HPV DNA testing in population-based cervical screening (VUSA-Screen study): results and implications.

BACKGROUND: Human papillomavirus (HPV) testing is more sensitive than cytology for detecting high-grade cervical intraepithelial neoplasia (CIN). We evaluated the performance of high-risk HPV (hrHPV) testing in routine screening. METHODS: In all, 25,871 women (29-61) enrolled in our population-based cohort study were offered both cytology and hrHPV testing. High-risk HPV-positive women with normal cytology and an age-matched subcohort of hrHPV-negative women with normal cytology were invited for repeat testing after 1 and/or 2 years and were referred for colposcopy if they presented with abnormal cytology and/or a positive hrHPV test. The hrHPV-positive women with borderline or mild dyskaryosis (BMD) and all women with moderate dyskaryosis or worse (>BMD) were directly referred for colposcopy. Women with BMD and an hrHPV-negative test were advised to repeat cytology at 6 and 18 months and were referred for colposcopy if the repeat cytology test was abnormal. The main outcome measure was CIN grade 3 or worse (CIN3+). Results were adjusted for non-attendance at repeat testing. RESULTS: The hrHPV-positive women with abnormal cytology had a CIN3+ risk of 42.2% (95% confidence interval (CI): 36.4-48.2), whereas the hrHPV-positive women with normal cytology had a much lower risk of 5.22% (95% CI: 3.72-7.91). In hrHPV-positive women with normal cytology, an additional cytology step after 1 year reduced the CIN3+ risk to only 1.6% (95% CI: 0.6-4.9) if the repeat test was normal. The CIN3+ risk in women with hrHPV-positive normal cytology was higher among women invited for the first time (29-33 years of age) (9.1%; 95% CI: 5.6-14.3) than among older women (3.0%; 95% CI: 1.5-5.5). CONCLUSION: Primary hrHPV screening with cytology triage in women aged 30 years is an effective way to stratify women on CIN3+ risk and seems a feasible alternative to cytological screening. Repeat cytology after 1 year for hrHPV-positive women with normal cytology is however necessary before returning women to routine screening.

Comparison of Hybrid capture 2 testing at different thresholds with cytology as primary cervical screening test.

BACKGROUND: We evaluated the performance of primary high-risk human papillomavirus (hrHPV) testing by hybrid capture 2 (HC2) with different thresholds for positivity, in comparison with conventional cytology. METHODS: We used data of 25,871 women (aged 30-60 years) from the intervention group of the VUSA-Screen study (VU University Medical Center and Saltro laboratory population-based cervical screening study), who were screened by cytology and hrHPV. Primary outcome measure was the number of cervical intraepithelial neoplasia grade 3 or higher (CIN3+), detected within 3 years. We compared baseline cytology testing with three possible hrHPV screening strategies at different relative light unit/cutoff (RLU/CO) thresholds. RESULTS: Compared with baseline cytology testing, hrHPV DNA testing as a sole primary screening instrument did not yield a superior sensitivity, as well as lower colposcopy referral rate and lower false positivity rate at any RLU/CO threshold. The hrHPV screening at 1 RLU/CO threshold with cytology triage at baseline and at 12 months revealed the highest sensitivity for CIN3+ (relative sensitivity of 1.32), although still displaying a lower colposcopy referral rate than cytology testing (relative colposcopy rate of 0.94). Higher thresholds (>1 RLU/CO) yielded lower colposcopy rates, but resulted in substantial loss in sensitivity. CONCLUSIONS: The hrHPV testing at the commonly used threshold of 1 RLU/CO with cytology triage at baseline and at 12 months showed a much higher sensitivity with a lower colposcopy referral rate compared with cytology testing.

Stereopsis in medical virtual-learning-environments.

In surgical training a lot of effort is directed at developing virtual learning environments (VLE's). These environments usually include one or more of the following features: haptic feedback, the sending of a slightly different computer-generated image to both eyes (affording an user's experience of stereoptical depth), and a transparant interface that allows 'natural' interaction. Now that many of these VLE's are approaching a state of maturity, time has come to start evaluating these environments, as basic questions regarding the effectiveness of the features mentioned above remain unanswered. The purpose of this article is to present an experiment aimed at evaluating the effects of stereopsis during a learning phase on visuo-spatial reasoning in two subsequent tasks that are related to the medical practice of diagnosis.

Investigations into the stabilisation of drugs by sugar glasses: II. Delivery of an inulin-stabilised alkaline phosphatase in the intestinal lumen via the oral route.

