No abstract

Vaccination has been proposed for the prevention of disease due to enterohemorrhagic Escherichia coli (EHEC), but the immune response following human infection, including the choice of potential antigens, has not been well characterized. To study this, sera were obtained from five pediatric patients with acute diarrhea caused by E. coli O157:H7 0, 8, and 60 days after hospitalization. These sera were used to examine the immune response to four different EHEC virulence factors: Tir (translocated intimin receptor, which is inserted into the host cell membrane), intimin (bacterial outer membrane protein which binds to Tir), EspA (secreted protein which forms filamentous structures on EHEC surface), and EspB (inserted into the host membrane and cytoplasm). The response to O157:H7 lipopolysaccharide was also examined. Sera were assayed against purified recombinant proteins using immunoblot analysis and by enzyme-linked immunosorbent assay to determine the sera's titers to each of the antigens in all patients. We found that there was little reaction to EspA, EspB, and intimin in the acute-phase sera, although there was some reactivity to Tir. By day 8, titers of antibody to all four virulence factors were present in all patients, with a very strong response against Tir (up to a titer of 1:256,000), especially in hemolytic-uremic syndrome patients, and lesser strong responses to the other three antigens. The titer to the antigens 60 days after hospitalization was decreased but was still highest for Tir. These results suggest that there is a strong immune response to Tir, and to a lesser extent to the other three virulence factors, following EHEC disease, indicating that these bacterial molecules are potential vaccine candidates for preventing EHEC disease. They also suggest that bacterial virulence factors that are inserted into host cells during infection by type III secretion systems (Tir or EspB) are still recognized by the host immune response.Enterohemorrhagic Escherichia coli (EHEC) (also called verotoxigenic E. coli) is an important cause of diarrhea. This organism also produces a toxin which plays a role in disease (verotoxin or Shiga-like toxin [SLT]). Most patients with EHEC infection recover uneventfully within a few days, but in about 8% of cases (23) the diarrhea is followed by hemolyticuremic syndrome (HUS), a life-threatening complication with a substantial morbidity in survivors (19,25). HUS appears to be caused by the interaction of SLT with endothelial cells. Worldwide, many serotypes of EHEC have been described, but in North America one type predominates, E. coli O157:H7 (11, 12). Epidemiological evidence shows that EHEC O157:H7 strains are present in the feces of healthy cattle, providing indirect evidence that EHEC O157:H7 can colonize the bovine intestine without causing disease.Because of the potential seriousness of EHEC infection (4), vaccination has been proposed, either to prevent the disease in humans or to reduce colonization in cattle. However, the immunological response to EHEC infection ...

A double-blind, placebo-controlled study was conducted to document possible side effects associated with oral consumption of synthetic verotoxin (VT, shiga-like toxin) Pk-trisaccharide receptor sequences attached to Chromosorb (Synsorb-Pk) by healthy adult volunteers. Synsorb-Pk reclaimed from volunteer stool samples was also analyzed to determine if its VT-binding activity was affected by exposure to the pH extremes and digestive processes of the human gastrointestinal tract. No participant reported any Synsorb-Pk-related adverse reactions, and no clinically important trends in laboratory data were evident. Synsorb-Pk recovered from stools retained its ability to absorb VT in polymyxin extracts of VT-producing Escherichia coli and also neutralized VT when mixed in vitro with VT-positive stools from children with hemorrhagic colitis or hemolytic-uremic syndrome (HUS). These results suggest a potential use for Synsorb-Pk in preventing HUS in patients infected with VT-producing E. coli.

The neuronal apoptosis-inhibitory protein (NAIP) is the founding member of the mammalian family of inhibitor of apoptosis (IAP) proteins (also known as BIRC proteins) and has been shown to be antiapoptotic both in vivo and in vitro. The 160-kDa NAIP contains three distinct regions: an amino-terminal cluster of three baculoviral inhibitory repeat (BIR) domains, a central nucleotide binding oligomerization domain (NOD), and a carboxyl-terminal leucine-rich repeat (LRR) domain. The presence of the NOD and LRR domains renders NAIP unique among the IAPs and suggests that NAIP activity is regulated in a manner distinct from that of other members of the family. In this report, we examined the interaction of various regions of NAIP with caspase-9 and Smac. Recombinant NAIPs with truncations of the carboxyl-terminal LRR or NOD-LRR regions bound to caspase-9. In contrast, the full-length protein did not, suggesting some form of structural autoregulation. However, the association of the wild type fulllength protein with caspase-9 was observed when interaction analysis was performed in the presence of ATP. Furthermore, mutation of the NAIP ATP binding pocket allowed full-length protein to interact with caspase-9. Thus, we conclude that NAIP binds to caspase-9 with a structural requirement for ATP and that in the absence of ATP the LRR domain negatively regulates the caspase-9-inhibiting activity of the BIR domains. Interestingly, and in contrast to the X-chromosome-linked inhibitor of apoptosis protein (XIAP), NAIP-mediated inhibition of caspase-9 was not countered by a peptide containing an amino-terminal IAP binding motif (IBM). Consistent with this observation was the failure of Smac protein to interact with the NAIP BIR domains. These results demonstrate that NAIP is distinct from the other IAPs, both in demonstrating a ligand-dependent caspase-9 interaction and in demonstrating a distinct mechanism of inhibition.

Projections from the lateral hypothalamic area (LHA) innervate components of the mesolimbic dopamine (MLDA) system, including the ventral tegmental area (VTA) and nucleus accumbens (NAc), to modulate motivation appropriately for physiologic state. Neurotensin (NT)-containing LHA neurons respond to multiple homeostatic challenges and project to the VTA, suggesting that these neurons could link such signals to MLDA function. Indeed, we found that pharmacogenetic activation of LHA NT neurons promoted prolonged DA-dependent locomotor activity and NAc DA efflux, suggesting the importance of VTA neurotransmitter release by LHA NT neurons for the control of MLDA function. Using a microdialysis-mass spectrometry technique that we developed to detect endogenous NT in extracellular fluid in the mouse brain, we found that activation of LHA NT cells acutely increased the extracellular concentration of NT (a known activator of VTA DA cells) in the VTA. In contrast to the prolonged elevation of extracellular NAc DA, however, VTA NT concentrations rapidly returned to baseline. Intra-VTA infusion of NT receptor antagonist abrogated the ability of LHA NT cells to increase extracellular DA in the NAc, demonstrating that VTA NT promotes NAc DA release. Thus, transient LHA-derived NT release in the VTA couples LHA signaling to prolonged changes in DA efflux and MLDA function.