Long-term results of a randomized controlled trial analyzing the role of systematic pre-operative coronary angiography before elective carotid endarterectomy in patients with asymptomatic coronary artery disease.

OBJECTIVES: To evaluate the potential benefit of systematic preoperative coronary-artery angiography followed by selective coronary-artery revascularization on the incidence of myocardial infarction (MI) in patients undergoing carotid endarterectomy (CEA) without a previous history of coronary artery disease (CAD). METHODS: We randomised 426 patients who were candidates for CEA, with no history of CAD, a normal electrocardiogram (ECG), and a normal cardiac ultrasound. In group A (n = 216) all patients underwent coronary angiography before CEA. In group B (n = 210) CEA was performed without coronary angiography. Patients were not blinded for relevant assessments during follow-up. Primary end-point was the occurrence of MI at 3.5 years. The secondary end-point was the overall survival rate. Median length of follow-up was 6.2 years. RESULTS: In group A, coronary angiography revealed significant coronary artery stenosis in 68 patients (31.5%). Among them, 66 underwent percutaneous Intervention (PCI) prior to CEA and 2 received combined CEA and coronary-artery bypass grafting (CABG). Postoperatively, no MI was observed in group A, whereas 6 MI occurred in group B, one of which was fatal (p = .01). During the study period, 3 MI occurred in group A (1.4%) and 33 were observed in group B (15.7%), 6 of which were fatal. The Cox model demonstrated a reduced risk of MI for patients in group A receiving coronary angiography (HR,.078; 95% CI, 0.024-0.256; p < .001). In addition, patients with diabetes and patients <70 years presented with an increased risk of MI. Survival analysis at 6 years by Kaplan-Meier estimates was 95.6 ± 3.2% in Group A and 89.7 ± 3.7% in group B (Log Rank = 6.54, p = .01). CONCLUSIONS: In asymptomatic coronary-artery patients, systematic coronary angiography prior to CEA followed by selective PCI or CABG significantly reduces the incidence of late MI and increases long-term survival. (ClinicalTrials.gov number, NCT02260453).

A simple circuit for cerebral perfusion during cardiopulmonary bypass surgery of the ascending aorta and the aortic arch.

INTRODUCTION: A circuit was developed to allow for rapid reaction to the needs of perfusion during extracorporeal circulation (ECC) in surgery of the aortic arch and ascending aorta. METHOD: From January 2008 through January 2010, a home-designed circuit was used on 30 patients with aortic dissection who underwent surgery to replace the ascending aorta and the aortic arch and, in some cases, the aortic valve and re-implant of the coronary arteries using Bentall's technique.

Factors influencing immediate and long-term results after button's technique.