In this study the possibility to deliver the acid-sensitive enzyme alkaline phosphatase (AP) from calf intestine (CIAP) to the intestinal system by oral administration was investigated. Tablets were prepared and in vitro evaluated. Final proof of concept studies were performed in rats. This acid labile enzyme is potentially useful in the treatment of sepsis, a serious condition during which endotoxins can migrate into the blood stream. The CIAP was freeze-dried with inulin and subsequently compacted into round biconvex tablets with a diameter of 4mm and a weight of 25-30 mg per tablet. The tablets were coated with an enteric coating in order to ensure their survival in the stomach. In vitro evaluation of tablets containing alkaline phosphatase from bovine intestine (BIAP) was the first step in the development. It was found that tablets without enteric coating dissolved rapidly in 0.10 M HCl with total loss of enzymatic activity of the alkaline phosphatase. Tablets that were coated were stable for at least 2 h in 0.10 M HCl, but dissolved rapidly when the pH was increased to 6.8. Furthermore, it was shown that the enzymatic activity of the released BIAP was fully preserved. The in vivo test clearly showed that the oral administration of enteric coated tablets resulted in the release of enzymatically active CIAP in the intestinal lumen of rats. The location of the enhanced enzymatic activity of AP in the intestines varied with the time that had passed between the administration of the tablets and the sacrificing of the rats. Also, the level of enzymatic activity increased with an increasing number of tablets that were administered.

New immunoassay for the detection of Helicobacter pylori infection compared with urease test, 13C breath test and histology: validation in the primary care setting.

Helicobacter pylori plays a major role in peptic ulcer disease and, as a result, testing for H. pylori infection among patients with dyspepsia has often been advocated. The aim of the study was to determine the diagnostic accuracy, the analytical performance, and optimal cut-off point of a new serological assay, the Pyloriset EIA-G III for the detection of H. pylori infection in the primary care setting. For 113 primary care patients with dyspepsia urea breath test, CLO test, histology and serology tests were performed. Diagnostic accuracy of the Pyloriset EIA-G III was evaluated against a reference standard of a carbon urea breath test (CUBT), CLO test and histology (from gastric biopsies). Precision, linearity and correlation of the serological assay with the CUBT and former Pyloriset were also determined. At the optimal cut-off level of 40 U/ml, the positive predictive value was 92.1%, negative predictive value 96.3%, sensitivity 87.5%, and specificity 93.9%. The within-run precision was high. The recovery data were good. The correlation of both CUBT and the former Pyloriset EIA-G and the Pyloriset EIA-G III was high. At the cut-off level of 40 U/ml, the new Pyloriset EIA-G III is a reliable method to detect H. pylori infection in the primary care setting.

Nasal or intramuscular immunization of mice with influenza subunit antigen and the B subunit of Escherichia coli heat-labile toxin induces IgA- or IgG-mediated protective mucosal immunity.

Local mucosal IgA antibodies play a central role in protection of the respiratory tract against influenza virus infection. Therefore, new-generation influenza vaccines should aim at stimulating not only systemic, but also local antibody responses. Previously, we demonstrated that the recombinant B subunit of the Escherichia coli heat-labile toxin (LTB) is a potent adjuvant towards nasally administered influenza subunit antigen. Here, we investigated the protection conferred by LTB-supplemented influenza subunit antigen given intranasally (i.n.) or intramuscularly (i.m.) to mice. Both i.n. and i.m. immunization with subunit antigen and LTB completely protected the animals against viral infection. Protection upon i.n. immunization was associated with the induction of antigen-specific serum IgG and mucosal IgA, whereas protection upon i.m. immunization correlated with strong serum and mucosal IgG, but not IgA responses. We conclude that LTB-supplemented influenza subunit antigen, given either i.n. or i.m, induces protective antibody-mediated mucosal immunity and thus represents a promising novel flu vaccine candidate.

Standardized comparison of processing capacity and efficiency of five new-generation immunoassay analyzers.

