The sialyl-O-acetylesterase NanS of Tannerella forsythia encompasses two catalytic modules with different regiospecificity for O7 and O9 of sialic acid.

The periodontal pathogen Tannerella forsythia utilizes host sialic acids as a nutrient source. To also make O-acetylated sialyl residues susceptible to the action of its sialidase and sialic acid uptake system, Tannerella produces NanS, an O-acetylesterase with two putative catalytic domains. Here, we analyzed NanS by homology modeling, predicted a catalytic serine-histidine-aspartate triad for each catalytic domain and performed individual domain inactivation by single alanine exchanges of the triad nucleophiles S32 and S311. Subsequent functional analyses revealed that both domains possess sialyl-O-acetylesterase activity, but differ in their regioselectivity with respect to position O9 and O7 of sialic acid. The 7-O-acetylesterase activity inherent to the C-terminal domain of NanS is unique among sialyl-O-acetylesterases and fills the current gap in tools targeting 7-O-acetylation. Application of the O7-specific variant NanS-S32A allowed us to evidence the presence of cellular 7,9-di-O-acetylated sialoglycans by monitoring the gain in 9-O-acetylation upon selective removal of acetyl groups from O7. Moreover, we established de-7,9-O-acetylation by wild-type NanS as an easy and efficient method to validate the specific binding of three viral lectins commonly used for the recognition of (7),9-O-acetylated sialoglycans. Their binding critically depends on an acetyl group in O9, yet de-7,9-O-acetylation proved advantageous over de-9-O-acetylation as the additional removal of the 7-O-acetyl group eliminated ligand formation by 7,9-ester migration. Together, our data show that NanS gained dual functionality through recruitment of two esterase modules with complementary activities. This enables Tannerella to scavenge 7,9-di-O-acetylated sialyl residues and provides a novel, O7-specific tool for studying sialic acid O-acetylation.

Stress is a bad advisor. Stress primes poor decision making in deluded psychotic patients.

Stress is implicated in the onset of psychosis but the complex links between stress and psychotic breakdown are yet poorly understood. For the present study, we examined whether two prominent cognitive biases in psychosis, jumping to conclusions and distorted attribution, in conjunction with neuropsychological deficits play a role in this process. Thirty participants with schizophrenia and acute delusional symptoms were compared with 29 healthy controls across three conditions involving a noise stressor, a social stressor or no stressor. Under each condition participants had to perform parallel versions of cognitive bias tasks and neuropsychological tests including a probabilistic reasoning task (jumping to conclusions), the revised Internal, Personal and Situational Attributions Questionnaire (IPSAQ-R; attributional style), and the Corsi block-tapping task (nonverbal memory). Stress, particularly noise, aggravated performance differences of patients relative to controls on memory. Participants with psychosis demonstrated an escalated jumping to conclusion bias under stress. At a medium effect size, patients made more monocausal attributions, which increased under social stress. The present study is partially in line with prior studies. It suggests that stress negatively affects cognition in psychosis more than in controls, which is presumably insufficiently realized by patients and thus not held in check by greater response hesitance. Raising patients' awareness about these response tendencies and encouraging them to be more cautious in their judgments under conditions of increased stress may prove beneficial for improving positive symptoms.

Do People With Psychosis Have Specific Difficulties Regulating Emotions?