BACKGROUND: Aim of this study was to evaluate the factors influencing immediate and long-term results in patients undergoing aortic root replacement with a composite graft. METHODS: Between January 1989 and February 1999, 105 patients (83 males, 22 females) who underwent Bentall technique were studied. Preoperative diagnosis was annulo-aortic ectasia in 54, aortic dissection in 27, atherosclerotic aneurysm in 21, and aortitis in 3 cases. Seventeen patients were affected by Marfan's syndrome. All cases, elective, urgent, and emergency were included. Button technique was performed and the associated surgical procedures were coronary artery bypass grafting in 21, total aortic arch replacement in 15, proximal hemi arch in 5, and mitral valve replacement in 5 cases. RESULTS: The overall hospital mortality rate was 7.6% (n=8). Univariate analysis using chi(2) and/or two-sample "t"-test showed that dissection, aortitis, aneurysm rupture into-pleura or pericardium, emergency status, redo, prolonged pump times and circulatory arrest, were predictors influencing in-hospital mortality. Coagulopathy, low cardiac output, stroke, perioperative myocardial infarction, surgical bleeding leading to reoperation, were significantly related to in-hospital mortality (by correlation analysis). A multivariate analysis showed that, emergency status (p=0.027), aortic dissection (p=0.029), perioperative myocardial infarction (p=0.0021), reoperation for bleeding (p=0.0023), and pump time >180 min (p=0.011), were significant. The actuarial survival rate at 10 years follow-up was 84.7%. There were 8 late deaths. The Kaplan-Meier showed significant differences when considering dissection vs non-dissection (p=0.018), but did not reach significance in Marfan vs non-Marfan groups (p=0.83). NYHA class IV (p=0.052), previous cardiac surgery procedure (p=0.041), concomitant CABG (p=0.021), total aortic arch reconstruction (p=0.001), and mitral valve replacement (p=0.016), were identified as significant by Log Rank test. CONCLUSIONS: The Bentall procedure for aortic root replacement is safe and durable; in hospital mortality in elective status it was 1.28%; early and long-term mortality higher in patients with acute dissection. Six late deaths were procedures related. Sixty-six patients (76.4%) were in NYHA I class at follow-up. The incidence of late outcomes, thromboembolism (1.03%), graft infection (2.06%), pseudoaneurysm (0%), reoperation in ascending aorta or aortic valve (3.1%), operations on the remaining aorta (6.7%), and hemorrhage due to anticoagulant therapy (1.03%), are very low.

Left ventricular thrombosis after blunt chest trauma.

A 22-year-old man was admitted to our observation with left ventricular thrombus arising after blunt chest trauma occurring during a ski accident one year before. None was obtained from a review of instrumental and laboratory data at trauma time. Transesophageal echocardiography showed an intraventricular thrombus and severe hypokinesia at the apex. Standard cardiac surgery procedure was performed and postoperative period was uneventful. Echocardiography controls at 6/12 months showed a normal apex kinesia. This case shows the importance of hospitalization, hemodynamics monitorization and late serial echocardiographic controls for timely diagnosis and management of myocardial contusion and consecutive ventricular thrombus formation to prevent life-threatening complications.

Characterization of Porphyromonas gingivalis insertion sequence-like element ISPg5.

Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobe, is found in periodontitis lesions, and its presence in subgingival plaque significantly increases the risk for periodontitis. In contrast to many bacterial pathogens, P. gingivalis strains display considerable variability, which is likely due to genetic exchange and intragenomic changes. To explore the latter possibility, we have studied the occurrence of insertion sequence (IS)-like elements in P. gingivalis W83 by utilizing a convenient and rapid method of capturing IS-like sequences and through analysis of the genome sequence of P. gingivalis strain W83. We adapted the method of Matsutani et al. (S. Matsutani, H. Ohtsubo, Y. Maeda, and E. Ohtsubo, J. Mol. Biol. 196:445-455, 1987) to isolate and clone rapidly annealing DNA sequences characteristic of repetitive regions within a genome. We show that in P. gingivalis strain W83, such sequences include (i) nucleotide sequence with homology to tRNA genes, (ii) a previously described IS element, and (iii) a novel IS-like element. Analysis of the P. gingivalis genome sequence for the distribution of the least used tetranucleotide, CTAG, identified regions in many of the initial 218 contigs which contained CTAG clusters. Examination of these CTAG clusters led to the discovery of 11 copies of the same novel IS-like element identified by the repeated sequence capture method of Matsutani et al. This new 1,512-bp IS-like element, designated ISPg5, has features of the IS3 family of IS elements. When a recombinant plasmid containing much of ISPg5 was used in Southern analysis of several P. gingivalis strains, including clinical isolates, diversity among strains was apparent. This suggests that ISPg5 and other IS elements may contribute to strain diversity and can be used for strain fingerprinting.

Genetic characterization of a Streptococcus mutans LraI family operon and role in virulence.

