A case series of the dynamics of lipid mediators in patients with sepsis.

BACKGROUND: Bioactive lipid mediators play a crucial role during infection. Previously, we showed the expression level of FAAH mRNA in septic patients was lower than in healthy controls. CASE PRESENTATION: Four patients with a Sequential Organ Failure Assessment (SOFA) score of <7 recovered from sepsis. One patient with SOFA score of 12 on day 7 died on day 21. In the fatal case, eicosapentaenoic acid, docosahexaenoic acid, arachidonic acid, and linoleic acid-derived lipid mediators, including 9-hydroxyoctadecadienoic acid (9-HODE), 13-HODE, 9,10-dihydroxy-12-octadecenoic acid (9,10-DiHOME), and 12,13-DiHOME, were elevated on day 1. Increase in anti-inflammatory prostaglandin E1 ethanolamide together with persistently lower transcription level of FAAH mRNA was detected on day 7 in the fatal case. CONCLUSION: Lipidomic analysis on day 1 revealed elevated linoleic acid metabolites, whereas on day 7, elevated prostaglandin E1 ethanolamide and low level of FAAH mRNA transcription were observed in the fatal case of sepsis.

Structural basis of α-catenin recognition by EspB from enterohaemorrhagic E. coli based on hybrid strategy using low-resolution structural and protein dissection.

Enterohaemorrhagic E. coli (EHEC) induces actin reorganization of host cells by injecting various effectors into host cytosol through type III secretion systems. EspB is the natively partially folded EHEC effector which binds to host α-catenin to promote the actin bundling. However, its structural basis is poorly understood. Here, we characterize the overall structural properties of EspB based on low-resolution structural data in conjunction with protein dissection strategy. EspB showed a unique thermal response involving cold denaturation in the presence of denaturant according to far-UV circular dichroism (CD). Small angle X-ray scattering revealed the formation of a highly extended structure of EspB comparable to the ideal random coil. Various disorder predictions as well as CD spectra of EspB fragments identified the presence of α-helical structures around G41 to Q70. The fragment corresponding to this region indicated the thermal response similar to EspB. Moreover, this fragment showed a high affinity to C-terminal vinculin homology domain of α-catenin. The results clarified the importance of preformed α-helix of EspB for recognition of α-catenin.

Cytoskeleton-modulating effectors of enteropathogenic and enterohemorrhagic Escherichia coli: a case for EspB as an intrinsically less-ordered effector.

Enterohemorrhagic and enteropathogenic Escherichia coli produce various effector proteins that are directly injected into the host-cell cytosol through the type III secretion system. E. coli secreted protein (Esp)B is one such effector protein, and affects host-cell morphology by reorganizing actin networks. Unlike most globular proteins that have well-ordered, rigid structures, the structures of type III secretion system effectors from pathogenic Gram-negative bacteria, including EspB, are often less well-ordered. This minireview focuses on the functional relationship between the structural properties of these proteins and their roles in type III secretion system-associated pathogenesis.

Reduced acetaminophen-induced liver injury in mice by genetic disruption of IL-1 receptor antagonist.

Acetaminophen (APAP) induced increases in intrahepatic expression of interleukin (IL)-1 alpha, IL-1 beta, and IL-1 receptor antagonist (IL-1ra), when administered intraperitoneally. These observations prompted us to define the pathophysiological roles of IL-1ra in APAP-induced liver injury. Compared with wild-type (WT) mouse-derived hepatocytes, IL-1ra-deficient (IL-1ra KO)-derived hepatocytes exhibited more resistance against APAP but not APAP-derived major toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Moreover, the amounts of a major APAP adduct (selenium-binding protein), an indicator of NAPQI generation from APAP, was significantly lower in IL-1ra KO mice than WT mice with depressed intrahepatic expression of CYP1A2, CYP2E1, and CYP3A11, the enzymes crucially involved in NAPQI generation from APAP. These observations would indicate that IL-1ra deficiency impaired APAP metabolism. IL-1 alpha and IL-1 beta were expressed to similar extents in livers of untreated IL-1ra KO and WT mice. By contrast, the intranuclear amount of p65 of NF-kappaB, which can suppress the gene expression of CYP1A2, CYP2E1, and CYP3A11, was higher in untreated IL-1ra KO than WT mice. Moreover, when mice were intraperitoneally administered APAP (200 mg/kg), IL-1ra KO mice exhibited attenuated APAP-induced liver injury as evidenced by reductions in serum alanine transferase levels and histopathological changes such as centrilobular necrosis, hemorrhages, and leukocyte infiltration. Finally, when given 12 h before APAP challenge, IL-1 alpha repressed the intrahepatic expression of CYP1A2, CYP2E1, and CYP3A11, eventually reducing APAP-induced liver injury, along with reduction in APAP adducts. Collectively, NF-kappaB was activated without any stimuli by the genetic disruption of IL-1ra, and suppressed cytochrome P450 enzyme expression, thereby reducing APAP-induced liver injury.

