Pilot scale purification of alpha-galactosidase A from Cohn fraction IV-1 of human plasma.

Human plasma alpha-galactosidase A (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was purified 7000-fold over plasma levels from Cohn Fraction IV-1. The yield per kg starting material averaged 11 000 units (nmol galactose liberated per h) and the specific activity was about 600 units per mg protein with 4-methylumbelliferyl-alpha-D-galactoside. The ratio of 4-methylumbelliferyl-alpha-galactosidase to ceramide trihexosidase activities was 6.2. Both activities were heat labile and exhibited the same relative mobilities on polyacrylamide gel electrophoresis. Enzymatic activity was stable for at least 4 months at 4 and -20 degrees C. The endotoxin concentration of this preparation averaged 0.26 mg per mg protein.

Use of cardiac pacing after the Mustard operation for transposition of the great arteries.

The most frequent complication of the venous redirection (Mustard or Senning) operation for transposition of the great arteries is cardiac arrhythmia. Drug treatment of tachyarrhythmia often worsens bradyarrhythmia. Pacemakers can now treat both arrhythmias. The technique for implantation of pacemakers after redirection for transposition has changed over time from thoracotomy to subxiphoid to transvenous. Atrial pacing is almost always the mode of choice since the electrophysiologic abnormality is sinus node dysfunction with intact atrioventricular conduction. Twenty-nine patients aged 3 to 19 years (mean 9.6) had implantation of a pacemaker a mean of 5.5 years (range 1 to 14) after undergoing the Mustard operation for transposition of the great arteries. Symptoms referable to bradycardia were eliminated in each case. Four patients who received an antitachycardia pacemaker no longer have symptomatic tachycardia. Four patients have required reoperation, three because of lead problems and one because of traumatic erosion of the pacemaker. Pacemakers provide excellent relief of symptoms after the Mustard or Senning operation. Transvenous atrial automatic antitachycardia pacemakers offer the best combination of ease of implantation and symptomatic relief.

Treatment of atrial automatic tachycardia by ablation procedures.

Sixteen cases of atrial focus tachycardia are described clinically, electrophysiologically and hemodynamically. In each case multiple attempts at drug treatment (average 2.5 drugs) had failed. After delineation of the electrophysiologic mechanism, the patients were treated by surgical removal or cryoablation or catheter electroablation of the focus. In two of four patients catheter ablation was successful and without complication. Surgical treatment was successful in 13 of 14 patients. Left ventricular function, which had been abnormal in 10 patients, normalized in all but 1 patient whose echocardiographic shortening fraction improved from 10 to 27%. There have been no recurrences in a follow-up period of 6 months to 5 years (mean 2.2 years). It is recommended that any atrial automatic focus tachycardia that produces hemodynamic compromise undergo definitive treatment. Patients with chronic tachycardia rates of more than 140 beats/min should be followed up closely.

Atrial tracking (synchronous) pacing in a pediatric and young adult population.

One hundred pediatric and young adult patients underwent implantation of an atrial tracking pacemaker. Seventy-four pacemakers paced in an atrioventricular (AV) sequential mode at the lower rate limit (DDD) while 26 paced in a ventricular demand mode at the lower rate limit (VDD). Five patients required reoperation during follow-up of 1 month to 2.5 years (mean 1.5 years). Six additional patients required programming to ventricular demand (3) or AV sequential (3) pacing, because of development of sinus bradycardia (2), atrial sensing problems (1) or pacemaker-mediated tachycardia (3). Pulse generators that could sense atrial signals less than 1.0 mV and had a programmable atrial refractory period did not require reprogramming out of the atrial tracking mode. No patient developed atrial flutter or fibrillation. Sensing problems during exercise occurred in 37% of the first 60 pacemakers but in none of the last 40, which had improved electronic components. Atrial tracking pacing is feasible in pediatric and young adult patients.

Production of purified hepatitis B surface antigen from Alexander hepatoma cells grown in artificial capillary units.

An artificial capillary system was devised for growth of hepatoma cells that yields very high titers of hepatitis B surface antigen (HBsAg). High yield of antigen was facilitated by slowing cellular metabolism through reduction of incubation temperature and addition of 0.1 mM caffeine. Deletion of serum from the medium did not reduce the yield of antigen. HBsAg prepared from the culture fluid by affinity chromatography and additional chemical and enzymatic steps was essentially pure and was indistinguishable from HBsAg prepared from infected human plasma. Preparation of HBsAg from the cell culture source presents advantages over that of human plasma and might be a source of HBsAg for vaccine preparation.