Proteins belonging to the LraI (for "lipoprotein receptor antigen") family function as adhesins in several streptococci, as a virulence factor for endocarditis in at least one of these species, and potentially as metal transporters in many bacteria. We have identified and characterized the chromosomal locus containing the LraI family gene (designated sloC) from Streptococcus mutans, an agent of dental caries and endocarditis in humans. Northern blot analysis indicated that sloC is cotranscribed with three other genes. As with other LraI operons, the sloA and sloB genes apparently encode components of an ATP-binding cassette transport system. The product of the fourth gene, sloR, has homology to the metal-dependent regulator from Corynebacterium diphtheriae, DtxR. A potential binding site for SloR was identified upstream from the sloABCR operon and was conserved upstream from LraI operons in several other streptococci. Potential SloR homologs were identified in the unfinished genomic sequences from two of these, S. pneumoniae and S. pyogenes. Mutagenesis of sloC in S. mutans resulted in apparent loss of expression of the entire operon as assessed by Northern blot analysis. The sloC mutant was indistinguishable from its wild-type parent in a gnotobiotic rat model of caries but was significantly less virulent in a rat model of endocarditis. Virulence for endocarditis was restored by correction of the sloC mutation but not by provision of the sloC gene in trans, suggesting that virulence requires the expression of other genes in the sloC operon.

Arrhythmia prophylaxis after coronary artery bypass grafting: regimens of magnesium sulfate administration.

BACKGROUND: Arrhythmias are common after open heart surgery and may be related to hypomagnesaemia due to cardiopulmonary bypass. Although perioperative prophylactic Mg2+ administration may prevent arrhythmias after coronary artery bypass grafting (CABG), clear indications as well as the timing of Mg2+ substitution and dose regimen need to be clarified. Aim of this study was to evaluate the antiarrhythmic effects of Mg2+ infusion in patients who underwent elective CABG. METHOD: Ninety-seven patients who underwent elective CABG were divided in four Groups. In Group A 1 g of magnesium sulfate was added to the pump prime, Group B received 1 g in the pump prime plus 5 mmol/L in the cardioplegic solution, Group C received 5 mmol/L in the cardioplegic solution, and Group D was a placebo control Group. Groups A, B, and C also received 24 h continuous infusion of magnesium sulfate at 10 mmol/L. Three-channel electrocardiogram (II-V5-V6) continuous monitoring was performed 12 hours preoperatively and 48 hours postoperatively. Blood samples were taken for subsequent Serum magnesium measurements, at five different time points before, during and after CBP. RESULTS: In all Groups serum Mg2+ levels were reduced during CPB (Time 2) and statistically significant differences from pre-anaesthesia levels (Time 1) were noted (p <0.05). In Groups A, B, and C Serum Mg2+ levels increased progressively from Time 3 to Time 5; in Group D serum Mg2+ levels were still much lower at Time 5. Significant differences (p<0.05) were noted for Groups B and C vs Groups A and D in atrial ectopics, atrial fibrillation, and ventricular arrhythmic events. CONCLUSION: Our results demonstrate that Mg2+ sulfate administration regimens used in Group B and C reduce postoperative arrhythmic events in patients undergoing CABG.

Hemoglobinase activity of the lysine gingipain protease (Kgp) of Porphyromonas gingivalis W83.

Porphyromonas gingivalis, an important periodontal disease pathogen, forms black-pigmented colonies on blood agar. Pigmentation is believed to result from accumulation of iron protoporphyrin IX (FePPIX) derived from erythrocytic hemoglobin. The Lys-X (Lys-gingipain) and Arg-X (Arg-gingipain) cysteine proteases of P. gingivalis bind and degrade erythrocytes. We have observed that mutations abolishing activity of the Lys-X-specific cysteine protease, Kgp, resulted in loss of black pigmentation of P. gingivalis W83. Because the hemagglutinating and hemolytic potentials of mutant strains were reduced but not eliminated, we hypothesized that this protease played a role in acquisition of FePPIX from hemoglobin. In contrast to Arg-gingipain, Lys-gingipain was not inhibited by hemin, suggesting that this protease played a role near the cell surface where high concentrations of hemin confer the black pigmentation. Human hemoglobin contains 11 Lys residues in the alpha chain and 10 Lys residues in the beta chain. In contrast, there are only three Arg residues in each of the alpha and beta chains. These observations are consistent with human hemoglobin being a preferred substrate for Lys-gingipain but not Arg-gingipain. The ability of the Lys-gingipain to cleave human hemoglobin at Lys residues was confirmed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry of hemoglobin fragments resulting from digestion with the purified protease. We were able to detect several of the predicted hemoglobin fragments rendered by digestion with purified Lys-gingipain. Thus, we postulate that the Lys-gingipain of P. gingivalis is a hemoglobinase which plays a role in heme and iron uptake by effecting the accumulation of FePPIX on the bacterial cell surface.

