Direct operations for the management of life-threatening ischemic ventricular tachycardia

Between June 1978 and 1986, 93 consecutive patients underwent electrophysiologically guided operations for life-threatening recurrent sustained ventricular tachycardia mostly associated with other surgical procedures, such as left ventricular resection (aneurysmectomy) and coronary artery bypass grafting. Data: Eighty-seven percent of the surviving patients were free of spontaneous ventricular tachycardia return or sudden death 1 year after the operation and 77% at 5 years. The instantaneous risk of ventricular tachycardia return was highest immediately after operation, declined rapidly, and by 2 weeks postoperatively had merged with the constant hazard phase, which persisted as long as the patients were observed. Endocardial resection, rather than encircling endocardial myotomy, increased the risk of spontaneous ventricular tachycardia return/sudden death. Survival rates, including hospital deaths, were 95% at 30 days, 89% at 1 year, and 70% at 5 years after operation. The most prevalent mode of death was heart failure. The absence of anterolateral left ventricular aneurysms and the use of more extended encircling incisional techniques for ventricular tachycardia ablation increased the risk of early and late death. Survival was particularly poor in that subset of patients in whom recurrent sustained ventricular tachycardia returned after operation; the most prevalent mode of death in this group was also progressive left ventricular failure. Inferences: (1) Complete and partial encircling endocardial myotomy incisions are the most effective surgical techniques for malignant ventricular tachycardia ablation. (2) Because of their adverse effects on left ventricular structure and function, the arrhythmogenic tissues have to be localized as precisely as possible, and the encompassing incisions should be kept as limited as possible. (3) The late return of ventricular tachycardia may be more related to a progressive ischemic left ventricular cardiomyopathy than to an inadequate operation.     

Muscarinic Receptors Activate Calcium Channels and Calcium Dependent Potassium Channels in NIE-115 Neuroblastoma Cells

Abstract: Responses of NIE-115 neuroblastoma cells to application of carbachol were studied using intracellular recording techniques. Activation of muscarinic cholinergic receptors by carbachol resulted in a depolarization of the cells. The response was blocked by pirenzepine (1 μM) and by CoCl₂ (5 mM), verapamil (10 μM) and gallopamil (10 μM), and prolonged by quinine (5 mM). It is suggested that muscarinic receptors increase the membrane calcium permeability, and that the influx of calcium activates calcium dependent potassium channels.     

Neutralization assay using a modified vaccinia virus Ankara vector expressing the green fluorescent protein is a high-throughput method to monitor the humoral immune response against vaccinia virus

Vaccination against smallpox is again considered in order to face a possible bioterrorist threat, but the nature and the level of the immune response needed to protect a person from smallpox after vaccination are not totally understood. Therefore, simple, rapid, and accurate assays to evaluate the immune response to vaccinia virus need to be developed. Neutralization assays are usually considered good predictors of vaccine efficacy and more informative with regard to protection than binding assays. Currently, the presence of neutralizing antibodies to vaccinia virus is measured using a plaque reduction neutralization test, but this method is time-consuming and labor-intensive and has a subjective readout. Here, we describe an innovative neutralization assay based on a modified vaccinia virus Ankara (MVA) vector expressing the green fluorescent protein (MVA-gfp). This MVA-gfp neutralization assay is rapid and sensitive and has a high-throughput potential. Thus, it is suitable to monitor the immune response and eventually the efficacy of a large campaign of vaccination against smallpox and to study the vector-specific immune response in clinical trials that use genetically engineered vaccinia viruses. Most importantly, application of the highly attenuated MVA eliminates the safety concern in using the replication-competent vaccinia virus in the standard clinical laboratory.     

Structure-function studies of the C3a-receptor: C-terminal serine and threonine residues which influence receptor internalization and signaling