UNLABELLED: Difficulties in emotion regulation (ER) are present in psychotic disorders, but their precise nature is not yet fully understood and it is unclear which difficulties are unique to psychosis compared with other disorders. This study investigated whether ER difficulties in psychosis are more prominent for the ability to modify emotions or for the ability to tolerate and accept them. Furthermore, it investigated whether ER difficulties occur for sadness, anxiety, anger and shame likewise. ER skills were assessed in participants with psychotic disorders (n = 37), participants with depression (n = 30) and healthy controls (n = 28) using the Emotion Regulation Skill Questionnaire that asks participants to rate the intensity of different emotions over the past week and the skills employed to handle each of them. Compared with healthy controls, participants with psychosis showed reduced skills related to awareness, understanding and acceptance of potentially distressing emotions, but not in the ability to modify them. These differences remained significant after controlling for depression. Participants with psychosis showed reduced ER skills in regard to all of the assessed emotions compared with the healthy controls, despite the fact that they only reported sadness as being significantly more intense. The participants with depression showed a similar pattern of ER skills to the psychosis sample, although with a tendency towards even more pronounced difficulties. It is concluded that psychosis is characterized by difficulties in using specific ER skills related to awareness, understanding and acceptance to regulate anger, shame, anxiety and sadness. These difficulties are not unique to psychosis but nevertheless present a promising treatment target. KEY PRACTITIONER MESSAGE: The participants with psychosis found it more difficult to be aware of their emotions, to understand them and to accept them than the healthy control group. However, they reported equal skills when it came to actively modifying emotions. The difficulties in emotion regulation reported by the participants with psychosis were comparable with those reported by the participants with depression, and they occurred for all types of negative emotions likewise. The difficulties in using specific ER skills related to awareness, understanding and acceptance are a promising target for psychological treatment of psychosis. Interventions that are aimed specifically at increasing these skills need to be further developed.

Polysialylation of the synaptic cell adhesion molecule 1 (SynCAM 1) depends exclusively on the polysialyltransferase ST8SiaII in vivo.

Polysialic acid is a unique carbohydrate polymer specifically attached to a limited number of glycoproteins. Among them is synaptic cell adhesion molecule 1 (SynCAM 1), a member of the immunoglobulin (Ig) superfamily composed of three extracellular Ig-like domains. Polysialylation of SynCAM 1 is cell type-specific and was exclusively found in NG2 cells, a class of multifunctional progenitor cells that form specialized synapses with neurons. Here, we studied the molecular requirements for SynCAM 1 polysialylation. Analysis of mice lacking one of the two polysialyltransferases, ST8SiaII or ST8SiaIV, revealed that polysialylation of SynCAM 1 is exclusively mediated by ST8SiaII throughout postnatal brain development. Alternative splicing of the three variable exons 8a, 8b, and 8c can theoretically give rise to eight transmembrane isoforms of SynCAM 1. We detected seven transcript variants in the developing mouse brain, including three variants containing exon 8c, which was so far regarded as a cryptic exon in mice. Polysialylation of SynCAM 1 was restricted to four isoforms in perinatal brain. However, cell culture experiments demonstrated that all transmembrane isoforms of SynCAM 1 can be polysialylated by ST8SiaII. Moreover, analysis of domain deletion constructs revealed that Ig1, which harbors the polysialylation site, is not sufficient as an acceptor for ST8SiaII. The minimal polypeptide required for polysialylation contained Ig1 and Ig2, suggesting an important role for Ig2 as a docking site for ST8SiaII.

Synaptic cell adhesion molecule SynCAM 1 is a target for polysialylation in postnatal mouse brain.

Among the large set of cell surface glycan structures, the carbohydrate polymer polysialic acid (polySia) plays an important role in vertebrate brain development and synaptic plasticity. The main carrier of polySia in the nervous system is the neural cell adhesion molecule NCAM. As polySia with chain lengths of more than 40 sialic acid residues was still observed in brain of newborn Ncam(-/-) mice, we performed a glycoproteomics approach to identify the underlying protein scaffolds. Affinity purification of polysialylated molecules from Ncam(-/-) brain followed by peptide mass fingerprinting led to the identification of the synaptic cell adhesion molecule SynCAM 1 as a so far unknown polySia carrier. SynCAM 1 belongs to the Ig superfamily and is a powerful inducer of synapse formation. Importantly, the appearance of polysialylated SynCAM 1 was not restricted to the Ncam(-/-) background but was found to the same extent in perinatal brain of WT mice. PolySia was located on N-glycans of the first Ig domain, which is known to be involved in homo- and heterophilic SynCAM 1 interactions. Both polysialyltransferases, ST8SiaII and ST8SiaIV, were able to polysialylate SynCAM 1 in vitro, and polysialylation of SynCAM 1 completely abolished homophilic binding. Analysis of serial sections of perinatal Ncam(-/-) brain revealed that polySia-SynCAM 1 is expressed exclusively by NG2 cells, a multifunctional glia population that can receive glutamatergic input via unique neuron-NG2 cell synapses. Our findings sug-gest that polySia may act as a dynamic modulator of SynCAM 1 functions during integration of NG2 cells into neural networks.

