Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro.

The intracellular signaling involved in the mechanism of action of zonula occludens toxin (ZOT) was studied using several in vitro and ex vivo models. ZOT showed a selective effect among various cell lines tested, suggesting that it may interact with a specific receptor, whose surface expression on various cells differs. When tested in IEC6 cell monolayers, ZOT-containing supernatants induced a redistribution of the F-actin cytoskeleton. Similar results were obtained with rabbit ileal mucosa, where the reorganization of F-actin paralleled the increase in tissue permeability. In endothelial cells, the cytoskeletal rearrangement involved a decrease of the soluble G-actin pool (-27%) and a reciprocal increase in the filamentous F-actin pool (+22%). This actin polymerization was time- and dose-dependent, and was reversible. Pretreatment with a specific protein kinase C inhibitor, CGP41251, completely abolished the ZOT effects on both tissue permeability and actin polymerization. In IEC6 cells ZOT induced a peak increment of the PKC-alpha isoform after 3 min incubation. Taken together, these results suggest that ZOT activates a complex intracellular cascade of events that regulate tight junction permeability, probably mimicking the effect of physiologic modulator(s) of epithelial barrier function.

A hybrid promoter and portable Shine-Dalgarno regions of Escherichia coli.

A gene expression system of Escherichia coli is described here that contains a portable Shine-Dalgarno region. Transcription of this system is under the direction of a hybrid promoter derived from trp and lac-UV5 promoter sequences which is followed by a region that encodes the portable Shine-Dalgarno (PSD) region. Using a series of synthetic PSD regions we varied the length (from 4 to 13 bases) of the Shine-Dalgarno region by increasing the number of bases on the mRNA that are complementary to the 3'-end of 16S rRNA. We found that increase of the Shine-Dalgarno region to 8 or 13 bases decreases the translation efficiency of the chimaeric leucocyte interferon messenger by 40%. We also varied in another series of PSD regions the four bases that follow the Shine-Dalgarno region. We found that the presence of four A residues or four T residues in this region results in the highest translation efficiency. The presence of four C residues reduces the translation efficiency by 50% as compared with PSD regions with A or T residues. The presence of four G residues following the Shine-Dalgarno region lowers the translation efficiency by 75% with respect to PSD regions with A or T residues.

Reliability, validity and responsiveness of a revised scoring system for the Liverpool Seizure Severity Scale.

This report examines the reliability, validity and responsiveness of a revised scoring system for the Liverpool Seizure Severity Scale (LSSS). The revised scoring system was validated using archival data from an observational study and a randomized controlled study. Factor analyses confirmed that a single dimension captured how patients evaluate the severity of their most severe seizures occurring during a recall period. The revised scoring system repositions the severity score to range from 0 (no seizures) to 100 (most severe possible). Scores based on the new system were reliable, had construct validity (known-groups validity), and were responsive to changes in the patients' epilepsy as noted by their physicians. Results suggest that future epilepsy studies assessing seizure severity should incorporate the revised LSSS scoring system and a modified version of the questionnaire that simplifies self-assessment and analyses. The modified version of the LSSS and its scoring system are appended to this report.

The lifetime cost of bipolar disorder in the US: an estimate for new cases in 1998.

OBJECTIVE: To develop a cost model that estimates the total and per case lifetime cost of bipolar disorder for 1998 incident cases in the US. STUDY DESIGN: Lifetime cost simulation model. PERSPECTIVE: Societal. METHODS: Age- and gender-specific incidence of bipolar disorder in 1998 was estimated by simulation based on existing prevalence data. The course of illness and mental health service cost of 6 clinically defined prognostic groups was estimated based on the research literature and the judgement of panels of experts. Excess cost of general medical care was estimated based on claims data from a large insurer. Indirect cost was projected including excess unemployment and reduced earnings reported in the National Comorbidity Survey. Comorbidity treatment and indirect cost related to alcohol (ethanol) and drug abuse was added based on a National Institute on Drug Abuse study. RESULTS: The present value of the lifetime cost of persons with onset of bipolar disorder in 1998 was estimated at 24 billion US dollars ($US). Average cost per case ranged from $US11,720 for persons with a single manic episode to $US624,785 for persons with nonresponsive/chronic episodes. CONCLUSION: The model indicates the potential cost savings of preventing a case of bipolar disorder and underscores the importance of achieving a stable outcome in new cases to limit the economic consequences of the disorder.