Construction and immunologic evaluation of a Porphyromonas gingivalis subsequence peptide fused to hepatitis B virus core antigen.

Several proteins of Porphyromonas gingivalis contain multiple copies of a 47 amino acid conserved repeated sequence. A fusion protein was constructed in which the P. gingivalis peptide was fused to the carboxy terminus of the hepatitis B core protein. This fusion protein was expressed in Escherichia coli, purified, and used to vaccinate mice that were later challenged with P. gingivalis W83 using the mouse abscess model. Although the mice were not protected against bacterial challenge, Western blot analysis showed that sera from the mice and from rabbits immunized with the fusion protein reacted with a number of vesicle proteins from P. gingivalis W83. These data suggested that this peptide is recognized by the host's immune system but that the antibodies are not protective.

Localization of HArep-containing genes on the chromosome of Porphyromonas gingivalis W83.

We have mapped a group of virulence genes of Porphyromonas gingivalis to a single large fragment of the genome. These genes (rgpA, kgp, and hagA) all contain a consensus repeat sequence (HArep). rgpA and kgp encode cysteine proteases with Arg-X and Lys-X specificity, respectively, and hagA encodes a hemagglutinin. Genomic DNA fragments separated by pulse-field gel electrophoresis were blotted and probed in order to localize the genes to a 0.25-Mb NheI fragment of the P. gingivalis W83 genome. Further hybridization analyses with single- and double-restriction digestion allowed us to generate a physical map of the fragment and determine the precise locations of the protease and hemagglutinin genes. In addition, we found an insertion-like sequence, IS195, near the ends of the 0. 25-Mb NheI fragment. A similarly sized fragment carrying HArep sequences was also demonstrated in the P. gingivalis W12 and W50 genomes.

Effects of bimakalim on human cardiac action potentials: comparison with guinea pig and nicorandil and use-dependent study.

Electrophysiologic effects of K(ATP) channel openers (KCOs) are rarely studied for tissue and species specificity, and use-dependent investigations in human tissues are lacking. We therefore investigated in vitro the concentration-dependent effects of the KCO bimakalim [from 10 nM to 10 microM, at 1,000 ms of cycle length (CL) and 37 degrees C] on human (atrium, n = 4, and ventricle, n = 6) and guinea pig (atrium, n = 7, and ventricle, n = 6) transmembrane action potential (AP). The frequency relation (from CL 1,600 to 300 ms, 31 degrees C) of human atrial AP duration 90% (APD90) shortening (10 microM vs. baseline, n = 7) also was determined. A parallel study was performed with the KCO nicorandil (from 10 nM to 1 mM, n = 3) in human atrial APs, at 31 degrees C. Resting membrane potential and maximal upstroke velocity of AP were not modified by bimakalim at maximal concentration, whereas AP amplitude was decreased in both guinea pig preparations (p < 0.05); APD90 was shortened in all tissues (p < 0.01). Median effective concentration (EC50) for APD90 shortening at 37 degrees C was 0.54 and 2.74 microM in atrial and ventricular human tissue, respectively, and 8.55 and 0.89 microM in atrial and ventricular guinea pig tissue, respectively. In human atrial tissue at 31 degrees C, EC50 with bimakalim was 0.39 microM; a much higher value was seen with nicorandil (210 microM). Bimakalim (10 microM)-induced APD90 shortening as a function of stimulation rate was greatest at longest CL. Evidence is provided for (a) species (human vs. guinea pig) and tissue (atrium vs. ventricle) differential AP sensitivity to bimakalim; (b) an approximately 500-fold higher efficacy of bimakalim versus nicorandil to shorten human atrial APD90; and (c) normal use-dependence of human atrial APD90 shortening with bimakalim at 10 microM.

