Bidirectional cavopulmonary shunt with right ventricular outflow patency: the impact of pulsatility on pulmonary endothelial function.

OBJECTIVE: Although in vitro studies have suggested the importance of flow pulsatility in endothelial function, few reports have focused on pulmonary endothelial function under decreased pulsatile flow after a bidirectional cavopulmonary shunt with or without an additional pulmonary flow source. The purpose of the present study was to assess the pulmonary endothelial function after bidirectional cavopulmonary shunt. METHODS AND RESULTS: Pulmonary vasodilating response was evaluated in 10 patients 0.4 to 7.0 years (median 1.6 years) after bidirectional cavopulmonary shunt who were provided an additional flow source by retaining the pulmonary outflow tract and in 8 control subjects. Average pulmonary flow velocity was measured with a Doppler flow wire placed in the segmental lower lobe pulmonary artery during incremental infusion of acetylcholine (10(-8), 10(-7), 10(-6), and 10(-5) mol/L) and then of nitroglycerin (0.5 and 1.0 microg. kg(-1). min(-1)) after recovery. In the control subjects, a dose-dependent increase in flow velocity was observed in response to acetylcholine (maximum increase was 155% +/- 17% of baseline) and to nitroglycerin (maximum increase was 151% +/- 20% of baseline). In contrast, patients showed a significantly impaired response to acetylcholine (maximum increase was 124% +/- 17% of baseline; P <.01 vs control), whereas the response to nitroglycerin was preserved (138% +/- 12% of baseline; P =.09 vs control). In addition, the maximum response to acetylcholine correlated significantly with the pulmonary pulse pressure (r = 0.89, P <.01) and with the pulmonary flow pulsatility (r = 0.88, P <.01). CONCLUSIONS: These results clearly suggest that patients after bidirectional cavopulmonary shunt show pulmonary endothelial functional attenuation and, of more importance, that decreased pulsatility of cavopulmonary flow is mainly responsible for this endothelial abnormality.

Reversal of intrapulmonary arteriovenous shunting detected by two-dimensional contrast-enhanced echocardiography after liver transplantation.

BACKGROUND: Intrapulmonary arteriovenous shunting (IPS), occasionally associated with advanced liver disease, may reverse after liver transplantation (LTx). Two-dimensional contrast-enhanced echocardiography, a convenient noninvasive study, has never been used to demonstrate disappearance of IPS after LTx. METHODS: For an 8-month-old girl undergoing living-related LTx, two-dimensional contrast-enhanced echocardiography was performed with the microbubble injection. The opacification of the microbubble in the left heart emerging within 3-6 beats after detection in the right heart was compared with that in the right heart. RESULTS: Microbubble opacification in the left heart was almost the same as that in the right heart (grade 3) shortly after LTx. However, the contrast in the left heart diminished (grade 1) as the respiratory condition improved and subsequently disappeared (grade 0). CONCLUSIONS: Two-dimensional contrast-enhanced echocardiography may be a feasible noninvasive method to evaluate the degree of IPS in the peritransplant period and observe disappearance of IPS after LTx.

Nicking sites in a subunit of cholera toxin and Escherichia coli heat-labile enterotoxin for Vibrio cholerae hemagglutinin/protease.

We analyzed the nicking site of the A subunit of Escherichia coli heat-labile enterotoxin for hemagglutinin/protease produced by Vibrio cholerae non-O1 (NAG-HA/P). The determined nicking site was the Thr193-Ile194 junction, which was distinct from that for a protease of V. cholerae (Ichinose et al., European Journal of Epidemiology 8, 743-747, 1992). We further analyzed proteolytic cleavage by NAG-HA/P of a synthetic peptide corresponding to the nicking region of cholera toxin A subunit and determined the cleavage site to be preferentially between Ser194 and Met195, and in addition between Ser193 and Ser194.

Quantitative, non-invasive assessment of ventricular septal defect shunt flow by measuring proximal isovelocity surface area on colour Doppler mapping.