BACKGROUND: With the trend toward laboratory and workstation consolidation, more studies are necessary to evaluate instrumentation, solutions for coping with workflow and test diversity, and opportunities for increasing the overall efficiency of laboratory testing. We assessed the processing capacity and efficiency of new-generation immunoassay analyzers by determining productivity parameters of five commercially available systems. METHODS: A workload protocol was developed and used to assess processing capacity and efficiency parameters of five immunoassay analyzers under standardized conditions in a real-life routine situation. We studied the ACS:Centaur((R)) (analyzer A), Architect(TM) i2000 (analyzer B), Elecsys((R)) 2010 tandem (analyzer C), Immulite((R)) 2000 (analyzer D), and Vitros ECi (analyzer E) on the basis of a standardized workload protocol that reflected a routine laboratory situation. This workload encompassed reflex and STAT testing, dilutions, and in-run calibration of a new reagent lot number. The analyzers were compared for hands-on labor time, unattended time (UT), throughput, and differentiated relative productivity indexes [RPI((UT)); number of reportable results/(processing time - sum of unattended time)]. The RPI data for analyzers linked to an automated (aut) sample-handling system [RPI((aut))] were also calculated. RESULTS: The evaluation produced a set of parameters for the productivity of the instruments. An overview of the most important parameters revealed the following: the throughput was 193, 123, 97, 109, and 46 tests/hour for instruments A, B, C, D and E, respectively; the RPI((10)) was 425, 238, 161, 445, and 151 tests/operator-hour; the RPI((30)) was 229, 136, 118, 264, and 86 tests/operator-hour; the RPI((10,aut)) was 1701, 637, 235, 964, and 223 tests/operator-hour; and the RPI((30, aut)) was 298, 150, 174, 400, and 114 tests/operator-hour. CONCLUSIONS: The combination of a standardized workload protocol and determination of parameters for productivity and labor efficiency, especially the differentiated RPIs, made it possible to make an objective comparison of the organizational consequences of the use of these instruments. The described parameters allow for a scientifically based choice, given a certain workflow and a particular laboratory organization.

A standardised and reproducible model of intraabdominal infection and abscess formation in rats.

OBJECTIVE: To develop a standardised and reproducible model of intra-abdominal infection and abscess formation in rats. DESIGN: Experimental study. SETTING: University hospital, The Netherlands. SUBJECTS: 36 adult male Wistar rats. INTERVENTIONS: In 32 rats, peritonitis was produced using two different concentrations of Escherichia coli (E. coli) and Bacteroides fragilis (B. fragilis) incorporated in fibrin clots (E. coii 1 x 10(5) colony forming units (CFU)/ml or 1 x 10(8) CFU/ml, B. fragilis: 1 x 10(8) CFU/ml). Four rats with fibrin clots without bacteria served as uninfected controls. MAIN OUTCOME MEASUREMENTS: Macroscopy and bacterial counts in peritoneal fluid, blood, and fibrin clots after 24 hours, 4 days, 7 days, and 4 weeks. RESULTS: Macroscopically, there were signs of intra-abdominal infection and abscesses. With the higher starting concentration of E. coli, macroscopic signs were more pronounced and in nearly all rats bacterial counts in peritoneal fluid and fibrin clots showed persistently high numbers of E. coli and B. fragilis for at least 7 days (E. coli = 2 x 10(3) to 1 x 10(6) CFU/ml and 5 x 10(7) to 9 x 10(8) CFU/clot; B. fragilis = 1 x 10(3) to 1 x 10(6) CFU/ml and 5 x 10(7) to 6 x 10(8) CFU/clot). CONCLUSION: This standardised and reproducible model of intra-abdominal infection and abscess formation seems well suited for further use and development in experiments on the pathophysiology of intra-abdominal infection and abscesses.

Characterization of the angR gene of Vibrio anguillarum: essential role in virulence.