Molecular basis of actin reorganization promoted by binding of enterohaemorrhagic Escherichia coli EspB to alpha-catenin.

EspB is a multifunctional protein associated with the type III secretion system of enterohaemorrhagic Escherichia coli, and interacts with various biomolecules including alpha-catenin in the host cell. The binding of EspB to alpha-catenin is thought be involved in actin reorganization during bacterial infection, although the precise mechanism of this phenomenon is still unclear. Recent research shows that dimerization of alpha-catenin dissociates it from E-cadherin/beta-catenin/alpha-catenin complexes, and that the dimer suppresses Arp2/3-mediated actin branching or polymerization. These results inspired us to evaluate the effect of EspB on the functions of alpha-catenin. Based on a series of in vitro biochemical approaches, including pull-down, co-sedimentation and pyrene-actin polymerization assays combined with transmission electron microscopy, we conclude that EspB promotes all the functions of dimeric alpha-catenin described above. These results clarified the molecular basis of reorganization of actin filaments during infection with enterohaemorrhagic Escherichia coli.

The mRNA expression of fatty acid amide hydrolase in human whole blood correlates with sepsis.

An excessive accumulation of anandamide (N-archidonoylethanolamine, AEA) is associated with septic shock. Results of previous studies have suggested that mRNA coding for the AEA degrading enzyme fatty acid amide hydrolase (FAAH), which converts AEA into arachidonic acid and ethanolamine, might be down-regulated in septic shock. We used real-time reverse transcription PCR assays to measure relative FAAH mRNA concentrations in the whole blood of 30 healthy donors and eight sepsis patients to ascertain whether such down-regulation takes place. Our results suggest that concentrations of FAAH mRNA in male and female samples from healthy donors are similar, but that concentrations are significantly lower in sepsis patients. These findings indicate that mRNA expression of FAAH in human whole blood correlates with sepsis, and may be an interesting biomarker for predicting the onset of septic shock.

Severe acute pancreatitis caused by organophosphate poisoning.

Organophosphate intoxication may be caused pancreatitis, but the role of diagnostic imaging for pancreatitis in these patients has not been well defined. We recently encountered a patient with organophosphate poisoning showing hyperamylasemia who was proven to have severe acute pancreatitis by CT findings. The patient was a 69-year-old woman who presented to a local hospital with disturbance of consciousness. She was initially treated for cerebral infarction, but an extremely low level of ChE was noted on Day 3. The patient was then referred to our institution. Examination of the abdomen revealed weak intestinal peristalsis, blood chemistry showed an increased level of serum amylase, and the urinary organophosphate test was positive. Based on the findings obtained by abdominal CT scanning, severe acute pancreatitis was diagnosed. Clouding of her consciousness resolved on day 21, but a pancreatic pseudocyst was detected on day 41.

[Development of a simple method of detecting acetaminophen using 96 well microplate readers].

Acetaminophen (APAP: N-Acetyl-p-aminophenol) has been widely used as a relatively safe antipyretic and analgesic drug. However, APAP is known as a major causative agent of fulminant hepatic failure, in a dose and a blood concentration dependent manner, in Western countries. The APAP toxicity should be expected to increase in proportion as the increasing of the clinical use in Japan. Therefore, a simplified method which determines the concentration of APAP would be valuable for clinical use. We have modified the APAP determination method developed by Yasojima. Our method enabled to measure APAP concentration with a small volume of samples by using 96-well microtiter plate, which can handle multiple samples simultaneously. From 10 microg/ml to 320 microg/ml of APAP was quantitatively measured by our method, which is suitable for diagnostics of the APAP toxicity. APAP levels below the lethal concentration of 160 microg/ml can be determined with one plate.