OBJECTIVE: To determine whether the proximal isovelocity surface area (PISA) method could be applied to estimate the magnitude of ventricular septal defect (VSD) shunt flow. DESIGN: Prospective analysis of clinical, echocardiographic, and angiographic data. SETTING: University hospital. PATIENTS: 14 children with VSD. METHODS: Colour Doppler images of VSD shunt flow were obtained in parasternal long axis view, four chamber view or both, adjusted to provide the best imaging of flow. The VSD shunt flow rate and shunt volume were calculated as follows: shunt flow rate (SFR) = 2 pi r2 V/BSA in ml/s/m2; shunt volume = SFR x shunt duration time. The shunt volume, shunt fraction, and pulmonary to systemic flow ratio (Qp:Qs) were confirmed by cardiac catheterisation. RESULTS: There was a correlation between shunt variables determined by PISA and those by catheterisation, including shunt volume (r = 0.78, P = 0.001) and shunt fraction (r = 0.74, P = 0.003). Qp:Qs was also significantly correlated with SFR (r = 0.79, P = 0.0007). The SFR was significantly different between the four patients with Qp:Qs < 2.0 (mean (SD) 54 (33) ml/s/m2) and the 10 patients with Qp:Qs > 2.0 (186 (69) ml/s/m2) (P = 0.004). CONCLUSIONS: These data suggest that the PISA method is a reliable non-invasive investigation for the quantitative assessment of VSD shunt flow and provides important information for decisions regarding surgical repair.

Rapid and differential detection of two analogous enterotoxins of Vibrio cholerae and enterotoxigenic Escherichia coli by a modified enzyme-linked immunosorbent assay.

The principle of a novel ELISA (nylon-slip immuno-test, NSIT) was applied to the differential detection of two analogous enterotoxins, cholera toxin (CT) of Vibrio cholerae and heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli. The results obtained for CT and LT detection by a single test were sufficiently sensitive (87.9 and 100%) and specific (100 and 94.7%) in the differential detection test, when compared with the result of a colony hybridization test with DNA probes. The results suggest that the novel ELISA is applicable to the diagnosis of bacterial infections, by means of differential immunological detection of toxins in a single test.

In vitro proteolytic processing and activation of the recombinant precursor of El Tor cytolysin/hemolysin (pro-HlyA) of Vibrio cholerae by soluble hemagglutinin/protease of V. cholerae, trypsin, and other proteases.

Vibrio cholerae produces a cytolytic toxin named El Tor cytolysin/hemolysin which is encoded by the hlyA gene. This cytolysin is produced as a 79-kDa precursor form (pro-HlyA) into the culture supernatant after cleavage of the signal peptide of the hlyA product (prepro-HlyA). The pro-HlyA is then processed to a 65-kDa mature cytolysin (mature HlyA) after cleavage of the 15-kDa N-terminal peptide (pro region) of the 79-kDa precursor, usually at the bond between Ala-157 and Asn-158. We investigated whether proteases could process the recombinant 79-kDa pro-HlyA to the 65-kDa mature HlyA. We observed that the soluble hemagglutinin/ protease (HA/protease; a major protease of V. cholerae), trypsin, alpha-chymotrypsin, subtilisin BPN', papain, and thermolysin all processed the pro-HlyA to the 65-kDa mature form of the protein. Along with this, the protease-processed HlyA showed drastically increased hemolytic activity. The N-terminal amino acid of the mature form of cytolysin generated by HA/protease was Phe-151, and that due to trypsin was Ser-149. Other proteases also cleaved the pro-HlyA at a nearby site, between Leu-146 and Ser-153, and all the processed cytolysins showed increased hemolytic activity. These data suggest that the active El Tor cytolysin of V. cholerae could be derived from the C-terminal region of a pro-HlyA following proteolytic cleavage of the bonds in the vicinity of Leu-146 to Asn-158 by any of a wide variety of proteases.