Naturally induced biphasic phytoplankton spring bloom reveals rapid and distinct substrate and bacterial community dynamics.

Phytoplankton spring blooms are typical features in coastal seas and provide heterotrophic bacteria with a rich blend of dissolved substrates. However, they are difficult to study in coastal seas in-situ. Here, we induced a phytoplankton spring bloom and followed its fate for 37 days in four 600 L-mesocosms. To specifically investigate the significance of phytoplankton-born dissolved organic carbon (DOC) we used artificial seawater with low DOC background and inoculated it with a 100 µm-prefiltered plankton community from the North Sea. A biphasic bloom developed, dominated by diatoms and Phaeocystis globosa respectively. In between, bacterial numbers peaked, followed by a peak in virus-like particles, implying that virus infection caused the collapse. Concentrations of dissolved free amino acids exhibited rapid changes, in particular during the diatom bloom and until the peak in bacterial abundance. Dissolved combined amino acids and neutral monosaccharides accumulated continuously, accounting for 22% of DOC as a mean and reaching levels as high as 44%. Bacterial communities were largely dominated by Bacteroidetes, especially the NS3a marine group (family Flavobacteriaceae), but Rhodobacteraceae and Gammaproteobacteria were also prominent members. Our study shows rapid organic matter and community composition dynamics that are hard to trace in natural coastal ecosystems.

Inhibitors of the Actin-Bundling Protein Fascin-1 Developed for Tumor Therapy Attenuate the T-Cell Stimulatory Properties of Dendritic Cells.

BACKGROUND: Stimulated dendritic cells (DCs), which constitute the most potent population of antigen-presenting cells (APCs), express the actin-bundling protein Fascin-1 (Fscn1). In tumor cells, de novo expression of Fscn1 correlates with their invasive and metastatic properties. Therefore, Fscn1 inhibitors have been developed to serve as antitumor agents. In this study, we were interested in better understanding the impact of Fscn1 inhibitors on DCs. METHODS: In parallel settings, murine spleen cells and bone-marrow-derived DCs (BMDCs) were stimulated with lipopolysaccharide in the presence of Fscn1 inhibitors (NP-G2-044 and BDP-13176). An analysis of surface expression of costimulatory and coinhibitory receptors, as well as cytokine production, was performed by flow cytometry. Cytoskeletal alterations were assessed by confocal microscopy. The effects on the interactions of BMDCs with antigen-specific T cells were monitored by time lapse microscopy. The T-cell stimulatory and polarizing capacity of BMDCs were measured in proliferation assays and cytokine studies. RESULTS: Administration of Fscn1 inhibitors diminished Fscn1 expression and the formation of dendritic processes by stimulated BMDCs and elevated CD273 (PD-L2) expression. Fscn1 inhibition attenuated the interaction of DCs with antigen-specific T cells and concomitant T-cell proliferation. CONCLUSIONS: Systemic administration of Fscn1 inhibitors for tumor therapy may also modulate DC-induced antitumor immune responses.

ß2 Integrins on Dendritic Cells Modulate Cytokine Signaling and Inflammation-Associated Gene Expression, and Are Required for Induction of Autoimmune Encephalomyelitis.