The anaphylatoxic peptide C3a is a pro-inflammatory mediator generated during complement activation, whose specific G protein coupled receptor is expressed on granulocytes, monocytes, mast cells, activated lymphocytes, and in the nervous tissue. We have generated RBL-2H3 cell clones stably expressing mutants of the human C3a-receptor (C3aR) with combined alanine (Ala) substitutions of ten C-terminal serine (Ser) or threonine (Thr) residues, which may represent putative phosphorylation sites to characterize their role in ligand-induced C3aR internalization and signaling. Ser475/479 and Thr480/481 as well as Ser449 seemed not to be involved in ligand-induced receptor internalization. Either directly or by a conformational change they even "inhibit" C3aR internalization. In contrast, mutants with Ala substitutions at Ser465/470 and Thr463/466 were poorly internalized, and Thr463 seemed to be the most important C-terminal Thr or Ser residue directly effecting receptor internalization. However, it is likely that other C3aR regions additionally participate in this negative feed-back mechanism since even mutants with multiple Ala substitutions still internalized to a limited degree. Interestingly, in a mutant with a single exchange of Ser449 to Ala, the signal transduction assessed by a Ca(2+) assay and [(35)S]GTP gamma S-binding on HEK cells transiently co-transfected with G-alpha 16 or G-alpha O, respectively, was severely impaired, indicating that this residue of C3aR is involved in G protein coupling.     

Enhancement of neonatal innate defense: effects of adding an N-terminal recombinant fragment of bactericidal/permeability-increasing protein on growth and tumor necrosis factor-inducing activity of gram-negative bacteria tested in neonatal cord blood ex vivo

Innate defense against microbial infection requires the action of neutrophils, which have cytoplasmic granules replete with antibiotic proteins and peptides. Bactericidal/permeability-increasing protein (BPI) is found in the primary granules of adult neutrophils, has a high affinity for lipopolysaccharides (or "endotoxins"), and exerts selective cytotoxic, antiendotoxic, and opsonic activity against gram-negative bacteria. We have previously reported that neutrophils derived from newborn cord blood are deficient in BPI (O. Levy et al., Pediatrics 104:1327-1333, 1999). The relative deficiency in BPI of newborns raised the possibility that supplementing the levels of BPI in plasma might enhance newborn antibacterial defense. Here we determined the effects of addition of recombinant 21-kDa N-terminal BPI fragment (rBPI(21)) on the growth and tumor necrosis factor (TNF)-inducing activity of representative gram-negative clinical isolates. Bacteria were tested in citrated newborn cord blood or adult peripheral blood. Bacterial viability was assessed by plating assay, and TNF-alpha release was measured by enzyme-linked immunosorbent assay. Whereas adult blood limited the growth of all isolates except Klebsiella pneumoniae, cord blood also allowed logarithmic growth of Escherichia coli K1/r and Citrobacter koseri. Bacteria varied in their susceptibility to rBPI(21)'s bactericidal action: E. coli K1/r was relatively susceptible (50% inhibitory concentration [IC(50)], approximately 10 nM), C. koseri was intermediate (IC(50), approximately 1,000 nM), Klebsiella pneumoniae was resistant (IC(50), approximately 10,000 nM), and Enterobacter cloacae and Serratia marcescens were highly resistant (IC(50), >10,000 nM). All isolates were potent inducers of TNF-alpha activity in both adult and newborn cord blood. In contrast to its variable antibacterial activity, rBPI(21) consistently inhibited the TNF-inducing activity of all strains tested (IC(50), 1 to 1,000 nM). The antibacterial effects of rBPI(21) were additive with those of a combination of conventional antibiotics typically used to treat bacteremic newborns (ampicillin and gentamicin). Whereas ampicillin and gentamicin demonstrated little inhibition of bacterially induced TNF release, addition of rBPI(21) either alone or together with ampicillin and gentamicin profoundly inhibited release of this cytokine. Thus, supplementing newborn cord blood with rBPI(21) potently inhibited the TNF-inducing activity of a variety of gram-negative bacterial clinical pathogens and, in some cases, enhanced bactericidal activity. These results suggest that administration of rBPI(21) may be of clinical benefit to neonates suffering from gram-negative bacterial infection and/or endotoxemia.     

The prognostic value of serum troponin T in unstable angina.