Purification and partial characterization of a hemagglutinating factor (HAF): a possible adhesive factor of the diffuse adherent of Escherichia coli (DAEC).

The mannose-resistant hemagglutinating factor (HAF) was extracted and purified from a diffuse adherent Escherichia coli (DAEC) strain belonging to the classic enteropathogenic E. coli (EPEC) serotype (0128). The molecular weight of HAF was estimated to be 18 KDa by SDS-PAGE and 66 KDa by Sephadex G100, suggesting that the native form of HAF consists of 3-4 monomeric HAF. Gold immunolabeling with specific HAF antiserum revealed that the HAF is not a rigid structure like fimbriae on the bacterial surface. The immunofluorescence test using purified HAF on HeLa cells, in addition to the fact that the HAF is distributed among serotypes of EPEC, suggests that HAF is a possible adhesive factor of DAEC strains.

Optimal degree of pulmonary artery banding--adequate circumference ratio to calculated size from normal pulmonary valve dimensions.

These findings suggest that PA banding may be suitable in children with congenital heart disease and excessive pulmonary flow, and that best results are obtained when the band circumference is < 90% of the standard pulmonary valve-ring circumference, as calculated from an equation derived from normal pulmonary valve dimensions. This guideline applies equally well to small infants weighing < 3 kg and to larger patients.

A novel method to chemically immobilize antibody on nylon and its application to the rapid and differential detection of two Vibrio parahaemolyticus toxins in a modified enzyme-linked immunosorbent assay.

A new method of chemically immobilizing antibody on nylon was developed. The method consists of serial treatments with HCl, polyethylene imine, and maleic anhydride methylvinyl ether copolymer, which resulted in the stable immobilization of sufficient amounts of antibodies on nylon. This principle was used to differentially detect two immunologically related but nonidentical hemolysins (thermostable direct hemolysin [TDH] and TDH-related hemolysin [TRH]) of Vibrio parahaemolyticus in a modified enzyme-linked immunosorbent assay with antibodies immobilized on nylon slips (NSIT). The results (dark purple color on nylon slips) were easily evaluated by the naked eye. The results with NSIT were compatible with those obtained by using DNA probes or a conventional bacterial culture test, not only with cultured specimens but also with clinical specimens (diarrheal stool samples). Furthermore, the NSIT differentially detected TDH and TRH in a single test. The antibody immobilization method developed here is applicable to various immunological detection methods and may improve their sensitivity and specificity.

Sequencing of the gene encoding the major pilin of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli and evidence that CFA/III is related to type IV pili.

The plasmid-encoded structural gene cofA necessary for the production of the major pilin subunit of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli was identified, and the nucleotide sequence of the gene was determined. cofA consists of 714 nucleotides encoding a 238-amino-acid protein (molecular weight of 25,309). CofA seems to be a precursor of CFA/III pilin, because the first 23 residues of the N-terminal amino acid sequence of the purified CFA/III pili coincided with the deduced amino acid sequence for residues 32 to 54 of CofA. Western blot (immunoblot) analysis of CofA also indicated its processing to form mature pilin in the presence of the downstream region of cofA. These results suggest that the major pilin of CFA/III pili is produced as a precursor form which is posttranslationally modified to the mature pilin and forms morphological pili after cleavage of the Gly-30-Met-31 junction, probably by a protease encoded by an as-yet-unknown gene located downstream of cofA. Interestingly, the N-terminal 30-amino-acid sequence of mature CFA/III shows the highest identity (76.7%) to TcpA pilin of Vibrio cholerae, which is a type IV class B pilin.

Isolation of mutant toxins of Vibrio parahaemolyticus hemolysin by in vitro mutagenesis.