Heterodimeric ß2 integrin surface receptors (CD11a-d/CD18) are specifically expressed by leukocytes that contribute to pathogen uptake, cell migration, immunological synapse formation and cell signaling. In humans, the loss of CD18 expression results in leukocyte adhesion deficiency syndrome (LAD-)1, largely characterized by recurrent severe infections. All available mouse models display the constitutive and ubiquitous knockout of either α or the common ß2 (CD18) subunit, which hampers the analysis of the cell type-specific role of ß2 integrins in vivo. To overcome this limitation, we generated a CD18 gene floxed mouse strain. Offspring generated from crossing with CD11c-Cre mice displayed the efficient knockdown of ß2 integrins, specifically in dendritic cells (DCs). Stimulated ß2-integrin-deficient splenic DCs showed enhanced cytokine production and the concomitantly elevated activity of signal transducers and activators of transcription (STAT) 1, 3 and 5, as well as the impaired expression of suppressor of cytokine signaling (SOCS) 2-6 as assessed in bone marrow-derived (BM) DCs. Paradoxically, these BMDCs also showed the attenuated expression of genes involved in inflammatory signaling. In line, in experimental autoimmune encephalomyelitis mice with a conditional DC-specific ß2 integrin knockdown presented with a delayed onset and milder course of disease, associated with lower frequencies of T helper cell populations (Th)1/Th17 in the inflamed spinal cord. Altogether, our mouse model may prove to be a valuable tool to study the leukocyte-specific functions of ß2 integrins in vivo.

Impact of the rpoS genotype for acid resistance patterns of pathogenic and probiotic Escherichia coli.

BACKGROUND: Enterohemorrhagic E. coli (EHEC), a subgroup of Shiga toxin (Stx) producing E. coli (STEC), may cause severe enteritis and hemolytic uremic syndrome (HUS) and is transmitted orally via contaminated foods or from person to person. The infectious dose is known to be very low, which requires most of the bacteria to survive the gastric acid barrier. Acid resistance therefore is an important mechanism of EHEC virulence. It should also be a relevant characteristic of E. coli strains used for therapeutic purposes such as the probiotic E. coli Nissle 1917 (EcN). In E. coli and related enteric bacteria it has been extensively demonstrated, that the alternative sigma factor sigmaS, encoded by the rpoS gene, acts as a master regulator mediating resistance to various environmental stress factors. METHODS: Using rpoS deletion mutants of a highly virulent EHEC O26:H11 patient isolate and the sequenced prototype EHEC EDL933 (ATCC 700927) of serotype O157:H7 we investigated the impact of a functional rpoS gene for orchestrating a satisfactory response to acid stress in these strains. We then functionally characterized rpoS of probiotic EcN and five rpoS genes selected from STEC isolates pre-investigated for acid resistance. RESULTS: First, we found out that ATCC isolate 700927 of EHEC EDL933 has a point mutation in rpoS, not present in the published sequence, leading to a premature stop codon. Moreover, to our surprise, one STEC strain as well as EcN was acid sensitive in our test environment, although their cloned rpoS genes could effectively complement acid sensitivity of an rpoS deletion mutant. CONCLUSION: The attenuation of sequenced EHEC EDL933 might be of importance for anyone planning to do either in vitro or in vivo studies with this prototype strain. Furthermore our data supports recently published observations, that individual E. coli isolates are able to significantly modulate their acid resistance phenotype independent of their rpoS genotype.

Dealing with feeling: Specific emotion regulation skills predict responses to stress in psychosis.

Elevated negative affect is an established link between minor stressors and psychotic symptoms. Less clear is why people with psychosis fail to regulate distressing emotions effectively. This study tests whether subjective, psychophysiological and symptomatic responses to stress can be predicted by specific emotion regulation (ER) difficulties. Participants with psychotic disorders (n=35) and healthy controls (n=28) were assessed for ER-skills at baseline. They were then exposed to a noise versus no stressor on different days, during which self-reported stress responses, state paranoia and skin conductance levels (SCL) were assessed. Participants with psychosis showed a stronger increase in self-reported stress and SCL in response to the stressor than healthy controls. Stronger increases in self-reported stress were predicted by a reduced ability to be aware of and tolerate distressing emotions, whereas increases in SCL were predicted by a reduced ability to be aware of, tolerate, accept and modify them. Although paranoid symptoms were not significantly affected by the stressors, individual variation in paranoid responses was also predicted by a reduced ability to be aware of and tolerate emotions. Differences in stress responses in the samples were no longer significant after controlling for ER skills. Thus, interventions that improve ER-skills could reduce stress-sensitivity in psychosis.