Molecular genetics and nomenclature of proteases of Porphyromonas gingivalis.

The strategies used by bacterial pathogens to establish and maintain themselves in the host represent one of the fundamental aspects of microbial pathogenesis. Characterization of these strategies and the underlying molecular machinery offers new opportunities both to our understanding of how organisms cause disease in susceptible individuals and to the development of novel therapeutic measures designed to undermine or interfere with these determinants of successful survival. With respect to the microbial aetiology of the periodontal diseases, a growing body of evidence suggests that the proteolytic enzymes of Porphyromonas gingivalis represent key survival and, by extrapolation, virulence determinants of this periodontal bacterium. This in turn has led to international efforts to characterize these enzymes at the gene and protein level. Approximately 20 protease genes of P. gingivalis with different names and accession numbers have been deposited in the gene databases and a correspondingly heterogeneous nomenclature system is employed for the products of these genes in the literature. However, it is evident, through comparison of these gene sequences and through gene inactivation studies, that the genetic structure of the proteases of this organism, particularly those with specificity for arginyl and lysyl peptide bonds, is less complicated than originally thought. The major extracellular and surface associated arginine specific protease activity is encoded by 2 genes which we recommend be designated rgpA and rgpB (arg-gingipains A & B). Similarly we recommend that the gene encoding the major lysine specific protease activity is designated kgp (lys-gingipain). These three genes, which account for all the extracellular/surface arginine and lysine protease activity in P. gingivalis, belong to a family of sequence-related proteases and haemagglutinins.

Preoperative identification of viable myocardium: effectiveness of nitroglycerine-induced changes in myocardial Sestamibi uptake.

In order to predict tissue viability in infarcted myocardial areas, changes induced by nitroglycerine infusion on Sestamibi myocardial uptake were evaluated in 37 patients with previously confirmed myocardial infarction undergoing coronary artery bypass grafting, and compared with echocardiographic and perfusional changes occurring after the operation. The improvement of Sestamibi uptake after nitroglycerine correctly classified 24/26 (92%) patients showing postoperative improvement of wall motion in the infarcted area, whereas 24/31 (77%) patients with nitroglycerine-induced increase in Sestamibi uptake had improved wall motion after operation. The presence of collateral flow to the infarcted area was associated with a significantly (P < 0.01) higher increase in Sestamibi uptake both during nitroglycerine infusion and postoperatively. An increase in wall motion score after operation was associated with a significantly higher (P < 0.05) increase in Sestamibi uptake score during nitroglycerine infusion. Thus, the results of this study suggest that Sestamibi perfusional myocardial scintigraphy during nitroglycerine infusion is capable of assessing viable but chronically hypoperfused myocardium and predicting postoperative wall motion and perfusional improvement, to yield the best results in patients with evidence of collateral circulation that supplies the infarcted area.

IS195, an insertion sequence-like element associated with protease genes in Porphyromonas gingivalis.