Thermostable direct hemolysin produced by Vibrio parahaemolyticus is a major virulence factor of the organism. The hemolysin has a variety of biological activities such as lethality to mice, cytotoxicity to cultured cells, cardiotoxicity, and fluid accumulating activity in rabbit ileal loop test. In this study, we attempted to isolate less hemolytic mutant toxins of the thermostable direct hemolysin to use them for analysis of mode of action of the hemolysin. Six mutant toxins were obtained by in vitro mutagenesis of the cloned gene for the hemolysin. Characterization of the mutant toxins demonstrated that single amino acid substitutions at Gly62, Trp65, Thr67, Gly86, Glu116 and Glu138 resulted in a loss or lowering of the hemolytic activity. Two of the mutant toxins inhibited hemolysis by wild-type toxin on rabbit blood agar plates, while their hemolytic activity was below the detectable level. These mutant toxins would be useful for identifying the as yet unknown receptor for the hemolysin on the target cell membrane.

Monoclonal antibody developed against a hemolysin of Bacillus thuringiensis.

A total of five hybridoma cell lines that produced monoclonal antibodies (MAb) against a hemolysin (Bt-hemolysin) produced by Bacillus thuringiensis were established and characterized. All of these monoclonal antibodies reacted similarly not only to Bt-hemolysin but also to a hemolysin (Bc-hemolysin) produced by B. cereus, suggesting that the two hemolysins are immunologically indistinguishable. The MAb developed in this study was also successfully applied for rapid and simple purification of both Bt- and Bc-hemolysins by immunoaffinity column chromatography. The partial N-terminal amino acid sequence of the purified hemolysins was determined to be Ile-Glu-Gln-Thr.

The effect on enterotoxicity of protease purified from Vibrio cholerae O1.

The effect on enterotoxicity of protease purified from Vibrio cholerae O1 was investigated by the inoculation of live vibrio cells into protease-treated loops of the ileal loop model. Fluid accumulation ratios in the protease-treated loops were elevated in a dose-dependent manner by challenge with live vibrio cells but not by that with toxin. An enhancement effect of protease on enterotoxicity was observed in both serotypes of V. cholerae O1 and V. cholerae non-O1. It is suggested, therefore, that the enterotoxicity was enhanced by treatment with protease when live vibrio cells were inoculated into the ileal loops of rabbits.

Demonstration and characterization of simultaneous production of a thermostable direct hemolysin (TDH/I) and a TDH-related hemolysin (TRHx) by a clinically isolated Vibrio parahaemolyticus strain, TH3766.

Simultaneous production of a thermostable direct hemolysin (TDH)-like toxin (TDHx) and a TDH-related hemolysin (TRH)-like toxin (TRHx) by a clinical isolate (strain TH3766) of Kanagawa phenomenon-positive Vibrio parahaemolyticus was demonstrated and characterized. The two hemolysins were differentially purified by column chromatography on hydroxyapatite and immunoaffinity columns. The molecular weight of the two hemolysins were estimated to be 23,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). The purified TDHx was indistinguishable from the previously reported TDH/I (from strain TH012) but was different from the authentic TDH of a Kanagawa phenomenon-positive strain (T4750) physicochemically. The mobility of TRHx in nondenaturing PAGE differed from all the known TDHs and TRHs. The genes (tdhX and trhX) coding for TDHx and TRHx were cloned and sequenced. Homologies of nucleotide sequences of the coding regions between tdhX and tdhA (a gene for the authentic TDH) and between trhX and trh (a gene for the authentic TRH) were 98.1 and 99.1%, respectively, and homology between tdhX and trhX was 68.1%. At the amino acid level, TdhX was completely identical to TDH/I, although two base differences were found in the nucleotide sequences between tdhX and tdh/I. Two amino acid differences were observed between TrhX and Trh. Thus, these findings suggest that the TH3766 strain produces two types of hemolysins simultaneously. This is the first evidence that a strain of V. parahaemolyticus produces two types of toxins of the TDH-TRH family at the same time.