The application of molecular biology to the development of novel vaccines.

In summary, we have shown that yeast is the preferred host for the expression of recombinant-derived hepatitis B vaccines, and that a yeast expression system which is productive, stable and scaleable can be developed for each of the three HBV envelope proteins. The versatility of regulated and integrated yeast expression systems in the production of foreign polypeptides with biomedical utility also has been highlighted. We also have shown that careful attention to the development of recombinant clones helps to optimize the entire production process leading to highly purified products which share many biochemical properties with the plasma-derived vaccine. Furthermore, immunization with PreS2 sequences is capable of protecting chimpanzees from HBV infection. The availability of PreS2 + S and PreS1 + PreS2 + S proteins expressed in yeast now provides the opportunity for establishing the relevance of such candidate vaccines in preventing human disease, thereby highlighting the utility of molecular biology in modern vaccine development.

Effectiveness of smokeless ashtrays.

Most environmental tobacco smoke (ETS) issues from the tips of smoldering cigarettes between puffs. Smokeless ashtrays are designed to reduce ETS exposure by removing particulate and/or gas-phase contaminants from this plume. This paper describes an experimental investigation of the effectiveness of four smokeless ashtrays: two commercial devices and two prototypes constructed by the authors. In the basic experimental protocol, one or more cigarettes was permitted to smolder in a room. Particulate or gas-phase pollutant concentrations were measured in the room air over time. Device effectiveness was determined by comparing pollutant concentrations with the device in use to those obtained with no control device. A lung deposition model was applied to further interpret device effectiveness for particle removal. The commercial ashtrays were found to be substantially ineffective in removing ETS particles because of the use of low-quality filter media and/or the failure to draw the smoke through the filter. A prototype ashtray using HEPA filter material achieved better than 90% particle removal efficiency. Gas-phase pollutant removal was tested for only one prototype smokeless ashtray, which employed filters containing activated carbon and activated alumina. Removal efficiencies for the 18 gas-phase compounds measured (above the detection limit) were in the range of 70 to 95%.

Regulation of a metabolic system in vitro: synthesis of threonine from aspartic acid.

Six enzymes involved in the conversion of aspartate to threonine have been extracted from Escherichia coli and separated from each other. Two of these enzymes, aspartokinase and homoserine dehydrogenase, have also been partially purified from Rhodopseudomonas spheroides. In an attempt to determine whether small changes in the kinetic properties of individual enzymes are important to the regulation of metabolic flux through a coupled reaction system, the partially purified enzymes were recombined in a variety of ways under reaction conditions designed to resemble the in vivo situation. These conditions include: use of an entire metabolic system rather than a single reaction; high enzyme concentrations at the same relative concentrations as found in the cell; and low, steady-state concentrations of substrates and products. Metabolic flux was followed spectrophotometrically and the concentrations of aspartic semialdehyde, hemoserine, O-phosphohomoserine, and threonine were measured. The results indicate that the threonine concentration is of major importance in regulating metabolic flux by inhibiting aspartokinase, the first reaction in threonine in the pathway. When threonine-insensitive aspartokinases were used, concentrations reached higher levels and the rate of NADPH oxidation remained higher. The fact that neither aspartic semialdehyde nor homoserine accumulated as the threonine concentration increased and the lack of correlation between changes in metabolic flux and ADP/ATP or NADPH/NADP ratios indicate that more subtle forms of metabolic regulation, such as "reverse cascade", secondary feedback sites, or "energy charge", are of little regulatory importance in this isolated, metabolic system. The results also emphasize the need for caution in projecting in vivo control mechanisms from in vitro experiments.

Rabbit factor VIII: identification of size heterogeneity.