Porphyromonas gingivalis is recognized as an important etiologic agent in adult and early-onset periodontal disease. Proteases produced by this organism contribute to its virulence in mice. Protease-encoding genes have been shown to contain multiple copies of repeated nucleotide sequences. These conserved sequences have also been found in hemagglutinin genes. In the process of studying the genetic loci containing the conserved repeated sequences, we have characterized a prtP gene homolog from P. gingivalis W83 encoding a cysteine protease with Lys-X specificity. However, this prtP gene was interrupted by an insertion sequence-like element which we designated IS195. Furthermore, IS195 and another element, IS1126, were present downstream of prtP gene homologs (kgp) found in P. gingivalis H66 and 381. IS195, a 1,068-bp insertion sequence-like element, contained 11-bp inverted repeats at its termini and was bordered by 9-bp direct repeats presumed to be a transposition-mediated target site duplication. Its central region contained one large open reading frame encoding a predicted 300-amino-acid protein which appeared to be a transposase. We isolated two naturally occurring variants of P. gingivalis W83, one carrying IS195 within the coding region of the prtP gene and another containing an intact prtP gene. Biochemical characterization revealed a lack of trypsin-like Lys-X specific proteolytic activity in the P. gingivalis W83 variant carrying the disrupted prtP gene. Studies using a mouse model revealed a reduction of virulence resulting from insertion of IS195 into the coding region of the prtP gene. An allelic-exchange mutant defective in the prtP gene also was constructed and tested in vivo. It displayed intermediate virulence compared to that of the wild-type and prtP::IS195 mutant strains. We conclude that the Lys-X cysteine protease contributes to virulence in soft tissue infections.

Nucleotide sequence of the Porphyromonas gingivalis W83 recA homolog and construction of a recA-deficient mutant.

Degenerate oligonucleotide primers were used in PCR to amplify a region of the recA homolog from Porphyromonas gingivalis W83. The resulting PCR fragment was used as a probe to identify a recombinant lambda DASH phage (L10) carrying the P. gingivalis recA homolog. The recA homolog was localized to a 2.1-kb BamHI fragment. The nucleotide sequence of this 2.1-kb fragment was determined, and a 1.02-kb open reading frame (341 amino acids) was detected. The predicted amino acid sequence was strikingly similar (90% identical residues) to the RecA protein from Bacteroides fragilis. No SOS box, characteristic of LexA-regulated promoters, was found in the 5' upstream region of the P. gingivalis recA homolog. In both methyl methanesulfonate and UV survival experiments the recA homolog from P. gingivalis complemented the recA mutation of Escherichia coli HB101. The cloned P. gingivalis recA gene was insertionally inactivated with the ermF-ermAM antibiotic resistance cassette to create a recA-deficient mutant (FLL33) by allelic exchange. The recA-deficient mutant was significantly more sensitive to UV irradiation than the wild-type strain, W83. W83 and FLL33 showed the same level of virulence in in vivo experiments using a mouse model. These results suggest that the recA gene in P. gingivalis W83 plays the expected role of repairing DNA damage caused by UV irradiation. However, inactivation of this gene did not alter the virulence of P. gingivalis in the mouse model.

Left ventricular aneurysmectomy; comparison between two techniques; early and late results.

OBJECTIVE: The aim of the present study was to evaluate early and late results of two different surgical techniques for left ventricular aneurysms repair. The conventional aneurysmectomy and direct closure of the ventricular wall and the endoventricular patch plasty. METHODS: We retrospectively reviewed 118 patients operated on for postinfarction left ventricular aneurysm from 1981 to 1994. Eighty-seven patients (Group A) were operated upon between 1981 and 1991 with the conventional technique and 31 patients (Group B) between 1992 and 1994 with the endoventricular patch plasty technique. Preoperative clinical, hemodynamic and echocardiographic evaluation with operative procedures and early postoperative results of all patients are reported. We also analyzed results of late clinical and echocardiographic controls of 34 patients of Group A and all patients of Group B after a mean follow-up of 42 and 28 months, respectively. RESULTS: Mean number of by-pass grafts was 1.9 in Group A and 2.6 in Group B (P = 0.01). The left anterior descending coronary artery was revascularized in 27 patients of Group A (34.6%) and 26 of Group B (89.7%) (P < 0.001); the left internal mammary artery was used in seven patients of Group A (8.9%) and 24 of Group B (82.8%) (P < 0.001). Hospital mortality in Group A was 10.3% (9/87), in Group B there was no hospital mortality (P > 0.05). Thirty-two patients of Group A (36.8%) and 3 of Group B (9.7%) suffered of low cardiac output syndrome (P = 0.01). At late control, improvements observed in NYHA and CCS classes, left ventricular ejection fraction (all P < 0.001 in both groups versus preoperative values) and left ventricular end-diastolic diameter (P > 0.05 in Group A and P < 0.001 in Group B) proved to be statistically higher in patients of Group B. CONCLUSIONS: Endoventricular patch plasty associated with a complete myocardial revascularization, in particular of the anterior descending coronary, and a larger use of the internal mammary artery, permits, by means of reconstruction of the left ventricular geometry, a better outcome for patients undergoing left ventricular aneurysmectomy.