Responses to stress in patients with psychotic disorders compared to persons with varying levels of vulnerability to psychosis, persons with depression and healthy controls.

BACKGROUND AND OBJECTIVES: An experimental design was used to test whether self-reported, psychophysiological and symptomatic stress-responses increase as a function of the underlying vulnerability to psychosis as proposed by vulnerability-stress-models. METHODS: Stress-responses of participants with psychotic disorders (PSY, n = 35) were compared to those of participants with attenuated positive symptoms (AS, n = 29), first-degree relatives of persons with psychotic disorders (REL, n = 26), healthy controls (HC, n = 28) and controls with depression (DEP, n = 30). Using a repeated measures design, participants were assigned to a noise stressor, a social stressor and a no stress condition in random order. Stress-responses were assessed via self-report, salivary cortisol levels, heart rate and skin conductance levels. State-paranoia and depression were assessed with clinical scales. RESULTS: PSY reported to be significantly more stressed than HC, AS and REL across all conditions which went along with increased heart rate and decreased overall cortisol release. In contrast, AS showed elevated levels of cortisol. PSY showed a stronger response of self-reported stress to the noise condition compared to the no stress condition than HC, but no stronger response than the other samples. Furthermore, the stressors did not trigger stronger psychophysiological responses or symptom-increases in PSY. LIMITATIONS: The social stressor was brief and not individualized and did not have an effect on cortisol. CONCLUSIONS: The findings support the notion that subjective stress-responsiveness increases with vulnerability, but not the assumption that symptoms arise directly as a function of stress and vulnerability. Also, the generally high levels of arousal seem to be more relevant to psychosis than the responsiveness to specific stressors.

Bridging psychophysiological and phenomenological characteristics of psychosis--Preliminary evidence for the relevance of emotion regulation.

In psychosis, the alleged increased subjective stress-sensitivity is reflected in abnormal physiological arousal such as higher heart rate, elevated skin conductance levels, decreased vagal activity, and unusual cortisol levels. Despite ongoing research, possible mechanisms that explain the interplay between the phenomenological (i.e., subjective stress and symptoms) and psychophysiological processes are not thoroughly understood. Building on the model of neurovisceral integration by Thayer and Lane (2000) that focuses on regulative mechanisms, we postulate that emotion regulation will be associated with vagal activity, and with both subjective and physiological stress. In the present analysis, we used data from a baseline relaxation period including a 5-minute assessment of heart rate variability (HRV), salivary cortisol, and momentary subjective stress ratings from a sample of 19 participants with psychosis (mean age=40.9, SD=11.1; 36.8% female). Emotion regulation modification skills were assessed for specific emotions (i.e., stress and arousal, anxiety, anger, sadness, shame) if these were present during the previous week. Vagal HRV was significantly and moderately associated with emotion regulation. Both stress parameters (i.e., cortisol, subjective stress) were significantly associated with emotion regulation, but not with HRV. We provide preliminary support for the notion that emotion regulatory processes represent a crucial link between phenomenological and psychophysiological phenomena in psychosis. A potential model that ascribes emotion regulation a central role in the restoration of homeostasis is discussed. Future studies are needed to verify its generalizability and predictive value.

9-O-Acetylation of sialic acids is catalysed by CASD1 via a covalent acetyl-enzyme intermediate.

