CB2) receptor-selective agonist JWH-015 significantly reduced colitis scores following cytokine incubation, as evidenced by a reduction in mucosal crypt and luminal epithelial damage and lymphocyte density in the lamina propria. The effect of JWH-015 was reversed in the presence of the CB2 receptor inverse agonist JTE-907. Anandamide was also protective in the cytokine-incubated explant colitis model in a manner reversible with JTE-907, while CB1 receptor agonism with ACEA was without effect. TNF-α and IL-1ß together evoked an increase in paracellular epithelial permeability in Caco-2 cell monolayers over 48h of incubation. However, neither CB2 nor CB1 receptor activation altered the cytokine-evoked increase in permeability. These findings support a discrete role for CB2 receptors in the attenuation of detrimental pro-inflammatory cytokine-mediated mucosal damage in the human colon without directly affecting mucosal epithelial barrier function.

Vertical transmission of Citrobacter diversus documented by DNA fingerprinting.

OBJECTIVE: To confirm the vertical transmission of Citrobacter diversus from a mother to her infant and to evaluate the epidemiologic usefulness of a new automated procedure for analysis of polymerase chain reaction (PCR)-generated DNA fingerprints. DESIGN: Repetitive element-based PCR (rep-PCR) analysis of C diversus isolates from the blood and amniotic fluid of a mother and the blood of her infant was performed. Unrelated C diversus isolates also were characterized and compared with the isolates from mother and infant. DNA fingerprints were generated by gel electrophoresis of PCR products derived from either unlabeled standard repetitive sequence-based oligonucleotide primers or fluorescent primers. The standard rep-PCR fingerprints were analyzed by visual inspection. The fluorescent primers were used in fluorophore-enhanced rep-PCR (FERP), and the FERP DNA fingerprints were analyzed by an Applied BioSystems (ABI) Model 373A laser scanning unit equipped with Genescan 672 software (Applied Biosystems, Inc, Foster City, CA). SETTING AND PATIENTS: A mother and her newborn infant, both with invasive disease due to C diversus, in an urban tertiary-care hospital. RESULTS: The DNA fingerprints of the maternal blood, amniotic fluid, and infant blood isolates of C diversus were identical by both visual inspection of ethidium bromide-stained agarose gels and computer-aided analysis of FERP patterns. These strains appeared to differ from all but one control isolate, which had been collected 7 years earlier in the same city in which the infant was born. CONCLUSIONS: Vertical transmission of C diversus from mother to infant can occur in utero. Automated analysis of rep-PCR-generated DNA fingerprints derived using fluorescent primers is an objective means for comparing isolates of C diversus and in all likelihood would be useful for other species of bacteria that possess repetitive elements.

Contributions of complement and immunoglobulin to neutrophil-mediated killing of enterococci.

Enterococci have become a frequent causative agent in neonatal sepsis. The relative contributions of antibody and complement and their interactions in the neutrophil-mediated bacterial killing of 11 Enterococcus strains from neonates were investigated. Polymorphonuclear leukocytes (PMNL) from adult and term newborn infants were tested with normal human serum, adult hypogammaglobulinemic serum, and normal newborn serum in a neutrophil bactericidal assay. Neutrophil bactericidal activity for enterococci was not influenced by the serum source but was essentially ablated after heat inactivation of complement in all sera. No differences were observed in the killing capacity of healthy newborn versus adult PMNL regardless of serum source. Representative Enterococcus strains were then tested with agammaglobulinemic serum or C4-deficient serum, resulting in neutrophil bactericidal activities consistently exceeding 90%. A neutrophil bactericidal assay performed with normal rabbit serum and hyperimmune rabbit serum against enterococci showed that antibodies to enterococci enhanced neutrophil-mediated killing of this organism. Thus, neutrophil killing of enterococci appears to be mediated primarily by complement, with antibody playing a less essential but potentially important role. PMNL from adult and healthy term infants functioned with equal efficiency in the neutrophil killing of enterococci.