Immunization with FimA protects against Streptococcus parasanguis endocarditis in rats.

FimA, a surface-associated protein of Streptococcus parasanguis, is associated with initial colonization of damaged heart tissue in an endocarditis model (D. Burnette-Curley, V. Wells, H. Viscount, C. Munro, J. Fenno, P. Fives-Taylor, and F. Macrina, Infect. Immun. 63:4669-4674, 1995). We have evaluated the efficacy of recombinant FimA as a vaccine in the rat model of endocarditis and investigated in vitro the mechanism for the protective role of immunization. FimA-immunized and nonimmunized control animals were catheterized to induce heart valve damage and infected intravenously with 10(7) CFU of wild-type S. parasanguis FW213 bacteria. The presence of bacteria associated with platelet-fibrin vegetations 24 h postchallenge was evaluated. Immunized rats were significantly less susceptible to endocarditis (2 cases among 34 animals) than the control group (21 cases among 33 animals) (P < 0.001). Incubation of S. parasanguis FW213 with rabbit anti-FimA immune serum decreased the mean percent adherence (0.34% of added cells) to platelet-fibrin matrix in vitro compared with that of preimmune normal serum (5.04% of added cells; P < 0.001). Adsorption of immune serum with FimA-positive S. parasanguis FW213 yielded antiserum that failed to block adherence to the platelet-fibrin matrix. We assessed the vaccine potential of FimA as a common immunogen able to provide cross-protection in streptococcal endocarditis by determining the occurrence and expression of fimA in the viridans group streptococci and enterococci. We detected the presence of fimA homologs by Southern hybridization and PCR amplification analyses and determined by immunoblotting the expression of FimA-like proteins among a variety of streptococci and enterococci that frequently cause endocarditis. Eighty-one percent (26 of 32) of streptococcal and enterococcal strains isolated from bacteremic patients expressed proteins that comigrated with FimA and were reactive with polyclonal anti-FimA serum. Streptococcal DNA from strains that were positive by Western blot (immunoblot) analysis hybridized to the full-length fimA probe. Our studies suggest that FimA immunization results in antibody-mediated inhibition of bacterial adherence, a critical early event in the pathogenesis of endocarditis. Our data demonstrate that a majority of streptococcal strains associated with endocarditis have genes that encode FimA-like proteins. Taken together, these results suggest that FimA is a promising candidate for an endocarditis vaccine.

Nitroglycerin-induced changes in myocardial sestamibi uptake to detect tissue viability: radionuclide comparison before and after revascularization.