Validation of the side effect and life satisfaction (SEALS) inventory.

Diminished quality of life (QOL) is a common feature of epilepsy. It is generally more severe among patients with poor seizure control but prevalent, to a clinically significant degree, even among those whose seizures are well controlled. People with epilepsy frequently report diminished socialization, negative self image, feelings of stigmatization, reduced earnings potential, and diminished hope and ambition. Problems with antiepileptic drug (AED) therapy are common, and AED therapy is recognized as an important determinant of health-related quality of life (HRQOL). A clinically efficient psychometric instrument is needed to measure its impact. The Side Effect and Life Satisfaction (SEALS) inventory is a 38-item, patient-completed questionnaire designed to measure satisfaction with AED therapy. We tested its construct validity in comparison with three widely used psychometric instruments of similar design, the Profile of Mood States (POMS), the Hospital Anxiety and Depression (HAD) scale, and the Medical Outcomes Study-Cognitive Functioning (MOS-COG) scale. All four instruments were completed by 307 epilepsy patients. A matrix of Pearson's correlations was produced for the SEALS inventory and the comparative instruments. A statistically significant correlation was found for each planned comparison. We conclude that the SEALS inventory is a valid psychometric instrument, well suited for use in clinical investigations of AED therapy and in the practical, long-term management of epilepsy.

A double-blind comparison of lamotrigine and carbamazepine in newly diagnosed epilepsy with health-related quality of life as an outcome measure.

The aim of this study was to compare the effect of treatment with lamotrigine (LTG) or carbamazepine (CBZ) on health-related quality of life (HRQOL) and to demonstrate the use of the SEALS Inventory as a comparative tool in clinical trials. Two hundred and sixty patients with newly diagnosed epilepsy were randomized to 48 weeks of treatment with LTG (n = 131) or CBZ (n = 129). HRQOL was measured at baseline and weeks 4, 12, 24, and 48 using the modified Side Effect and Life Satisfaction (SEALS) Inventory-a 38-item questionnaire divided into five subscales: Worry, Temper, Cognition, Dysphoria, and Tiredness. Overall, SEALS scores in the LTG group decreased (improved) significantly from baseline (P = 0.001). The LTG group had improvement in all five subscales over the 48 weeks of the study. CBZ patients had significantly worse SEALS scores than LTG patients at week 4 (P < 0.038). There was no significant change (positive or negative) in subsequent SEALS assessments. Analysis of SEALS data by subscale showed that the the CBZ group experienced more cognitive side-effects in general and more general changes in energy levels and affect during the first 4 weeks of treatment. These changes may help explain the difference in study completion rate: LTG 65%, CBZ 51% (P = 0.018). LTG offers the patient with newly diagnosed epilepsy significant benefits of greater tolerability and better health-related quality of life compared with CBZ. The SEALS Inventory is an effective tool for use in clinical trials of AEDs; it was a better predictor of trial completion than seizure counts, and used as a covariate enabled better detection of treatment effects. In general practice, the use of the SEALS Inventory to assess HRQOL has the potential to improve quality of care for people with epilepsy.

Penetration of endothelial cell monolayers by Borrelia burgdorferi.

The ability of Borrelia burgdorferi, the agent of Lyme disease, to penetrate cultured human umbilical vein endothelial cell monolayers was investigated. After 4 h of coincubation, approximately 7.7% of added bacteria passed through the host cell monolayers. Electron microscopy revealed that the borreliae entered the endothelial cells and suggested that the organisms penetrated the host monolayers primarily by passing through them.

Interaction of Lyme disease spirochetes with cultured eucaryotic cells.

The association of the Lyme disease spirochete, Borrelia burgdorferi, with cultured human endothelial cells was investigated. Attachment was time and temperature dependent, with optimal adherence occurring after 4 h of incubation at 37 degrees C. Pretreatment of borreliae with heat, immune human serum, or monoclonal antibodies directed against outer surface protein B (OspB) reduced the attachment of organisms to host cell monolayers. These results suggest that the adherence of B. burgdorferi may be mediated, at least in part, by borrelial surface proteins.