Two forms of rabbit factor VIII procoagulant activity, distinguishable by size on gel filtration and ultracentrifugation, have been identified in normal rabbit plasma. These studies have been carried out with citrate-anticoagulated rabbit plasma obtained by cardiac puncture. Two peaks of factor VIII activity were obtained on agarose gel chromatography, using physiologic ionic strength buffers: a high molecular weight peak eluting at the void volume and a second peak eluting with smaller plasma proteins. The presence of high and low molecular weight factor VIII activities was confirmed by sucrose density gradient centrifugation. The two peaks of factor VIII activity remained distinct when proteolytic inhibitors were added to the plasma and eluting buffers. Both the high and low molecular weight factor VIII procoagulant activities were inhibited by antibodies to human and rabbit factor VIII, and both were activated by thrombin. The identification of size heterogeneity of factor VIII in normal rabbit plasma, in the absence of any modification by ionic strength, may permit more satisfactory study of the relationship of factor VIII size to function.

Procollagen: intermediate forms containing several types of peptide chains and non-collagen peptide extensions at NH2 and COOH ends.

A population of procollagen molecules has been isolated from the culture medium of a clonal line of calf dermatosparactic cells and shown to have the amino-acid composition, physical properties, and molecular structure consistent with collagen precursors. Although this procollagen population shares immunologic determinants with the procollagen obtained from dermatosparactic skin, it differs from the latter in aminoacid composition, in subunit properties, and by its content of both amino and carboxyl terminal non-collagen peptide appendages. We propose that the cell culture procollagen contains earlier biosynthetic forms of dermatosparactic procollagen.

Use of atrial pacing in a young population.

Forty pediatric and young adult patients (1-20 years; mean, 11 years of age) had implantation of atrial demand (AAI) pacemakers over a 5.5 year period. Nineteen were implanted by epicardial technique and 21 transvenously. Standard lithium single chamber pulse generators and standard atrial leads were used. Operative stimulation thresholds were better for transvenous leads than for epicardial (0.75 volts vs. 1.5 volts at 0.5 ms) (p less than 0.05). Pulse width thresholds at a mean of 1.5 years were not significantly different (0.11 ms for transvenous vs. 0.18 for epicardial). No patient developed AV block. Eight patients (four epicardial and four endocardial) required reoperation during the mean 3.5 year follow-up--four because of lead problems and four because of persistent tachycardia. Of the six patients who received an automatic antitachycardia pacemaker, only one had persistent symptoms while seven of eight who received a standard unit continued to have symptoms. Twenty-eight of 29 symptomatic patients without tachycardia became asymptomatic. Atrial pacing appears to be a safe and effective therapy for children with sick sinus syndrome.

Multiple chemical forms of hepatitis B surface antigen produced in yeast.

Hepatitis B surface antigen (HBsAg) has been extracted from yeast cells that produce HBsAg. These cells contain the gene for surface antigen carried on a plasmid that replicates in the cells. Analysis of the yeast-derived HBsAg by sucrose gradient centrifugation and by polyacrylamide gel electrophoresis shows that the antigen that is initially released from yeast cells is a high molecular weight aggregate of the fundamental Mr 25,000 subunit. Unlike HBsAg derived from human plasma, the yeast antigen is held together by noncovalent interactions and can be dissociated in 2% NaDodSO4 without the use of reducing agents. During in vitro purification of the yeast antigen, some disulfide bonds form spontaneously between the antigen subunits, resulting in a particle composed of a mixture of monomers and disulfide-bonded dimers. Treatment with 3 M thiocyanate converts the 20-nm particles into a fully disulfide-bonded form that is not disrupted in NaDodSO4 unless a reducing agent is added. This disulfide-bonded particle resembles the naturally occurring, plasma-derived surface antigen particle, and the in vitro formed particle has been used to prepare a vaccine for humans against hepatitis B virus infection.

Cardiac pacing in children and young adults.

Pacemakers are being used with increasing frequency in children. New pacemakers are smaller, more sophisticated, can be programmed, have output telemetry, are more reliable, and are longer lasting. Implant techniques have been modified, so that they are less traumatic to the patient and result in better electrical measurements. Transvenous implants seem to be the preferred method in children who weigh 13.5 kg or more. Physiologic dual-chamber pacemakers make the patients feel better. The time between hospitalizations for pacemaker replacements or revisions has increased from 14.7 months in 1974 to 22 months in this study.

Long-term follow-up of dysrhythmias following the Mustard procedure.