BACKGROUND: Nitroglycerin (NTG) is known to increase the blood supply to the myocardium, and would thus increase the delivery of a perfusional tracer such as sestamibi (MIBI) to the tissue. The latter, in turn, would take up and concentrate the tracer to a greater extent than in basal conditions only if energy-dependent mechanisms were still available-that is, only if the cells were still viable. METHODS: We evaluated the changes that intravenous administration of NTG induced on the uptake of MIBI by akinetic myocardial areas, using tomographic perfusional imaging in 23 patients with previously ascertained anterior myocardial infarction who were undergoing myocardial revascularization procedures. Changes in uptake were compared with echocardiographic and perfusional changes occurring after operation. RESULTS: The improvement of MIBI uptake after NTG correctly identified 12 of the 16 patients (75%) showing postoperative wall motion improvement; they comprised 12 of the 14 (86%) patients with NTG-induced increase in MIBI uptake who showed improved wall motion after operation. A close correlation (r = 0.88, P < 0.001) was found between the increase in myocardial MIBI uptake induced by NTG infusion and that induced by revascularization. The presence of collaterals to the akinetic area was associated with a significantly (P < 0.01) greater increase in MIBI uptake both during NTG infusion and after operation. CONCLUSIONS: The results of this study suggest that MIBI perfusional myocardial scintigraphy during infusion of NTG is capable of detecting viable but chronically hypoperfused myocardium, predicting postoperative wall motion and perfusional improvement, and reflecting the postoperative pattern of perfusion. The best results were achieved in patients with evidence of collateral circulation supplying the infarcted area.

Characterization of IS199 from Streptococcus mutans V403.

The complete nucleotide sequence of the insertion sequence IS199 has been determined. This novel element was found in Streptococcus mutans V403 where DNA hybridization studies suggested that it was present in multiple copies on the genome. IS199 is 1220 bp and appears unique to the mutans streptococci. A limited epidemiological survey revealed this element to occur infrequently in this group (4/50 strains examined). IS199 belongs to the IS3 family of procaryotic insertion sequence elements. It contained tandem open reading frames (ORFs) reminiscent of the structure of the IS3 class. Additionally, the larger of the two ORF products (ORFB) exhibited amino acid sequence similarities to comparable proteins from IS3 and other elements of this family. Transposition of IS199 gave rise to a 3-bp target site duplication. We were able to insert an antibiotic resistance gene into IS199 enabling us to follow the transposition of this element in S. mutans V403.

Immunologic characteristics of a Streptococcus mutans glucosyltransferase B sucrose-binding site peptide-cholera toxin B-subunit chimeric protein.

Glucosyltranferases (Gtfs) produced by the mutans streptococci are recognized as virulence factors in dental caries, and the inhibition of Gtfs by secretory immunoglobulin A is predicted to provide protection against this disease. The basis of such mucosal immunity is linked to the ability to reliably stimulate production of secretory immunoglobulin A against Gtfs. In this regard, we are exploring the immunogenicities of various Gtf peptides genetically fused to the B subunit of cholera toxin (CTB), a known mucosal adjuvant. In this work, we have created a gene fusion linking the GtfB active-site (AS) peptide DANFDSIRVDAVDNVDADLLQIA to the amino terminus of CTB. This sequence, deduced from the nucleotide sequence of gtfB from Streptococcus mutans GS5, has been found to be strongly conserved in Gtfs from several mutans streptococci. We have purified this recombinant protein (AS:CTB) from Escherichia coli carrying the fusion gene under the control of the lactose operon promoter. This protein was immunogenic in rabbits and produced specific serum antibodies against both the Gtf peptide and the CTB moiety. The antiserum was tested for its ability to inhibit GtfB activity obtained from a mutant of S. mutans able to make only this enzyme and none of the other usual Gtfs or fructosyltransferase. Approximately 50% of the GtfB activity was inhibited in such assays. These results suggest that the AS of this enzyme is accessible to antibody binding and that this region of the protein may be considered a vulnerable target for vaccine design and development. The AS:CTB was able to bind GM1, ganglioside in enzyme-linked immunosorbent assays, indicating that the recombinant protein retained this property, which is though to be critical to the mucosal immunoadjuvant properties of CTB. Thus, this protein may be promising as a candidate anticaries vaccinogen alone or in combination with other Gtf peptides or conjugates.