Persistence of the efficacy of zoster vaccine in the shingles prevention study and the short-term persistence substudy.

BACKGROUND: The Shingles Prevention Study (SPS; Department of Veterans Affairs Cooperative Study 403) demonstrated that zoster vaccine was efficacious through 4 years after vaccination. The Short-Term Persistence Substudy (STPS) was initiated after the SPS to further assess the persistence of vaccine efficacy. METHODS: The STPS re-enrolled 7320 vaccine and 6950 placebo recipients from the 38 546-subject SPS population. Methods of surveillance, case determination, and follow-up were analogous to those in the SPS. Vaccine efficacy for herpes zoster (HZ) burden of illness, incidence of postherpetic neuralgia (PHN), and incidence of HZ were assessed for the STPS population, for the combined SPS and STPS populations, and for each year through year 7 after vaccination. RESULTS: In the STPS as compared to the SPS, vaccine efficacy for HZ burden of illness decreased from 61.1% to 50.1%, vaccine efficacy for the incidence of PHN decreased from 66.5% to 60.1%, and vaccine efficacy for the incidence of HZ decreased from 51.3% to 39.6%, although the differences were not statistically significant. Analysis of vaccine efficacy in each year after vaccination for all 3 outcomes showed a decrease in vaccine efficacy after year 1, with a further decline thereafter. Vaccine efficacy was statistically significant for the incidence of HZ and the HZ burden of illness through year 5. CONCLUSIONS: Vaccine efficacy for each study outcome was lower in the STPS than in the SPS. There is evidence of the persistence of vaccine efficacy through year 5 after vaccination but, vaccine efficacy is uncertain beyond that point.

In vivo tissue sampling of embryonic resorption sites using ultrasound guided biopsy.

In the polytocous European brown hare (Lepus europaeus) more than 23% of all successful implantations undergo embryonic resorption. The objective of the study was to establish a minimally invasive ultrasound guided biopsy technique to collect embryonic resorption tissue in vivo. The sampled material was genetically analysed to determine paternity and the sex of the embryo. Female hares were either mated or artificially inseminated and pregnancy was confirmed by ultrasound on day six post ovulation. Subsequent embryonic development was ultrasonographically monitored on a regular basis to detect embryos undergoing resorption. Cell material of the resorption site was collected under ultrasonographic control via transabdominal biopsy of the placenta or aspiration of resorption fluid. To avoid breathing movements during the biopsy, the animals were intubated and a short apnoea was evoked by assisted ventilation. The presence of embryonic cells in the biopsy material was confirmed by microsatellite analysis in 11 of the fluid samples (n = 28) and six of the placental samples (n = 8). The lower success rate in the fluid samples was attributed to the abundance of maternal cells which was confirmed by the analysis of fluid sample smears. Male sex of the embryos undergoing resorption was detected by SRY analysis for ten of the fluid samples and for one of the placental samples. The two biopsy techniques did not have any negative impact on the prenatal development of the healthy siblings nor did it influence the future breeding performance of the females that were biopsied.

Early reoperation for acute dysphagia following laparoscopic fundoplication.

BACKGROUND: A small number of patients develop acute severe dysphagia for which reoperation is necessary within 10 days of laparoscopic fundoplication. The aim of this study was to identify clinical variables that might predict the likelihood of this condition occurring, such that it could be avoided in the future. METHODS: This was a prospective cohort study from three tertiary referral centres, using reoperation for acute dysphagia as the main outcome variable. Gastrointestinal symptom rating scale, and psychological well-being index questionnaires were undertaken before laparoscopic fundoplication, and dysphagia scores were determined before operation and 1 year later. Standard preoperative assessment included gastroscopy, oesophageal manometry and pH studies. RESULTS: Twelve (1.9 per cent) of the 617 patients suffered acute dysphagia, which was predicted by older age and female sex, and resulted in a longer duration of hospital stay. This condition was not predicted by any other demographic, clinical, investigative or operative variables. CONCLUSION: The study did not identify useful criteria by which severe acute dysphagia could be anticipated and thereby avoided following laparoscopic fundoplication.

Chronic dysphagia following laparoscopic fundoplication.