Sialic acids, terminal sugars of glycoproteins and glycolipids, play important roles in development, cellular recognition processes and host-pathogen interactions. A common modification of sialic acids is 9-O-acetylation, which has been implicated in sialoglycan recognition, ganglioside biology, and the survival and drug resistance of acute lymphoblastic leukaemia cells. Despite many functional implications, the molecular basis of 9-O-acetylation has remained elusive thus far. Following cellular approaches, including selective gene knockout by CRISPR/Cas genome editing, we here show that CASD1--a previously identified human candidate gene--is essential for sialic acid 9-O-acetylation. In vitro assays with the purified N-terminal luminal domain of CASD1 demonstrate transfer of acetyl groups from acetyl-coenzyme A to CMP-activated sialic acid and formation of a covalent acetyl-enzyme intermediate. Our study provides direct evidence that CASD1 is a sialate O-acetyltransferase and serves as key enzyme in the biosynthesis of 9-O-acetylated sialoglycans.

Altered autonomic arousal in psychosis: an analysis of vulnerability and specificity.

Vulnerability-stress models implicate that alterations of the autonomous nervous system contribute to the development of psychosis. Previous research has found autonomic arousal alterations in psychotic disorders and at-risk individuals that are not explained by medication alone. To test whether these alterations are associated with the extent of an individual's vulnerability and whether they are specific to psychosis, we compared participants with psychosis (n=23), first-degree relatives of individuals with psychosis (n=21), and healthy participants with attenuated positive symptoms (n=23) to participants with depression (n=24) and healthy controls (n=24). At rest, skin conductance level was assessed and photoplethysmography was applied to measure time- and frequency-domain heart rate variability (HRV). Univariate and multivariate analyses of covariance with perceived stress and psychophysiological values as dependent variables showed significant between-group differences for perceived stress (p=.010), heart rate (p=.022), time-domain HRV indices (all ps≤.027), and vagal activity (p=.017). Group differences in sympathetic activity were nonsignificant (p=.069). In an additional analysis with medication as a second between-group factor, the physiological between-group differences remained significant or trend significant (all ps≤.060). With the exception of sympathetic activity, participants with psychosis exhibited more extreme arousal than the control groups. First-degree relatives and participants with attenuated symptoms showed comparable autonomic activity to healthy controls. Thus, the hypothesized association of an alteration of arousal and vulnerability to psychosis was not confirmed. However, particularly low time-domain HRV was found for psychosis, with significant differences to healthy controls (all ps≤.007) and to depression (all ps≤.004), with the latter indicating a specificity to psychosis.

The extent and origin of discordance between self- and observer-rated depression in patients with psychosis.

It is assumed that patients with psychosis have difficulties indicating clinical symptoms accurately in self-reported measures. The present study investigated the ability of self-rating scales to detect symptoms of depression in patients with psychosis and aimed at identifying demographic, clinical and neurocognitive factors that predict the discordance between self-ratings and observer ratings. Inpatients and outpatients with psychosis (n=118) were assessed for depression by applying two observer rating and two self-rating scales. We found reasonable correlation scores between the ratings by patients and observers (range: r=0.50-0.57). In half of the patients (49.2%) the self-ratings corresponded well with the ratings of clinicians. Patients who rated their depressive symptoms as less severe than the clinicians demonstrated more negative symptoms such as blunted affect and poor affective rapport. Patients who rated their depression symptoms as being more severe were characterized by more self-reported general psychopathology. The concordance rates indicate that self-ratings of depression can be a valid additional tool in clinical assessment of patients with psychosis. However, clinicians should be attentive to the fact that some patients might have a general tendency to over-report symptoms and that patients with negative symptoms tend to be rated as more depressed in observer ratings compared with self-assessments.

Improving health from the inside: Use of engineered intestinal microorganisms as in situ cytokine delivery system.

The anti-inflammatory cytokine interleukin-10 and its viral homologs were chosen as model proteins for the development of drug delivery systems based on probiotic carriers like E. coli Nissle 1917, E. coli G3/10, and Saccharomyces boulardii. Exterior cytokine secretion was achieved by a modified E. coli hemolysin transporter. Release of interleukin-10 transported to the periplasm via the OmpF signal peptide was enabled by a T4 phage lysis system under control of the araC PBAD activator-promoter. The yield of interleukin-10 delivered by the phage lysis system was too low for functional analysis whereas the fusion protein secreted by the hemolysin transporter proved to be biologically inactive. Moreover, partial processing of the fusion protein by the E. coli membrane protease OmpT had no effect on the protein's functionality. Using the α-mating factor signal sequence, the yeast S. boulardii proved to be suitable for secretory expression of biologically active viral interleukin-10.