The ability to utilize the iron bound by high-affinity iron-binding proteins in the vertebrate host is an important virulence factor for the marine fish pathogen Vibrio anguillarum. Virulence in septicemic infections is due to the presence of a highly efficient plasmid-encoded iron transport system. AngR, a 110-kDa protein component of this system, appears to play a role in both regulation of the expression of the iron transport genes fatDCBA and the production of the siderophore anguibactin. Therefore, study of the expression of the angR gene and the properties of its product, the AngR protein, may contribute to the understanding of the mechanisms of virulence of this pathogen. In this work, we present genetic and molecular evidence from transposition mutagenesis experiments and RNA analysis that angR, which maps immediately downstream of the fatA gene, is part of a polycistronic transcript that also includes the iron transport genes fatDCBA and angT, a gene located downstream of angR which showed domain homology to certain thioesterases involved in nonribosomal peptide synthesis of siderophores and antibiotics. In order to dissect the specific domains of AngR associated with regulation of iron transport gene expression, anguibactin production, and virulence, we also generated a panel of site-directed angR mutants, as well as deletion derivatives. Both virulence and anguibactin production were dramatically affected by each one of the angR modifications. In contrast to the need for an intact AngR molecule for anguibactin production and virulence, the regulation of iron transport gene expression does not require the entire AngR molecule, since truncation of the carboxy terminus carrying the nonribosomal peptide synthetase cores, as well as the site-directed mutations, resulted in derivatives that retained their ability to regulate gene expression which was only abolished after truncation of amino-terminal sequences containing helix-turn-helix and leucine zipper motifs and a specialized heterocyclization and condensation domain found in certain nonribosomal peptide synthetases. The evidence, while not rigorously eliminating the possibility that a separate regulatory polypeptide exists and is encoded somewhere within the 5'-end region of the angR gene, strongly supports the idea that AngR is a bifunctional protein and that it plays an essential role in the virulence mechanisms of V. anguillarum. We also show in this study that the angT gene, found downstream of angR, intervenes in the mechanism of anguibactin production but is not essential for virulence or iron transport gene expression.

Mucosal immunogenicity and adjuvant activity of the recombinant A subunit of the Escherichia coli heat-labile enterotoxin.

The Escherichia coli heat-labile enterotoxin (LT) is an exceptionally effective mucosal immunogen and mucosal immunoadjuvant towards coadministered antigens. Although, in general, the molecular basis of these properties is poorly understood, both the toxic ADP-ribosylation activity of the LTA subunit and the cellular toxin receptor, ganglioside, GM1-binding properties of the LTB-pentamer have been suggested to be involved. In recent studies we found that GM1-binding is not essential for the adjuvanticity of LT, suggesting an important role for the LTA subunit in immune stimulation. We now describe the immunomodulatory properties of recombinant LTA molecules with or without ADP-ribosylation activity, LTA(His)10 and LTA-E112K(His)10, respectively. These molecules were expressed as fusion proteins with an N-terminal His-tag to allow simple purification on nickel-chelate columns. Their immunogenic and immunoadjuvant properties were assessed upon intranasal administration to mice, and antigen-specific serum immunoglobulin-isotype and -subtype responses and mucosal secretory immunoglobulin A (IgA) responses were monitored using enzyme-linked immunosorbent assay. With respect to immunogenicity, both LTA(His)10 and LTA-E112K(His)10 failed to induce antibody responses. On the other hand, immunization with both LT and the non-toxic LT-E112K mutant not only induced brisk LTB-specific, but also LTA-specific serum and mucosal antibody responses. Therefore, we conclude that linkage of LTA to the LTB pentamer is essential for the induction of LTA-specific responses. With respect to adjuvanticity, both LTA(His)10 and LTA-E112K(His)10 were found to stimulate serum and mucosal antibody responses towards coadministered influenza subunit antigen. Remarkably, responses obtained with LTA(His)10 were comparable in both magnitude and serum immunoglobulin isotype and subtype distributions to those observed after coimmunization with LT, LT-E112K, or recombinant LTB. We conclude that LTA, by itself, can act as a potent adjuvant for intranasally administered antigens in a fashion independent of ADP-ribosylation activity and association with the LTB pentamer.

Mucosal immunoadjuvant activity of recombinant Escherichia coli heat-labile enterotoxin and its B subunit: induction of systemic IgG and secretory IgA responses in mice by intranasal immunization with influenza virus surface antigen.