BACKGROUND: Many surgeons practise tailored laparoscopic antireflux surgery in an attempt to prevent postoperative dysphagia. The aim of this study was to determine the effect of 360 degrees fundoplication (Nissen) or 270 degrees fundoplication (Toupet), and the influence of abnormal oesophageal peristalsis, upon postoperative dysphagia. METHODS: This was a cohort study from three tertiary referral centres, using dysphagia before laparoscopic fundoplication and 1 year after operation as the main outcome variable. Preoperative oesophageal manometry was performed on all patients. RESULTS: Some 761 patients underwent Nissen and 85 underwent Toupet fundoplication. Only 2 per cent reported severe postoperative dysphagia. There was a significant selection bias towards the Toupet operation for patients with abnormal oesophageal motility (P < 0.001). For patients whose oesophageal manometric findings were normal there was a significant improvement in dysphagia after Nissen fundoplication (P = 0.02), and no significant change following Toupet fundoplication. There was no significant change in the rate of dysphagia following either method of fundoplication amongst other subgroups in which oesophageal manometry was stratified as non-specific motor disorder, low-amplitude peristalsis, or aperistalsis. CONCLUSION: A tailored approach to the degree of fundoplication is unnecessary as patients with dysmotility suffer no more dysphagia after full laparoscopic Nissen fundoplication than those who have a partial Toupet wrap.

Staphylococcus aureus alpha-toxin induces apoptosis in endothelial cells.

The internalization of Staphylococcus aureus by cultured human umbilical vein endothelial cells was recently shown to induce apoptosis. We examined the role of alpha-toxin, a major pore-forming toxin secreted by S. aureus, in causing apoptosis in vitro. Purified alpha-toxin, at sublytic concentrations, induced apoptosis in endothelial cell monolayers. Comparisons of two alpha-toxin (hla)-positive S. aureus strains and their isogenic hla-deficient mutants in the invasion assay of endothelial cells demonstrated that the capacity to produce alpha-toxin was associated with a greater propensity for apoptosis in endothelial cells. These results demonstrate for the first time that expression of alpha-toxin during endothelial cell invasion by S. aureus enhances apoptosis.

Internalization of Staphylococcus aureus by endothelial cells induces apoptosis.

The ability of Staphylococcus aureus to invade and survive within endothelial cells is believed to contribute to its propensity to cause persistent endovascular infection with endothelial destruction. In the present study, we show that following invasion of human umbilical vein endothelial cells, intracellular S. aureus organisms remain viable over a 72-h period and, as determined by transmission electron microscopic examination, that the bacteria exist within vacuoles and free within the cytoplasm. We also demonstrate that endothelial cell death following S. aureus invasion occurs at least in part by apoptosis as shown by DNA fragmentation and changes in nuclear morphology. Apoptotic changes were evident as early as 1 h after infection of endothelial cells. Internalization of S. aureus rather than adherence appears to be necessary, since use of the phagocytosis inhibitor cytochalasin D prevented apoptosis. UV-killed staphylococci, although retaining the capacity to be internalized, were not capable of inducing apoptosis, suggesting that apoptosis is dependent upon a factor associated with viable organisms. The studies demonstrate that viable intracellular S. aureus induces apoptosis of endothelial cells and that internalized staphylococci can exist free within the cytoplasm.

Recombinant expression and characterization of the major beta-lactamase of Mycobacterium tuberculosis.

New antibiotic regimens are needed for the treatment of multidrug-resistant tuberculosis. Mycobacterium tuberculosis has a thick peptidoglycan layer, and the penicillin-binding proteins involved in its biosynthesis are inhibited by clinically relevant concentrations of beta-lactam antibiotics. beta-Lactamase production appears to be the major mechanism by which M. tuberculosis expresses beta-lactam resistance. beta-Lactamases from the broth supernatant of 3- to 4-week-old cultures of M. tuberculosis H37Ra were partially purified by sequential gel filtration chromatography and chromatofocusing. Three peaks of beta-lactamase activity with pI values of 5.1, 4.9, and 4.5, respectively, and which accounted for 10, 78, and 12% of the total postchromatofocusing beta-lactamase activity, respectively, were identified. The beta-lactamases with pI values of 5.1 and 4.9 were kinetically indistinguishable and exhibited predominant penicillinase activity. In contrast, the beta-lactamase with a pI value of 4.5 showed relatively greater cephalosporinase activity. An open reading frame in cosmid Y49 of the DNA library of M. tuberculosis H37Rv with homology to known class A beta-lactamases was amplified from chromosomal DNA of M. tuberculosis H37Ra by PCR and was overexpressed in Escherichia coli. The recombinant enzyme was kinetically similar to the pI 5.1 and 4.9 enzymes purified directly from M. tuberculosis. It exhibited predominant penicillinase activity and was especially active against azlocillin. It was inhibited by clavulanic acid and m-aminophenylboronic acid but not by EDTA. We conclude that the major beta-lactamase of M. tuberculosis is a class A beta-lactamase with predominant penicillinase activity. A second, minor beta-lactamase with relatively greater cephalosporinase activity is also present.