Earlier reports have suggested that the incidence of dysrhythmias after the Mustard procedure can be reduced if the sinoatrial node (SAN) is protected during surgery. To determine if these initial differences continue after longer follow-up, we examined all ECGs available for three groups of patients operated upon from January, 1965, through December, 1977. Group A included 37 patients who survived the operation prior to January, 1972, when surgical modifications were initiated to protect the SAN; group B included 44 patients available for follow-up who were operated upon from 1972 through 1974; and group C consisted of the 39 patients available for follow-up operated upon from 1975 to 1977. Dysrhythmias were classified as passive (failure of initiation or propagation of the SAN impulse), active (atrial flutter or supraventricular tachycardia), or atrioventricular (AV) conduction defects. Results were expressed as the incidence per number of different rhythms during follow-up intervals. The incidence of sinus rhythm in groups B and C (80%) was much greater than in group A (27%) during the first 2 years. However, after 8 years, less than 50% of the rhythms were sinus. Both brady- and tachydysrhythmias were common. Seven patients (6%) required pacemaker insertion for symptomatic sick sinus syndrome. Therefore despite efforts to protect the sinus node, late occurring dysrhythmias remain a significant problem in the postoperative Mustard patient.

Human hepatitis B vaccine from recombinant yeast.

The worldwide importance of human hepatitis B virus infection and the toll it takes in chronic liver disease, cirrhosis and hepatocarcinoma, make it imperative that a vaccine be developed for worldwide application. Human hepatitis B vaccines are presently prepared using hepatitis B surface antigen (HBsAg) that is purified from the plasma of human carriers of hepatitis B virus infection. The preparation of hepatitis B vaccine from a human source is restricted by the available supply of infected human plasma and by the need to apply stringent processes that purify the antigen and render it free of infectious hepatitis B virus and other possible living agents that might be present in the plasma. Joint efforts between our laboratories and those of Drs W. Rutter and B. Hall led to the preparation of vectors carrying the DNA sequence for HBsAg and antigen expression in the yeast Saccharomyces cerevisiae. Here we describe the development of hepatitis B vaccine of yeast cell origin. HBsAg of subtype adw was produced in recombinant yeast cell culture, and the purified antigen in alum formulation stimulated production of antibody in mice, grivet monkeys and chimpanzees. Vaccinated chimpanzees were totally protected when challenged intravenously with either homologous or heterologous subtype adr and ayw virus of human serum source. This is the first example of a vaccine produced from recombinant cells which is effective against a human viral infection.

Vaccine against human hepatitis B virus prepared from antigen derived from human hepatoma cells in culture.

Vaccine against human hepatitis B was prepared using antigen derived from hepatitis B carrier hepatoma cells grown in the interstices of a Diaflo hollow filter unit. Hepatitis B surface antigen (HBsAg) produced by these cells was purified by immune affinity chromatography, digestion with DNase and pepsin, and Sephadex G-150 separation. The Formalin-treated antigen was formulated in 20-micrograms dose on alum adjuvant with thimerosal added as a preservative. This cell culture vaccine was as potent as human plasma-derived vaccine as measured in a mouse potency assay. The vaccine proved safe in tests in chimpanzees and in human subjects who were in late stages of cancer of the central nervous system and who were receiving therapy for their condition. None of five subjects who received the vaccine developed untoward clinical reactions. Two of the subjects who received all three doses of vaccine developed antibody against HBsAg. Three persons, two given only the primary doses and one who was given all three doses but was lost to follow-up, demonstrated no response. The slow and relatively low antibody responses to the vaccine were similar to those in other immunosuppressed persons who were given vaccine of human plasma origin.

Characterization and evaluation of a recombinant hepatitis B vaccine expressed in yeast defective for N-linked hyperglycosylation.

The hepatitis B (HB) virus preS2 + 2 polypeptide (the M or middle envelope polypeptide) is N-glycosylated at the N4 residue of the preS2 domain when expressed in recombinant yeast. Hyperglycosylation at this amino acid residue (the addition of a large number of mannose residues to the core oligosaccharide), which occurs in common yeast strains, results in an HB vaccine with diminished immunogenicity. Hyperglycosylation can be prevented by expressing the preS2 + S polypeptide in mutant yeast strains (e.g. mnn9) which limit N-linked glycosylation to the addition of only core saccharide residues. An HB vaccine prepared from recombinant yeast expressing the non-hyperglycosylated preS2 + 2 polypeptide was of similar immunogenicity in mice to a licensed HB vaccine and was much more immunogenic in humans than the hyperglycosylated preS2 + 2 vaccine.