The Escherichia coli heat-labile enterotoxin (LT) is a very potent mucosal immunogen. LT also has strong adjuvant activity towards coadministered unrelated antigens and is therefore of potential interest for development of mucosal vaccines. However, despite the great demand for such mucosal vaccines, the use of LT holotoxin as an adjuvant is essentially precluded by its toxicity. LT is composed of an A subunit, carrying the toxic ADP-ribosylation activity, and a pentamer of identical B subunits, which mediates binding to ganglioside GM1, the cellular receptor for the toxin. In this paper, we demonstrate that recombinant enzymatically inactive variants of LT, including the LTB pentamer by itself, retain the immunoadjuvant activity of LT holotoxin in a murine influenza model. Mice were immunized intranasally (i.n.) with influenza virus subunit antigen, consisting mostly of the isolated surface glycoprotein hemagglutinin (HA), supplemented with either recombinant LTB (rLTB), a nontoxic LT mutant (E112K, with a Glu112-->Lys substitution in the A subunit), or LT holotoxin, and the induction of systemic IgG and local S-IgA responses was evaluated by direct enzyme-linked immunosorbent assay (ELISA). Immunization with subunit antigen alone resulted in a poor systemic IgG response and no detectable S-IgA. However, supplementation of the antigen with E112K or rLTB resulted in a substantial stimulation of the serum IgG level and in induction of a strong S-IgA response in the nasal cavity. The adjuvant activity of E112K or rLTB under these conditions was essentially the same as that of the LT holotoxin. The present results demonstrate that nontoxic variants of LT, rLTB in particular, represent promising immunoadjuvants for potential application in an i.n. influenza virus subunit vaccine. Nontoxic LT variants may also be used in i.n. vaccine formulations directed against other mucosal pathogens. In this respect, it is of interest that LT(B)-stimulated antibody responses after i.n. immunization were also observed at distant mucosal sites, including the urogenital system. This, in principle, opens the possibility to develop i.n. vaccines against sexually transmitted infectious diseases.

Role of GM1 binding in the mucosal immunogenicity and adjuvant activity of the Escherichia coli heat-labile enterotoxin and its B subunit.

Escherichia coli (E. coli) heat-labile toxin (LT) is a potent mucosal immunogen and immunoadjuvant towards co-administered antigens. LT is composed of one copy of the A subunit, which has ADP-ribosylation activity, and a homopentamer of B subunits, which has affinity for the toxin receptor, the ganglioside GM1. Both the ADP-ribosylation activity of LTA and GM1 binding of LTB have been proposed to be involved in immune stimulation. We investigated the roles of these activities in the immunogenicity of recombinant LT or LTB upon intranasal immunization of mice using LT/LTB mutants, lacking either ADP-ribosylation activity, GM1-binding affinity, or both. Likewise, the adjuvant properties of these LT/LTB variants towards influenza virus subunit antigen were investigated. With respect to the immunogenicity of LT and LTB, we found that GM1-binding activity is essential for effective induction of anti-LTB antibodies. On the other hand, an LT mutant lacking ADP-ribosylation activity retained the immunogenic properties of the native toxin, indicating that ADP ribosylation is not critically involved. Whereas adjuvanticity of LTB was found to be directly related to GM1-binding activity, adjuvanticity of LT was found to be independent of GM1-binding affinity. Moreover, a mutant lacking both GM1-binding and ADP-ribosylation activity, also retained adjuvanticity. These results demonstrate that neither ADP-ribosylation activity nor GM1 binding are essential for adjuvanticity of LT, and suggest an ADP-ribosylation-independent adjuvant effect of the A subunit.

The role of ADP-ribosylation and G(M1)-binding activity in the mucosal immunogenicity and adjuvanticity of the Escherichia coli heat-labile enterotoxin and Vibrio cholerae cholera toxin.

The mucosal route of vaccination has attracted a great deal of attention recently. Not only is mucosal application of vaccines, for example, orally or intranasally, particularly convenient, it also offers the possibility to induce locally produced and secreted S-IgA antibodies in addition to systemic IgG antibodies. These IgA antibodies are known to play a key role in protection against pathogens that invade the host through mucosal surfaces. Induction of such responses is not readily achieved by currently used vaccination strategies, which generally involve intramuscular or subcutaneous injection with inactivated pathogens or antigens thereof. For the induction of a mucosal immune response, the vaccine needs to be applied locally. However, local vaccination with non-replicating antigens is usually ineffective and may result in tolerance unless a mucosal immunoadjuvant is included. The most potent mucosal immunoadjuvants known to date are probably cholera toxin (CT) and the closely related Escherichia coli heat-labile enterotoxin (LT). Although CT and LT have become standard adjuvants for experimental mucosal vaccines, the intrinsic toxicity has thus far precluded their use as adjuvants for human vaccine formulations. In the present review, the mucosal immunogenic and adjuvant properties of LT and CT are described, with special emphasis on the functional role of the individual subunits on their immune-stimulatory properties.

Mutational analysis of the role of ADP-ribosylation activity and GM1-binding activity in the adjuvant properties of the Escherichia coli heat-labile enterotoxin towards intranasally administered keyhole limpet hemocyanin.