Elucidating the black box from stress to paranoia.

Sensitivity to stress has long been implicated in the pathogenesis of schizophrenia. It remains unclear, however, which exact mechanisms underlie the progression from vulnerability to psychotic breakdown. For the present study, we hypothesized that the induction of stress would aggravate cognitive biases in schizophrenia. A total of 20 acute and remitted schizophrenia patients and 15 healthy controls were tested with parallel versions of cognitive biases paradigms under 2 laboratory conditions: stress (loud noise, 75 dB) vs no-stress. In the course of both conditions, participants had to fill out a questionnaire that assessed depressive, obsessive-compulsive, and paranoid symptoms. For the patients with acute psychotic symptoms, paranoid but not other psychiatric symptoms were elevated under stress in comparison with no-stress. In contrast, stress somewhat diminished subclinical paranoid symptoms in healthy participants. Jumping to conclusions was evident in schizophrenia under both conditions but significantly more pronounced when stress was applied first in the acute group. A tendency emerged in both acute and remitted patients to attribute events to other people under stress which was not seen in healthy subjects. The present study may serve as a starting point for further research investigating how stress translates vulnerability into acute paranoia and to pinpoint cognitive risk factors that can be modified by treatment.

Comparison of a shiga toxin enzyme-linked immunosorbent assay and two types of PCR for detection of shiga toxin-producing Escherichia coli in human stool specimens.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of sporadic cases of disease as well as serious outbreaks worldwide. The spectrum of illnesses includes mild nonbloody diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome. STEC produces one or more Stxs, which are subdivided into two major classes, Stx1 and Stx2. The ingestion of contaminated food or water, person-to-person spread, and contact with animals are the major transmission modes. The infective dose of STEC may be less than 100 organisms. Effective prevention of infection is dependent on rapid detection of the causative bacterial pathogen. In the present study, we examined 295 stool specimens for the presence of Stx-producing E. coli by three different methods: an Stx enzyme-linked immunosorbent assay, a conventional PCR assay, and a LightCycler PCR (LC-PCR) assay protocol recently developed by our laboratory at the Institute of Medical Microbiology at Hannover Medical School. Our intent was to compare these three methods and to examine the utility of the STEC LC-PCR protocol in a clinical laboratory. The addition of a control DNA to each sample to clearly discriminate inhibited specimens from negative ones enhanced the accuracy of the LC-PCR protocol. From our results, it can be concluded that LC-PCR is a very useful tool for the rapid and safe detection of STEC in clinical samples.

Molecular and functional analysis of Shiga toxin-induced response patterns in human vascular endothelial cells.

Enterohemorrhagic Escherichia coli (EHEC) is the major cause of hemolyticuremic syndrome (HUS) characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. EHEC produces one or more Shiga toxins (Stx1 and Stx2), and it was assumed that Stx's only relevant biologic activity was cell destruction through inhibition of protein synthesis. However, recent data indicate that in vivo the cytokine milieu may determine whether endothelial cells survive or undergo apoptosis/necrosis when exposed to Stxs. In this study, we analyzed the genome-wide expression patterns of human endothelial cells stimulated with subinhibitory concentrations of Stxs in order to characterize the genomic expression program involved in the vascular pathology of HUS. We found that Stxs elicited few, but reproducible, changes in gene expression. The majority of genes reported in this study encodes for chemokines and cytokines, which might contribute to the multifaceted inflammatory response of host endothelial cells observed in patients suffering from EHEC disease. In addition, our data provide for the first time molecular insights into the epidemiologically well-established higher pathogenicity of Stx2 over Stx1.