Characterization of Borrelia burgdorferi invasion of cultured endothelial cells.

Borrelia burgdorferi can adhere to cultured endothelial cells and penetrate through cell monolayers by passing through intercellular tight junctions and through the host cell cytoplasm. Borrelia burgdorferi strains which were isolated from different sources and areas of the U.S. all demonstrated similar invasive capabilities. Bacterial penetration from the apical to the basal surface of the monolayer was 20 times more efficient than from the basal to the apical surface. Borreliae which were non-viable as a result of either heat treatment or ultraviolet (UV) irradiation showed reduced association with the endothelial cell monolayer and loss of invasive capabilities. Borreliae were able to invade when protein synthesis was inhibited with streptomycin or chloramphenicol. When assays were conducted at 4 degrees C, bacterial penetration of the monolayer was completely inhibited. Treatment of borreliae with proteases affecting outer surface proteins greatly reduced cell association and bacterial invasion.

The way we teach : students to care for patients.

This article describes several techniques used at the University of New Mexico School of Medicine in an attempt to overcome the problem of dehumanization of young physicians in training. Finding the use of questionnaires, interview evaluators and simulated patients insufficient, the authors developed a caring skills curriculum. Practical skills in communication, understanding, listening and awareness are developed through a study programme utilizing videotapes, work-books, interrupted audiotapes and simulated patients.

Genetic diversity of the capsular polysaccharide C biosynthesis region of Bacteroides fragilis.

A genetic approach was used to assess the heterogeneity of the capsular polysaccharide C (PS C) biosynthesis locus of Bacteroides fragilis and to determine whether distinct loci contain genes whose products are likely to be involved in conferring charged groups that enable the B. fragilis capsular polysaccharides to induce abscesses. A collection of 50 B. fragilis strains was examined. PCR analysis demonstrated that the genes flanking the PS C biosynthesis region are conserved, whereas the genes within the loci are heterogeneous. Only cfiA(+) B. fragilis strains, which represent 3% of the clinical isolates of B. fragilis, displayed heterogeneity in the regions flanking the polysaccharide biosynthesis genes. Primers were designed in the conserved regions upstream and downstream of the PS C locus and were used to amplify the region from 45 of the 50 B. fragilis strains studied. Fourteen PS C genetic loci could be differentiated by a combination of PCR and extended PCR. These loci ranged in size from 14 to 26 kb. Hybridization analysis with genes from the PS C loci of strains 9343 and 638R revealed that the majority of strains contain homologs of wcgC (N-acetylmannosamine dehydrogenase), wcfF (putative dehydrogenase), and wcgP (putative aminotransferase). The data suggest that the synthesis of polysaccharides that have zwitterionic characteristics rendering them able to induce abscesses is common in B. fragilis.

The tac promoter: a functional hybrid derived from the trp and lac promoters.

Two hybrid promoters that are functional in Escherichia coli have been constructed. These hybrid promoters, tacI and tacII, were derived from sequences of the trp and the lac UV5 promoters. In the first hybrid promoter (tacI), the DNA upstream of position -20 with respect to the transcriptional start site was derived from the trp promoter. The DNA downstream of position -20 was derived from the lac UV5 promoter. In the second hybrid promoter (tacII), the DNA upstream of position -11 at the Hpa I site within the Pribnow box was derived from the trp promoter. The DNA downstream of position -11 is a 46-base-pair synthetic DNA fragment that specifies part of the hybrid Pribnow box and the entire lac operator. It also specifies a Shine-Dalgarno sequence flanked by two unique restriction sites (portable Shine-Dalgarno sequence). The tacI and the tacII promoters respectively direct transcription approximately 11 and 7 times more efficiently than the derepressed parental lac UV5 promoter and approximately 3 and 2 times more efficiently than the trp promoter in the absence of the trp repressor. Both hybrid promoters can be repressed by the lac repressor and both can be derepressed with isopropyl beta-D-thiogalactoside. Consequently, these hybrid promoters are useful for the controlled expression of foreign genes at high levels in E. coli. In contrast to the trp and the lac UV5 promoters, the tacI promoter has not only a consensus -35 sequence but also a consensus Pribnow box sequence. This may explain the higher efficiency of this hybrid promoter with respect to either one of the parental promoters.

