Conditioned fear and extinction learning performance and its association with psychiatric symptoms in active duty Marines.

BACKGROUND: Posttraumatic Stress Disorder (PTSD) is a major public health concern, especially given the recent wars in Iraq and Afghanistan. Nevertheless, despite a sharp increase in the incidence of psychiatric disorders in returning veterans, empirically based prevention strategies are still lacking. To develop effective prevention and treatment strategies, it is necessary to understand the underlying biological mechanisms contributing to PTSD and other trauma related symptoms. METHODS: The "Marine Resiliency Study II" (MRS-II; October 2011-October 2013) Neurocognition project is an investigation of neurocognitive performance in Marines about to be deployed to Afghanistan. As part of this investigation, 1195 Marines and Navy corpsmen underwent a fear conditioning and extinction paradigm and psychiatric symptom assessment prior to deployment. The current study assesses (1) the effectiveness of the fear potentiated startle paradigm in producing fear learning and extinction and (2) the association of performance in the paradigm with baseline psychiatric symptom classes (healthy: n=923, PTSD symptoms: n=42, anxiety symptoms: n=37, and depression symptoms: n=12). RESULTS: Results suggest that the task was effective in producing differential fear learning and fear extinction in this cohort. Further, distinct patterns emerged differentiating the PTSD and anxiety symptom classes from both healthy and depression classes. During fear acquisition, the PTSD symptom group was the only group to show deficient discrimination between the conditioned stimulus (CS+) and safety cue (CS-), exhibiting larger startle responses during the safety cue compared to the healthy group. During extinction learning, the PTSD symptom group showed significantly less reduction in their CS+ responding over time compared to the healthy group, as well as reduced extinction of self-reported anxiety to the CS+ by the end of the extinction session. Conversely, the anxiety symptom group showed normal safety signal discrimination and extinction of conditioned fear, but exhibited increased baseline startle reactivity and potentiated startle to CS+, as well as higher self-reported anxiety to both cues. The depression symptom group showed similar physiological and self-report measures as the healthy group. DISCUSSION: These data are consistent with the idea that safety signal discrimination is a relatively specific marker of PTSD symptoms compared to general anxiety and depression symptoms. Further research is needed to determine if deficits in fear inhibition vs. exaggerated fear responding are separate biological "domains" across anxiety disorders that may predict differential biological mechanisms and possibly treatment needs. Future longitudinal analyses will examine whether poor learning of safety signals provides a marker of vulnerability to develop PTSD or is specific to symptom state.

Psychophysiology in the study of psychological trauma: where are we now and where do we need to be?

Posttraumatic stress disorder (PTSD) is a major public health concern, which has been seeing increased recent attention partly due to the wars in Iraq and Afghanistan. Historically, research attempting to understand the etiology and treatment of PTSD has made frequent use of psychophysiological measures of arousal as they provide a number of advantages in providing objective, non-self-report outcomes that are closely related to proposed neurobiological mechanisms and provide opportunity for cross-species translation. Further, the ongoing shift in classification of psychiatric illness based on symptom clusters to specific biological, physiological, and behavioral constructs, as outlined in the US National Institute of Mental Health (NIMH) Research Domain Criteria project (RDoC), promises that psychophysiological research will continue to play a prominent role in research on trauma-related illnesses. This review focuses on the current state of the knowledge regarding psychophysiological measures and PTSD with a focus on physiological markers associated with current PTSD symptoms, as well as markers of constructs thought to be relevant to PTSD symptomatology (safety signal learning, fear extinction), and psychophysiological markers of risk for developing PTSD following trauma. Future directions and issues for the psychophysiological study of trauma including traumatic brain injury (TBI), treatment outcome studies, and new wearable physiological monitoring technologies are also discussed.

Shiga toxins induce, superinduce, and stabilize a variety of C-X-C chemokine mRNAs in intestinal epithelial cells, resulting in increased chemokine expression.