BACKGROUND. Cardiac troponin T is a regulatory contractile protein not normally found in blood. Its detection in the circulation has been shown to be a sensitive and specific marker for myocardial cell damage. We used a newly developed enzyme immunoassay for troponin T to determine whether its presence in the serum of patients with unstable angina was a prognostic indicator. METHODS. We screened 109 patients with unstable angina (25 with accelerated or subacute angina and 84 with acute angina at rest) for serum creatine kinase activity, creatine kinase isoenzyme MB activity, and troponin T every eight hours for two days after admission to the hospital. The outcomes of interest during the hospitalization were death and myocardial infarction. RESULTS. Troponin T was detected (range, 0.20 to 3.64 micrograms per liter; mean, 0.78; median, 0.50) in the serum of 33 of the 84 patients (39 percent) with acute angina at rest. Only three of these patients had elevated creatine kinase MB activity (two were positive for troponin T, and one was negative). Of the 33 patients who were positive for troponin T, 10 (30 percent) had myocardial infarction (3 after coronary-artery bypass surgery), and 5 of these died during hospitalization. In contrast, only 1 of the 51 patients with angina at rest who were negative for troponin T had an acute myocardial infarction (P less than 0.001), and this patient died (P = 0.03). Thus, 10 of the 11 patients with myocardial infarctions had detectable levels of troponin T; only 1 had elevated creatine kinase MB activity. Troponin T was not detected in any of the 25 patients with accelerated or subacute angina, and none of these patients died. CONCLUSIONS. Cardiac troponin T in serum appears to be a more sensitive indicator of myocardial-cell injury than serum creatine kinase MB activity, and its detection in the circulation may be a useful prognostic indicator in patients with unstable angina.     

Rapid PCR Detection of Helicobacter pylori-Associated Virulence and Resistance Genes Directly from Gastric Biopsy Material

We have developed a PCR-based method to detect macrolide resistance and the virulence gene cagA in Helicobacter pylori within 24 h, thereby improving the lengthy process of culture-based approaches. Total DNA was prepared directly from stomach biopsy specimens. The procedure proved to be rapid and reliable and could be utilized for diagnostic purposes.     

Prophylactic and therapeutic efficacy of antibodies to a capsular polysaccharide shared among vancomycin-sensitive and -resistant enterococci

Enterococci are important nosocomial pathogens that are increasingly difficult to treat due to intrinsic and acquired resistance to antibiotics, including vancomycin. A recently described capsular polysaccharide (CP) isolated from Enterococcus faecalis 12030 was used to evaluate the potential efficacy of active or passive immunotherapy regimens as adjunctive treatments. Evaluation of protective efficacy was carried out in immunocompetent mice challenged intravenously (i.v.) with live enterococci. In nonimmune mice, i.v. inoculations resulted in high levels of bacteria in kidneys, spleens, and livers 5 days after challenge. Mice immunized with four 10-microg doses of CP antigen/mouse were protected against challenge with the homologous E. faecalis strain. High-titer opsonic immunoglobulin G was also induced by immunizing rabbits with the purified CP, and passive transfer of this antiserum to mice produced significantly lower bacterial counts in organs than did normal rabbit serum or sterile saline. Antibodies to the polysaccharide isolated from E. faecalis 12030 were protective against Enterococcus faecalis OG1RF and against two serologically related, vancomycin-resistant Enterococcus faecium clinical isolates. Antibodies to this CP antigen were also effective as a therapeutic reagent in mice when passive therapy was initiated 48 h after live bacterial challenge. These data indicate that CP antigens from enterococci are potential targets of protective antibodies and that these antibodies may be useful for prophylaxis and treatment of enterococcal infections.     

Cytotoxicity in vitro of preleukaemic lymphoid cells on syngeneic monolayers of embryo or thymus cells

Lymphoid cells from preleukaemic AKR mice were cytotoxic for monolayers of syngeneic embryo and thymus target cells in tissue culture. This reactivity was detectable with cells from mice aged 3–36 weeks but was not present with cells from younger mice. Cytotoxic reactions to syngeneic embryo tissues were also seen with lymphoid cells from high leukaemia strain C3H mice carrying Moloney virus but not with lymphoid cells from normal low leukaemic strain C3H/Bi or DBA/2 mice. Lymphoid or lymphoma cells from leukaemic AKR mice showed reduced reactivity. Phytohaemagglutinin was not necessary for the reaction of preleukaemic AKR cells against AKR monolayers and cytotoxicity was inhibited by preincubation of target cells with an antiserum directed against AKR G+ cells.

The reactivity of preleukaemic AKR and C3H lymphoid cells against syngeneic monolayers may represent some type of allogeneic inhibition due to acquired antigenic differences between aggressor and target cell but the data fit best an interpretation that some lymphoid cells in preleukaemic AKR and C3H mice acquire immunological reactivity to virus-induced G+ or M+ antigens exhibited by the target cells.