Hyperproduction of alpha-toxin by Staphylococcus aureus results in paradoxically reduced virulence in experimental endocarditis: a host defense role for platelet microbicidal proteins.

Staphylococcal alpha-toxin targets several cell types which are important components of cardiac vegetations in endocarditis, including platelets, erythrocytes, and endothelial cells. We evaluated the in vivo role of Staphylococcus aureus alpha-toxin in experimental endocarditis by using isogenic strains differing in the capacity to produce functional alpha-toxin, including 8325-4 (wild-type strain), DU-1090 (a mutant strain with allelic replacement of the alpha-toxin gene [hla]), DU1090(pH35L) (a mutant strain producing a target cell-binding but nonlytic toxin), DU1090(pDU1212) (a variant of DU1090 carrying the cloned hla gene on a multicopy plasmid), and DU1090(pCL84::hla) (a variant of DU1090 with a single copy of the hla gene cloned into the chromosomal lipase locus). In vitro, wild-type alpha-toxin (from parental strain 8325-4) extensively lysed both erythrocytes and platelets. In contrast, mutant alpha-toxin [from strain DU1090(pH35L)] lysed neither cell type. Following exposure to the wild-type alpha-toxin, platelet lysates were found to contain microbicidal activity against Bacillus subtilis (but not against Micrococcus luteus), as well as against the parental and alpha-toxin variant S. aureus strains noted above. Furthermore, lysate microbicidal activity was heat stable, neutralized by polyanionic filters or compounds, and recoverable from anionic filter membranes by hypertonic saline elution. These characteristics are consistent with those of cationic platelet microbicidal proteins (PMPs). Reverse-phase high-pressure liquid chromatography and polyacrylamide gel electrophoresis confirmed the presence of three distinct PMPs (1, 2, and 3) in platelet lysates. In experimental endocarditis, the two variant staphylococcal strains producing either minimal alpha-toxin or nonlytic alpha-toxin in vitro [strains DU1090 and DU1090(pH35L), respectively] exhibited significantly lower virulence in vivo than the parental strain (decreased intravegetation staphylococcal densities). Paradoxically, the two variant staphylococcal strains producing alpha-toxin at supraparental levels in vitro [strains DU1090(p1212) and DU1090(pCL84::hla)] also exhibited significantly decreased induction rates and intravegetation staphylococcal densities in experimental endocarditis versus the parental strain. The reduced in vivo virulence of the latter variant staphylococcal strains could not be explained by differences in bacteremic clearance or initial adherence to sterile vegetations (compared to the parental strain). These findings suggest that the reduced virulence exhibited by the variant staphylococcal strains in this model was related to pathogenetic events subsequent to bacterial adherence to the damaged endocardium. Excess intravegetation secretion of alpha-toxin, leading to increased PMP release (secondary to either increased platelet secretion or lysis), may well explain the reduced virulence observed in experimental endocarditis.

Expression of an antisense hla fragment in Staphylococcus aureus reduces alpha-toxin production in vitro and attenuates lethal activity in a murine model.

Isogeneic bacterial strains that differ only in the production of a single microbial factor have been invaluable in studying the pathogenesis of bacterial infections. The targeted, intentional inactivation of a gene encoding a potential virulence determinant generally requires homologous recombination to replace the gene with an inactivated allele. To determine whether the insertion and expression of a fragment of a bacterial gene in an antisense orientation could be used as a rapid alternative to allelic inactivation for producing paired isogeneic isolates, we inverted a 600-bp fragment of the Staphylococcus aureus gene encoding alpha-toxin, hla, behind its native promoter on an Escherichia coli-S. aureus shuttle vector. A transformant of an S. aureus strain carrying the antisense hla fragment produced antisense hla RNA and made 16-fold less alpha-toxin than either its parent or an isogeneic transformant containing vector DNA without hla. Also, intraperitoneal injection of 1.5 x 10(9) CFU of the antisense hla-containing transformant was significantly less lethal in a murine model than that of the parent (1 of 10 versus 7 of 10 mice expired [P < 0.02]) or the transformant without hla (1 of 10 versus 7 of 7 mice expired [P < 0.001]). We conclude that the expression of a fragment of hla in an antisense orientation in S. aureus on a plasmid vector reduces alpha-toxin production and the lethal activity of the strain in a murine model. The antisense strategy for creating isogeneic strains of bacteria may facilitate molecular investigations into the pathogenesis of infection. It also may be useful in creating novel live-attenuated strains of bacteria for use as vaccine candidates.