Exposure of humans to Shiga toxins (Stxs) is a risk factor for hemolytic-uremic syndrome (HUS). Because Stx-producing Escherichia coli (STEC) is a noninvasive enteric pathogen, the extent to which Stxs can cross the host intestinal epithelium may affect the risk of developing HUS. We have previously shown that Stxs can induce and superinduce IL-8 mRNA and protein in intestinal epithelial cells (IECs) in vitro via a ribotoxic stress response. We used cytokine expression arrays to determine the effect of Stx1 on various C-X-C chemokine genes in IECs. We observed that Stx1 induces multiple C-X-C chemokines at the mRNA level, including interleukin-8 (IL-8), GRO-alpha, GRO-beta, GRO-gamma, and ENA-78. Like that of IL-8, GRO-alpha and ENA-78 mRNAs are both induced and superinduced by Stx1. Furthermore, Stx1 induces both IL-8 and GRO-alpha protein in a dose-response fashion, despite an overall inhibition in host cell protein synthesis. Stx1 treatment stabilizes both IL-8 and GRO-alpha mRNA. We conclude that Stxs are able to increase mRNA and protein levels of multiple C-X-C chemokines in IECs, with increased mRNA stability at least one mechanism involved. We hypothesize that ribotoxic stress is a pathway by which Stxs can alter host signal transduction in IECs, resulting in the production of multiple chemokine mRNAs, leading to increased expression of specific proteins. Taken together, these data suggest that exposing IECs to Stxs may stimulate a proinflammatory response, resulting in influx of acute inflammatory cells and thus contributing to the intestinal tissue damage seen in STEC infection.

Shiga toxin translocation across intestinal epithelial cells is enhanced by neutrophil transmigration.

Shiga toxin-producing E. coli (STEC) is a food-borne pathogen that causes serious illness, including hemolytic-uremic syndrome (HUS). STEC colonizes the lower intestine and produces Shiga toxins (Stxs). Stxs appear to translocate across intestinal epithelia and affect sensitive endothelial cell beds at various sites. We have previously shown that Stxs cross polarized intestinal epithelial cells (IECs) via a transcellular route and remain biologically active. Since acute inflammatory infiltration of the gut and fecal leukocytes is seen in many STEC-infected patients and since polymorphonuclear leukocyte (PMN) transmigration across polarized IECs diminishes the IEC barrier function in vitro, we hypothesized that PMN transmigration may enhance Stx movement across IECs. We found that basolateral-to-apical transmigration of neutrophils significantly increased the movement of Stx1 and Stx2 across polarized T84 IECs in the opposite direction. The amount of Stx crossing the T84 barrier was proportional to the degree of neutrophil transmigration, and the increase in Stx translocation appears to be due to increases in paracellular permeability caused by migrating PMNs. STEC clinical isolates applied apically induced PMN transmigration across and interleukin-8 (IL-8) secretion from T84 cells. Of the 10 STEC strains tested, three STEC strains lacking eae and espB (eae- and espB-negative STEC strains) induced significantly more neutrophil transmigration and significantly greater IL-8 secretion than eae- and espB-positive STEC or enteropathogenic E. coli. This study suggests that STEC interaction with intestinal epithelia induces neutrophil recruitment to the intestinal lumen, resulting in neutrophil extravasation across IECs, and that during this process Stxs may pass in greater amounts into underlying tissues, thereby increasing the risk of HUS.

GT160-246, a toxin binding polymer for treatment of Clostridium difficile colitis.

GT160-246, a high-molecular-weight soluble anionic polymer, was tested in vitro and in vivo for neutralization of Clostridium difficile toxin A and B activities. Five milligrams of GT160-246 per ml neutralized toxin-mediated inhibition of protein synthesis in Vero cells induced by 5 ng of toxin A per ml or 1.25 ng of toxin B per ml. In ligated rat ileal loops, 1 mg of GT160-246 neutralized fluid accumulation caused by 5 microg of toxin A. At doses as high as 80 mg/loop, cholestyramine provided incomplete neutralization of fluid accumulation caused by 5 microg of toxin A. GT160-246 protected 80% of the hamsters from mortality caused by infection with C. difficile, whereas cholestyramine protected only 10% of animals. Treatment of C. difficile-infected hamsters with metronidazole initially protected 100% of the hamsters from mortality, but upon removal of treatment, 80% of the hamsters had relapses and died. In contrast, removal of GT160-246 treatment did not result in disease relapse in the hamsters. GT160-246 showed no antimicrobial activity in tests with a panel of 16 aerobic bacteria and yeast and 22 anaerobic bacteria and did not interfere with the in vitro activities of most antibiotics. GT160-246 offers a novel, nonantimicrobial treatment of C. difficile disease in humans.

A monkey model for enterohemorrhagic Escherichia coli infection.