The Escherichia coli heat-labile enterotoxin (LT) is known for its potent mucosal immunoadjuvant activity towards co-administered antigens. LT is composed of one A subunit, which has ADP-ribosylation activity, and a homopentameric B subunit, which has high affinity for the toxin receptor, ganglioside GM1. In previous studies, we have investigated the role of the LTA and LTB subunits in the adjuvanticity of LT towards influenza virus hemagglutinin (HA), administered intranasally to mice. We now studied the adjuvant properties of LT and LT variants towards keyhole limpet hemocyanin (KLH), which, in contrast to HA, does not bind specifically to mucosal surfaces. It is demonstrated that LT mutants without ADP-ribosylation activity, as well as LTB, retain mucosal immunoadjuvant activity when administered intranasally to mice in conjunction with KLH. As with influenza HA, adjuvanticity of LTB required GM1-binding activity, whereas GM1-binding was not essential for adjuvant activity of LT. Furthermore, we found that also recombinant LTA alone acts as a potent mucosal adjuvant, and that this adjuvanticity is independent of ADP-ribosylation activity. It is concluded that binding of the antigen to mucosal surfaces does not play an essential role in the immunostimulation by LT and LT variants, and that both recombinant LTA and LTB represent powerful nontoxic mucosal adjuvants.

Mutants of the Escherichia coli heat-labile enterotoxin with reduced ADP-ribosylation activity or no activity retain the immunogenic properties of the native holotoxin.

The Escherichia coli heat-labile enterotoxin (LT) is a potent inducer of mucosal immune responses. In a previous study (L. DeHaan, W. R. Verweij, M. Holtrop, E. Agsteribbe, and J. Wilschut, Vaccine 14:620-626, 1996), we have shown that efficient induction of an LTB-specific mucosal immune response by LT requires the presence of the LTA chain, suggesting a possible role of the enzymatic activity of LTA in the induction of these responses. In the present study, we generated LT mutants with altered ADP-ribosylation activities and evaluated their immunogenicity upon intranasal administration to mice. The results demonstrate that the mucosal immunogenicity of LT is not dependent on its ADP-ribosylation activity.

Mucosal immunogenicity of the Escherichia coli heat-labile enterotoxin: role of the A subunit.

The Escherichia coli heat-labile enterotoxin (LT) is a potent mucosal immunogen, inducing high secretory as well as systemic antibody responses upon oral or intranasal (i.n.) administration. In addition, LT has the capacity to act as an adjuvant in antibody responses against coadministered other antigens. To investigate the role of the individual subunits of LT in the mucosal immunogenicity and adjuvanticity of LT, the LT holotoxin and the non-toxic B subunit (LTB) were cloned separately and purified from overproducing E. coli cultures. Mice were immunized i.n. with the recombinant LT, LTB and combinations of the two and the induction of LTB-specific serum IgG and IgA as well as mucosal S-IgA was monitored. Intranasal administration of 2 micrograms LTB by itself induced a moderate systemic and a low mucosal antibody response, the latter being restricted to the site of immunization. However, addition of a trace amount (50 ng) of LT holotoxin to LTB strongly stimulated both serum antibody and mucosal S-IgA responses to LTB: the antibody levels induced by 2 micrograms LTB supplemented with 50 ng LT were similar to those seen after immunization with 2.9 micrograms of the LT holotoxin alone (representing an amount of 2 micrograms LTB). Furthermore, immunization with LT-supplemented LTB or with LT holotoxin alone, but not immunization with LTB alone, induced an S-IgA response in distant mucosal tissues including the lung, intestine and the urogenital system. Nicking of the LTA chain with trypsin did not enhance the immunogenicity of LT. These results indicate that, although the LTA chain plays an important role in the mucosal immunogenicity of LT including priming of the common mucosal immune system, extremely low amounts of the LT holotoxin suffice for the induction of high antibody responses to LTB, the trace LT and LTB acting in a synergistic fashion.

Transthoracic echo/Doppler in the identification of patients with chronic non-valvular atrial fibrillation at risk for thromboembolic events.