Laparoscopic Nissen fundoplication and postoperative dysphagia-can it be predicted?

Temporary swallowing difficulty has been reported in up to 100% of patients after laparoscopic Nissen fundoplication for gastro-oesophageal reflux, but the long-term prevalence of dysphagia is not known. If reliable predictors of persistent postoperative dysphagia exist, their identification may permit tailoring of the fundoplication for specific cases at risk. Of 475 consecutive cases of laparoscopic fundoplication, 202 have undergone detailed symptom scoring at 1 year postoperatively. In each case, a short, 360 degrees loose wrap was constructed over an intra-oesophageal bougie, and short gastric vessels were divided when insufficient fundal mobility was encountered. At one year postoperatively, 24% complained of grade 1 dysphagia (intermittent sensation of food sticking), 9% of grade 2 (food sticking requiring liquids to clear) and 1 patient had regular dysphagia for solids (grade 3). Gender, preoperative complaint of dysphagia, endoscopic grade of oesophagitis, oesophageal motility, lower oesophageal sphincter pressure, and division of short gastric vessels were reviewed in relation to dysphagia. Dysphagia is common after laparoscopic Nissen fundoplication, but is less prevalent than before operation. It is usually mild, intermittent and improves with time. Those with division of short gastric vessels had a reduced risk of postoperative dysphagia.

Passive immunization with antiserum to a nontoxic alpha-toxin mutant from Staphylococcus aureus is protective in a murine model.

A nonhemolytic, nonlethal variant of Staphylococcus aureus alpha-toxin constructed via oligonucleotide-directed mutagenesis and containing a single amino acid substitution (H-35 to L) was used to immunize a rabbit. The resulting antiserum was cross-reactive with wild-type alpha-toxin and neutralized its hemolytic activity in vitro. Passive immunization of mice with rabbit antiserum conferred protection against lethal challenge with wild-type alpha-toxin and against acute lethal challenge with a high-alpha-toxin -producing S. aureus strain. H35L alpha-toxin may be useful as a protective immunogen in S. aureus vaccine studies.

Laparoscopic Nissen fundoplication--200 consecutive cases.

PATIENTS: Laparoscopic Nissen fundoplication was undertaken in 200 patients between 1991 and 1994. METHODS: Pre-operative assessment included symptom score, endoscopy, manometry, and 24 hour pH monitoring of the oesophagus. Patients were evaluated at three and 12 months after surgery with symptom scoring and 96 patients also underwent 24 hour pH studies at three to six months postoperatively. RESULTS: In the first 100 patients median duration of operation was 155 minutes (range: 70-330), conversion rate to laparotomy was 7%, median hospital stay was three days (range: 2-57), and total morbidity was 16%. This compared with a median operation time of 120 minutes (60-240) (p = 0.0003, 95% CI 10, 40), a conversion rate of 2% (p = 0.2), a hospital stay of three days (1-18) (p = 0.0016, 95% CI 0, 1), and total morbidity of 7% (p = 0.15) in the second 100 patients. Median total symptom scores fell from 5/9 to 0/9 after fundoplication (< 0.0001) while median 24 hour oesophageal acid exposure in 96 patients was reduced from 10% to 1% (p < 0.001). CONCLUSIONS: Laparoscopic Nissen fundoplication is a safe and effective procedure for gastro-oesophageal reflux disease. With experience, the duration of operation falls and the hospital stay is shorter. Shortterm symptomatic and pH results are consistently improved by surgery.

Laparoscopic versus open appendectomy: prospective randomized trial.

A prospective randomized trial comparing laparoscopic appendectomy with open appendectomy in patients with a diagnosis of acute appendicitis was conducted between October 1992 and April 1994. Of the 158 patients randomized, 7 patients were excluded because of protocol violations (conversion to laparotomy in 4, appendix not removed in 3). The 151 patients randomized to either a laparoscopic (n = 79) or an open appendectomy (n = 72) showed no difference in sex, age, American Society of Anesthesiology (ASA) rating, or previous abdominal surgery. The histologic classification of normal, catarrhal, inflamed, suppurative, and gangrenous appendicitis was not different between the two groups. Conversion from laparoscopic to open appendectomy was necessary in seven patients (9%) who had advanced forms of appendiceal inflammation. When compared to open appendectomy the laparoscopic group had a longer median operating time (63 minutes versus 40 minutes), fewer wound infections (2% versus 11%), less requirement for narcotic analgesia, and an earlier return to normal activity (median 7 days versus 14 days). There was no difference in morbidity, and both groups had a median time to discharge of 3 days. Laparoscopic appendectomy is as safe as open appendectomy; and despite the longer operating time, the advantages such as fewer wound infections and earlier return to normal activity make it a worthwhile alternative for patients with a clinical diagnosis of acute appendicitis.