A survey of urease-positive Vibrio parahaemolyticus strains isolated from traveller's diarrhea, sea water and imported frozen sea foods.

The frequency of urease-positive Vibrio parahaemolyticus among isolates from patients, imported frozen sea foods and the environment (sea water) was studied. The highest isolation frequency of urease-positive V. parahaemolyticus was found in clinical isolates (11.2% out of 204 strains examined). Urease-positive V. parahaemolyticus was found in 5.7% of 88 frozen sea food-isolates examined, but no strains isolated from sea water were urease-positive. The isolates were further examined for the production of thermostable direct hemolysin (Vp-TDH) and its related hemolysin (Vp-TRH). Both are possible pathogenic toxins produced by mostly clinical isolates of V. parahaemolyticus. Urease-positive strains have a tendency to associate with clinical isolates producing both or neither Vp-TDH and Vp-TRH. Rabbit ligated ileal loops test was performed with several strains of urease-positive and -negative clinical isolates, and we found that some strains producing urease, even those which do not produce Vp-TDH or Vp-TRH, caused intestinal fluid accumulation.

The thermostable direct hemolysin of Vibrio parahaemolyticus is a pore-forming toxin.

The hemolytic mechanism of thermostable direct hemolysin (TDH), a possible virulence factor of Vibrio parahaemolyticus, was studied. We demonstrated that TDH acts as a "pore-forming toxin" in temperature-dependent and -independent steps. The first temperature-dependent step requires only about 1-2 min incubation at 37 degrees C and makes a "pore" with a functional diameter of approximately 2 nm. The pore size was deduced from the molecular diameter of the colloidal inhibitory polysaccharides. The formation of the pores on TDH-treated erythrocyte membranes was also demonstrated by electron microscopic examination. The second step, which is a temperature-independent lytic step, causes the erythrocytes to swell owing to a colloidal osmotic influx of water via the "pores" into cells, resulting in erythrocyte lysis (or rupture) owing to increased intracellular pressure.

Characterization of two forms of hemagglutinin/protease produced by Vibrio cholerae non-O1.

Two forms (34 kDa and 32 kDa) of hemagglutinin/protease produced by Vibrio cholerae non-O1 were characterized. The hemagglutinin/protease purified by immunoaffinity column chromatography using a monoclonal antibody was essentially a 34-kDa form. By incubation of the purified 34-kDa form at 37 degrees C, it was processed (autodigested) to the 32-kDa form. The N-terminal 20 amino acid sequences of both the 34- and 32-kDa forms were identical, suggesting that proteolytic processing at the C-terminal region of the 34-kDa hemagglutinin/protease resulted in the 32-kDa form. With this shift, protease activity increased, but hemagglutinating activity decreased, suggesting that the C-terminal region of the hemagglutinin/protease is related to hemagglutinating activity.

Enzyme-labeled oligonucleotide probes for detection of the genes for thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus.

Alkaline phosphatase conjugated oligonucleotide probes were developed to detect the genes (tdh and trh) coding for the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus. Using dot blot hybridization, probes were tested with 94 clinical isolates of V. parahaemolyticus. Results agreed well with those obtained using radio-labeled recombinant DNA probes for the genes tdh and trh. Specificity and sensitivity of enzyme tdh probes for detection of the trh gene were 100 and 93%, respectively, and those of the trh probes for trh gene detection were 93 and 86%, respectively. The tdh probes also hybridized with tdh-like genes processed by all strains of V. hollisae, and some strains of V. mimicus and V. cholerae non-O1, but neither tdh nor trh probes reacted with other bacterial species isolated from diarrheal stools. However, some V. parahaemolyticus strains that were negative with the enzyme trh probe hybridized weakly with a radio-labeled trh DNA fragment probe at medium stringency, and a few strains that were negative in high stringency conditions with a radio-labeled trh DNA fragment probe hybridized with the enzyme trh probe. This suggests that some strains of V. parahaemolyticus may carry another gene resembling trh.