UNLABELLED: Left atrial spontaneous echo contrast, detected by transoesophageal echocardiography in patients with non-valvular atrial fibrillation reflects slow blood flow and is associated with an increased risk of cardio-embolism. The purpose of this study was to find echo/Doppler predictors of left atrial spontaneous echo contrast by transthoracic examination. In a retrospective case control study, 17 patients with chronic non-valvular atrial fibrillation who had suffered a recent cerebral ischaemic event (group A) and 17 patients with chronic non-valvular atrial fibrillation who had not suffered such an event (group B) were studied. Both groups were matched for age and sex. All patients underwent standard transthoracic echocardiography with transmitral Doppler as well as transoesophageal echocardiography. Left atrial spontaneous echo contrast was demonstrated by transoesophageal echocardiography in nine group A patients and in two group B patients (P = 0.028); left atrial spontaneous echo contrast was not detected by transthoracic echocardiography in these patients. All patients with left atrial spontaneous echo contrast (11 patients) had a left atrial size, corrected for base index, exceeding 24 mm and a transmitral time velocity integral < 10 cm (sensitivity 100%). Left atrial spontaneous echo contrast was absent in six patients with both characteristics (specificity 74%). CONCLUSION: transthoracic echo/Doppler aids in the prediction of the presence of left atrial spontaneous echo contrast and the identification of patients with non-valvular atrial fibrillation with increased cardioembolic risk, thus avoiding transoesophageal echocardiography.

Migration of rat peritoneal cells after intra-abdominal infection with Bacteroides fragilis and Escherichia coli.

A fibrin clot model for intra-abdominal abscess formation was used to study the migratory properties of peritoneal cells from rats during the early stages of infection. Peritoneal cells and fibrin clot remnant were harvested 6 h after implantation of a sterile, singly infected (Escherichia coli or Bacteroides fragilis) or mixed infected (E. coli and B. fragilis) fibrin clot. Histological study of fibrin clots, removed 6 h after implantation, showed a deeper infiltration by host cells of B. fragilis infected clots compared to the others. This difference in infiltration by peritoneal cells was not due to differences in fibrinolytic activity of the bacterial strains. Differential cell counts of the peritoneal cells from rats implanted with sterile, singly and mixed infected fibrin clots showed distribution over subpopulations to be independent of the bacterial content of the infected clots used. In vitro migration assays showed no significant differences in migration by peritoneal cells from rats implanted with clots containing a different bacterial composition. Since B. fragilis infected fibrin clots were more deeply infiltrated by host defence cells than the other clots, and only mixed infected clots led to persistent abscesses in this model, we conclude that local conditions within the fibrin matrix rather than intrinsic cellular capacities of the host cells are important for the process of abscess formation.

In-vitro activity of peritoneal cells from rats after intra-abdominal infection with Bacteroides fragilis and Escherichia coli.

Peritoneal cells from rats infected intraperitoneally with Escherichia coli and Bacteroides fragilis, alone or in combination were examined in vitro. Cells were harvested 6 h after implantation of fibrin clots infected with E. coli or B. fragilis, separately or containing both species, and assayed for their bactericidal capacities, chemiluminescence and production of cidal metabolites. Peritoneal cell populations from rats with implants of any of the infected clots showed similar distribution of different subpopulations. Bactericidal activity of peritoneal cells did not differ with the bacterial species used. Chemiluminescence values of peritoneal cells from rats with mono-infected B. fragilis or mixed-infected implanted clots, after stimulation with either particles or chemical stimuli, were significantly higher than those of rats with mono-infected E. coli or sterile clots. The same tendency was seen with regard to the production of cidal metabolites such as hydrogen peroxide and superoxide anions although no significant differences were found.

Human immune response to an iron-repressible outer membrane protein of Bacteroides fragilis.

Under conditions of iron starvation, Bacteroides fragilis expresses various iron-repressible outer membrane proteins (IROMPs). A 44-kDa protein appears to be one of the major outer membrane proteins (OMPs) in B. fragilis under iron stress and plays a role in heme uptake by this bacterium. To determine whether the 44-kDa IROMP of B. fragilis is expressed in vivo and whether this protein is immunogenic, we used Western immunoblotting to examine serum samples from patients with an infection caused by Bacteroides species. All the serum samples from patients and from normal controls showed reactivity with several proteins of B. fragilis. Only serum samples from patients infected with B. fragilis showed immunoreactivity with the 44-kDa protein. We also used a rat infection model to study the immune response against this protein during the process of an intra-abdominal infection in these animals. During the first 8 days of infection a gradual increase of antibodies to the 44-kDa protein in the rat was detected. These results suggest that the 44-kDa IROMP is expressed in vivo, since it induces an antibody response in patients and animals. We also analyzed 85 strains of the B. fragilis group for the presence of proteins antigenically related to the B. fragilis 44-kDa protein. The data indicate that this protein was conserved in B. fragilis strains and was absent in the other bacterial strains tested.