Adult Macaca radiata (n=22) were infected intragastrically with 10(12) Escherichia coli O157:H7 strain 84-01, which produces Shiga toxins 1 and 2. Clinical symptoms and bacterial excretion were documented in each monkey for a specified time period before they were killed. At necropsy, samples were obtained for culture and histologic and ultrastructural examination. Seventeen monkeys had diarrhea: E. coli O157 was isolated from postinfection stool samples from all monkeys and from autopsy cultures for 14 of 22 monkeys. Histologic examination showed attaching-effacing lesions, which appeared at 12 h and persisted for 7 days, in 12 monkeys. Widening of the intercellular spaces, degeneration and vacuolization of the epithelial cells, epithelial tufting, extrusion of epithelial cells, and neutrophilic infiltration were characteristic features seen in 20 of the 22 infected monkeys but not in 4 control monkeys. This monkey model closely parallels the early stages of the disease produced by E. coli O157:H7 and would be useful in the further study of pathogenic mechanisms and prevention methods in enterohemorrhagic E. coli infections.

Prevalence, antibiotic susceptibility, and diversity of Escherichia coli O157:H7 isolates from a longitudinal study of beef cattle feedlots.

Prevalence, antibiotic susceptibility, and genetic diversity were determined for Escherichia coli O157:H7 isolated over 11 months from four beef cattle feedlots in southwest Kansas. From the fecal pat (17,050) and environmental (7,134) samples collected, 57 isolates of E. coli O157:H7 were identified by use of bacterial culture and latex agglutination (C/LA). PCR showed that 26 isolates were eaeA gene positive. Escherichia coli O157:H7 was identified in at least one of the four feedlots in 14 of the 16 collections by C/LA and in 9 of 16 collections by PCR, but consecutive positive collections at a single feedlot were rare. Overall prevalence in fecal pat samples was low (0.26% by C/LA, and 0.08% by PCR). No detectable differences in prevalence or antibiotic resistance were found between isolates collected from home pens and those from hospital pens, where antibiotic use is high. Resistant isolates were found for six of the eight antibiotics that could be used to treat E. coli infections in food animals, but few isolates were multidrug resistant. The high diversity of isolates as measured by random amplification of polymorphic DNA and other characteristics indicates that the majority of isolates were unique and did not persist at a feedlot, but probably originated from incoming cattle. The most surprising finding was the low frequency of virulence markers among E. coli isolates identified initially by C/LA as E. coli O157:H7. These results demonstrate that better ways of screening and confirming E. coli O157:H7 isolates are required for accurate determination of prevalence.

Risks of fracture of Björk-Shiley 60 degree convexo-concave prosthetic heart valves: long-term cohort follow up in the UK, Netherlands and USA.

BACKGROUND AND AIM OF THE STUDY: Approximately 82,000 Björk-Shiley convexo-concave (BSCC) 60 degree prosthetic heart valves were implanted in patients worldwide between 1979 and 1986. Outlet strut fractures (OSF) of some of the valves were first reported shortly after their introduction. Here, the determinants of OSF are examined, and the between-country variation and long-term risk are assessed. METHODS: Cohorts of patients in the UK, Netherlands and USA with 15,770 BSCC 60 degree heart valves were followed up to 18 years for the occurrence of OSF. RESULTS: Crude rates of OSF were highest in the UK (0.18% per year), intermediate in the Netherlands (0.13%), and lowest in the USA (0.06%), although risk factor adjustment reduced the inter-country differences. Furthermore, in the UK and Netherlands, OSF rates (particularly for mitral valves) declined with time since implantation, and between-country differences were considerably diminished 10 or more years post implantation. The risk of OSF decreased steadily with advancing patient age. Fracture rates were lower among women than men, and also varied significantly with valve size and position and OSF status of other valves in the same shoporder. CONCLUSION: This long-term follow up of BSCC 60 degree heart valve patients indicates that risk factors for valve fracture are generally similar in the UK, Netherlands and USA. It also identifies a strong association between fracture risk and age, newly reveals gender-related differences, and shows that the risk of valve fracture persisted, albeit at a reduced rate, into the 1990s.

Human neutrophils and their products induce Shiga toxin production by enterohemorrhagic Escherichia coli.