Growth of Staphylococcus aureus with nafcillin in vitro induces alpha-toxin production and increases the lethal activity of sterile broth filtrates in a murine model.

The morbidity and mortality of Staphylococcus aureus infections remain high despite antibiotic therapy. To investigate further the observation that penicillins increase the hemolytic activity of staphylococcal cultures, 37 strains were grown in broth with and without subinhibitory nafcillin. Nafcillin stimulated hemolytic activity in nafcillin-susceptible and -resistant isolates. Sterile broth filtrates of nafcillin-associated cultures injected intraperitoneally in mice were more rapidly lethal than filtrates of the same strain grown without nafcillin. Lethality was neutralized by anti-alpha-toxin antisera. DNA-RNA hybridization revealed a nafcillin-associated increase in alpha-toxin mRNA during the postexponential growth phase after the activation of agr. Isolates grown in slightly inhibitory nafcillin concentrations had more alpha-toxin mRNA than did nafcillin-free cultures, whereas agr RNAIII levels were comparable. This suggests that nafcillin-induced alpha-toxin production is not entirely attributable to agr. A supplemental regulatory mechanism may be involved.

Long-term bone loss after renal transplantation: comparison of immunosuppressive regimens.

Serial measurements of serum and urine markers of bone metabolism and of forearm bone density (BMD) by dual photon absorptiometry were performed in 22 patients undergoing renal transplantation in 1986. Patients were randomised to immunosuppression with (1) cyclosporin alone (CsA group, n = 10), (2) cyclosporin for 3 months followed by azathioprine-prednisone (CsA/AzP group, n = 3) or (3) long-term azathioprine-prednisone (LT AzP group, n = 9). As no reduction in bone mineral density (BMD) was noted in the first 6 months, groups 2 and 3 were considered together (AzP group, n = 12). Mean +/- SEM BMD fell by 19 +/- 2% at 36 months (n = 19, p < 0.01), with similar reductions seen in the CsA and AzP groups. At 60 months, BMD of the AzP group was 25 +/- 3% below baseline (p < 0.01), while the CsA group were only 5 +/- 4% below baseline (p = NS vs baseline, p < 0.05 vs AzP group). The degree of reduction in BMD over 5 years correlated with total glucocorticoid dose (r = 0.63, p < 0.05), but not with biochemical markers of bone turnover. Serum alkaline phosphatase fell post-transplant in patients treated with AzP, but not in the CsA group. These results demonstrate significant loss of forearm bone mineral with long-term follow-up after renal transplantation, but suggest that patients treated with cyclosporin monotherapy may be at lower risk of this complication.

Site-directed mutagenesis of the alpha-toxin gene of Staphylococcus aureus: role of histidines in toxin activity in vitro and in a murine model.

Staphylococcus aureus alpha-toxin is a membrane-damaging exoprotein that oligomerizes to form transmembrane pores. Chemical modification of histidines with diethylpyrocarbonate has been shown to reduce the hemolytic activity of alpha-toxin, suggesting that one or more of the histidine residues is important for toxin function. To individually assess the functional importance of each of the four histidine residues (residues 35, 48, 144, and 259), we used oligonucleotide-directed mutagenesis of the cloned alpha-toxin gene to replace each histidine with leucine. The mutant toxins were expressed in S. aureus and evaluated for hemolytic activity in vitro and for lethality in an intraperitoneal murine model. Substitution of histidine 35 with leucine produced a mutant toxin (H35L) without hemolytic or lethal activity. Mutant toxins H48L, H144L, and H259L exhibited 7, 16, and 46%, respectively, of the hemolytic activity of wild-type toxin. Immunoblotting of purified H35L toxin incubated with liposomal membranes demonstrated intact membrane binding and hexamer formation that was clearly detectable but reduced compared with that of the wild-type toxin. This suggests that hexamer formation alone is not sufficient for the expression of alpha-toxin activity. The nature of the defect underlying the lack of activity of the H35L mutant toxin remains to be elucidated but may involve failure of the hexamer to span the lipid bilayer to form a transmembrane pore or a change in the internal surface and permeability characteristics of the pore.