A monoclonal antibody to OspA inhibits association of Borrelia burgdorferi with human endothelial cells.

Previously, it has been shown that polyclonal antibodies to Borrelia burgdorferi and some monoclonal antibodies (MAbs) to borrelia major surface proteins caused inhibition of adherence of the bacteria to cultured human umbilical vein endothelial (HUVE) cells. In this study, fragment antigen binding (Fab) molecules generated from the immunoglobulin G fraction of rabbit anti-recombinant OspA serum were found to inhibit the adherence of B. burgdorferi to HUVE cells by 73%. Subsequently, MAbs were generated for use in determining whether or how B. burgdorferi outer surface proteins (Osps) A and/or B are involved in mediating attachment to, and/or invasion of, HUVE cells by B. burgdorferi. Twenty-two MAbs were generated to borrelial proteins with apparent molecular masses (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 19, 31 (OspA), 34 (OspB), and 35 kDa. Fab molecules from one anti-OspA MAb, 9B3D, demonstrated an inhibitory effect on bacterial association with HUVE cells. None of the other MAbs, including the other anti-OspA MAbs, showed an inhibitory effect on cell association of greater than 5%. This effect of Fab 9B3D was concentration dependent and plateaued at approximately 6 micrograms of Fab per ml (nearly 80% inhibition of the bacterial association with the monolayer). Penetration assays and cell association experiments performed by using immunofluorescence also suggested that the inhibitory action of 9B3D occurs at the level of adherence. MAb 9B3D recognized the OspA of every North American strain tested (n = 19) but only 3 [corrected] of 20 strains from western Europe, Russia, and Japan, suggesting that the North American strains and strains from other parts of the world may use different molecules and/or different OspA epitopes to interact with endothelial cells. Immunoblots of Escherichia coli expressing different OspA fusion peptides suggested that the 9B3D epitope resides in the carboxy-terminal half of OspA. MAb 9B3D promises to be a valuable tool for elucidating the domain or domains of OspA involved in the endothelial cell cytadherence of North American strains of B. burgdorferi.

Distribution of Zonula occludens toxin (zot) gene among clinical isolates of Vibrio cholerae O1 from Bangladesh and Africa.

Seventy-two clinical isolates of Vibrio cholerae O1 from Bangladesh, and 12 and 9 isolates respectively from Tanzania and Nigeria were screened for sequences homologous to zonula occludens toxin (zot) and cholera toxin (ctx) genes. As observed previously, all isolates in the present study also possessed sequences for both toxins which suggested that zot does not occur independent of ctx. It appears that along with the virulence genes located in the "virulence cassette" region of the bacterial chromosome, zot may play a role in the pathogenesis of cholera.

Interstrain variation of the polysaccharide B biosynthesis locus of Bacteroides fragilis: characterization of the region from strain 638R.

The sequence and analysis of the capsular polysaccharide biosynthesis locus, PS B2, of Bacteroides fragilis 638R are described, and the sequence is compared with that of the PS B1 biosynthesis locus of B. fragilis NCTC 9343. Two genes of the region, wcgD and wcgC, are shown by complementation to encode a UDP-N-acetylglucosamine 2-epimerase and a UDP-N-acetylmannosamine dehydrogenase, respectively.

Portable Shine-Dalgarno regions: a system for a systematic study of defined alterations of nucleotide sequences within E. coli ribosome binding sites.

We have developed a gene expression system in Escherichia coli that contains a portable Shine-Dalgarno region. Transcription of this system is under the direction of a hybrid promoter (tacII) derived from trp and lac-UV5 promoter sequences which is followed by a region that encodes a portable Shine-Dalgarno region (PSDR). Using a series of synthetic PSDRs, we varied the four bases that follow the Shine-Dalgarno (SD) region. We found that the presence of four A residues or four T residues in this position gives the highest translational efficiency. The presence of four C residues reduces the translation efficiency by 50% as compared with PSDRs with A or T residues. The presence of four G residues following the SD region lowers the translational efficiency by at least 75% with respect to PSDRs with A or T residues.