The Shiga toxins (Stx) are critical virulence factors for Escherichia coli O157:H7 and other serotypes of enterohemorrhagic E. coli (EHEC). These potent toxins are encoded in the genomes of temperate lambdoid bacteriophages. We recently demonstrated that induction of the resident Stx2-encoding prophage in an O157:H7 clinical isolate is required for toxin production by this strain. Since several factors produced by human cells, including hydrogen peroxide (H2O2), are capable of inducing lambdoid prophages, we hypothesized that such molecules might also induce toxin production by EHEC. Here, we studied whether H2O2 and also human neutrophils, an important endogenous source of H2O2, induced Stx2 expression by an EHEC clinical isolate. Both H2O2 and neutrophils were found to augment Stx2 production, raising the possibility that these agents may lead to prophage induction in vivo and thereby contribute to EHEC pathogenesis.

Role for a phage promoter in Shiga toxin 2 expression from a pathogenic Escherichia coli strain.

Shiga toxins (Stxs), encoded by the stxA and stxB genes, are important contributors to the virulence of Escherichia coli O157:H7 and other Stx-producing E. coli (STEC) strains. The stxA and stxB genes in STEC strains are located on the genomes of resident prophages of the lambda family immediately downstream of the phage late promoters (p(R')). The phage-encoded Q proteins modify RNA polymerase initiating transcription at the cognate p(R') promoter which creates transcription complexes that transcend a transcription terminator immediately downstream of p(R') as well as terminator kilobases distal to p(R'). To test if this Q-directed processive transcription plays a role in stx(2)AB expression, we constructed a mutant prophage in an O157:H7 clinical isolate from which p(R') and part of Q were deleted but which has an intact pStx, the previously described stx(2)AB-associated promoter. We report that production of significant levels of Stx2 in this O157:H7 isolate depends on the p(R') promoter. Since transcription initiating at p(R') ultimately requires activation of the phage lytic cascade, expression of stx(2)AB in STEC depends primarily on prophage induction. By showing this central role for the prophage in stx(2)AB expression, our findings contradict the prevailing assumption that phages serve merely as agents for virulence gene transfer.

Glomerular injury induced in mice by intraperitoneal injection of Shiga-like toxins.

BACKGROUND AND OBJECTIVES: Shiga-like toxins I and II (Stx1 and Stx2) play an important role in the pathogenesis of renal disease by causing renal microvascular injury. A murine model was used to study glomerular lesions produced by Stx1 and Stx2. METHODS: Swiss albino mice of the Rockefeller strain were inoculated intraperitoneally with LD(50) doses of endotoxin-free Stx1 of Stx2 and observed for signs of disease. Samples of renal cortical tissue from mice were examined with the electron microscope. RESULTS: the mice developed systemic and neurological symptoms including hind limb paralysis and generalised convulsions. Renal arteriolar damage and glomerular endothelial cytoplasmic swelling, vacuolation, lysis and intravascular coagulation were present and resembled the microangiopathy seen in renal biopsies from patients. INTERPRETATION AND CONCLUSIONS: these experiments establish the role of Stx1 and Stx2 in glomerular vascular injury and provide a model for studying the pathogenesis of Shiga-like toxin related microangiopathy.

Random transposon mutagenesis of Campylobacter jejuni.

Genetic studies of Campylobacter jejuni have been limited due to the lack of a transposon mutagenesis method. Here, we describe a novel technique for random transposon mutagenesis using a mariner-based transposon into C. jejuni strain 480. Insertions were random, as demonstrated by Southern blot analysis and insertional junction sequencing. We have demonstrated, for the first time, random in vivo transposon mutagenesis of C. jejuni.

How does Escherichia coli O157:H7 testing in meat compare with what we are seeing clinically?

Escherichia coli O157:H7 is but one of a group of Shiga toxin-producing E. coli (STEC) that cause both intestinal disease such as bloody and nonbloody diarrhea and serious complications like hemolytic uremic syndrome (HUS). While E. coli O157: H7 is the most renowned STEC, over 200 different types of STEC have been documented in meat and animals, at least 60 of which have been linked with human disease. A number of studies have suggested that non-O157 STEC are associated with clinical disease, and non-O157 STEC are present in the food supply. Non-O157 STEC, such as O111 have caused large outbreaks and HUS in the United States and other countries. The current policy in the United States is to examine ground beef for O157:H7 only, but restricting the focus to O157 will miss other important human STEC pathogens.