Immunogenicity of biopharmaceuticals in laboratory animals.

The potential immunogenicity of new protein therapeutics raises concerns about the possibility of inducing untoward immune reactions in humans. It is generally assumed that all animals will make antibody to human proteins and therefore, there is sentiment among some scientists that this makes the issue of immunogenicity as a safety concern irrelevant. However, recent clinical trials with some proteins have detected the presence of autoantibodies that have resulted in clinical sequelae. These reactions were also observed in preclinical animal studies. In fact, non-human primate and transgenic mouse models can be useful for predicting the relative immunogenicity of human proteins. In addition, the characterization of the immunogenicity of biotechnology molecules provides a practical basis for determining the significance of antibody formation in preclinical safety studies.

Immunogenicity of tissue plasminogen activators in rhesus monkeys: antibody formation and effects on blood level and enzymatic activity.

The immunogenicity of a tissue-type plasminogen activator analog, mt-PA6, consisting of the second kringle and protease domains, was compared to that of the native-sequence protein (nt-PA) in rhesus monkeys. Antibody responses were compared in groups of eight monkeys that were treated by i.v. injection twice, 1 month apart, using doses and regimens chosen to mimic therapy (0.5 mg/kg mt-PA6 bolus, 1.25 mg/kg nt-PA bolus + infusion). An additional group was treated with a 0.5 mg/kg nt-PA bolus. A single positive response was obtained in a monkey treated with 0.5 mg/kg nt-PA after the primary injection. Following the secondary injection, responses were obtained in 1/8, 3/8, and 6/8 monkeys treated with mt-PA6, nt-PA as a bolus, or nt-PA as a bolus + infusion, respectively. Several monkeys were selected to determine whether circulating tPA antibody altered the pharmacokinetics of mt-PA6. Clearance was found to decrease without affecting peak blood levels as antibody concentrations increased from 0.02 to 100 micrograms/ml. In contrast, the peak blood level was reduced by 99% at an antibody concentration of 152 micrograms/ml in a monkey that had been exposed to mt-PA6 in adjuvant 14 months previously. Further, only the serum from this and three other hyperimmunized monkeys inhibited the enzymatic activity of tPA in vitro. It is concluded that mt-PA6 is not more immunogenic than nt-PA in rhesus, and that low levels of antibody are more likely to influence the pharmacokinetic properties of tPA than to inhibit its enzymatic activity. It is unlikely that mt-PA6 would present a serious immunogenic risk in humans.

Immunogenicity of biosynthetic human LysPro insulin compared to native-sequence human and purified porcine insulins in rhesus monkeys immunized over a 6-week period.

Development of insulin antibodies in rhesus monkeys was investigated after immunization with 3 forms of insulin in Freund's adjuvant. Insulins examined included: 1. biosynthetic LysPro insulin (LY275585), a new human insulin analog, 2. biosynthetic native-sequence human insulin, and 3. purified porcine insulin. Male monkeys, 4/insulin type, were immunized weekly over a 6-week period with increasing doses of insulin, ranging from 10 to 100 micrograms/monkey. An ELISA assay was used to measure IgG insulin antibodies in sera collected prior to immunization and 5, 10, and 16 days after final immunization. One monkey had detectable pretreatment levels of antibody. This monkey, which had been assigned to the LysPro insulin treatment group, responded to immunization with a peak antibody level of 20 micrograms/ml. IgG insulin antibody responses were not detected in any of the other monkeys. A passive cutaneous anaphylaxis (PCA) assay was used to measure IgE insulin antibodies in sera collected prior to immunization and 10 days after final immunization. No IgE antibodies were detected in any of the monkeys pre- or post-immunization. Considering that 1. an immunological adjuvant was used, 2. eleven of twelve monkeys failed to develop an antibody response, and 3. the IgG insulin antibody level observed in the single responding monkey was low, it was concluded that these insulins have an extremely weak immunogenic potential in rhesus monkeys. It is suggested that immunization of non-human primates with new therapeutic proteins in adjuvant may be a useful primary screen to determine their immunogenic potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Discovery, synthesis, and bioactivity of bis(heteroaryl)piperazines. 1. A novel class of non-nucleoside HIV-1 reverse transcriptase inhibitors.

A variety of analogues of 1-[4-methoxy-3,5-dimethylbenzyl]-4-[3-(ethylamino)-2-pyridyl]piperazine hydrochloride (U-80493E) were synthesized and evaluated for their inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Replacement of the substituted aryl moiety with various substituted indoles provided bis(heteroaryl)piperazines (BHAPs) that were 10-100-fold more potent than U-80493E. The pyridyl portion of the lead molecule was found to be very sensitive to modifications. Extensive preclinical evaluations of several of these compounds led to the selection of 1-[(5-methoxyindol-2-yl)carbonyl]-4-[3-(ethylamino)-2- pyridyl]piperazine methanesulfonate (U-87201E, atevirdine mesylate) for clinical evaluation.

Cleft lip and palate surgery in La Ceiba, Honduras.

The volume of experience in the surgical repair of cleft lip and palate deformities is limited in many areas of the United States. This deficit in experience exists not only for those in resident training programs but also for those who practice at universities or are in private practice. Physicians are performing too few operations to maintain their surgical skills in cleft lip and palate surgery. This deficit is also encountered by physicians in the major specialties of plastic surgery, oral surgery, and otolaryngology who perform cleft lip and palate surgery. Physicians from the United States can gain surgical experience in other countries through groups like the Medical Group Mission Christian Medical and Dental Society. Surgical experience is available in Central and South America, India, China, and Africa through similar organizations. The complexity of providing service to under-privileged people through organizations such as the Medical Group Mission Christian Medical and Dental Society is described.

Contribution of leukotriene B4 to airway inflammation and the effect of antagonists.

Inhalation of aerosols of ovalbumin in sensitized guinea pigs produced a marked, bronchoalveolar eosinophilia 24 hr after challenge. The lung eosinophilia was not prevented by the cyclooxygenase inhibitors, indomethacin or PAF antagonists (WEB-2086 and L-652731) but was inhibited by methylprednisolone, the 5-LO inhibitor, U-66858 and a series of structural analogs of LTB4, U-75302, U-77692, U-75485 and U-78489. The effectiveness of LTB4 antagonists but not PAF antagonists in vivo was consistent with in vitro studies in which LTB4 was shown to be far more chemotactic than PAF for guinea pig eosinophils. LTB4 elicited maximal directional migration of guinea pig eosinophils at concentrations from 10(-7) M to 10(-9) M while PAF showed no effect over the same concentration range. The structural analogs of LTB4 were shown to inhibit LTB4 induced chemotaxis of guinea pig eosinophils and produced a dose-related inhibition of binding of LTB4 to guinea pig eosinophil membranes. To add further proof to the hypothesis that LTB4 contributed to the antigen-induced lung eosinophilia we attempted to measure LTB4 release into BAL fluid immediately after and at various time points up to 24 hr after antigen inhalation. However, using a sensitive radioimmunoassay (detection limit 10 pg/ml) very low levels of LTB4 (24.9-67.9 pg/ml) or its metabolite, 20-OH LTB4 (24.9-98.2 pg/ml) were detected in BAL fluid and these levels did not increase significantly following antigen provocation. Inhalation of LTB4 aerosols in unsensitized Brown-Norway rats or inhalation of aerosols of ovalbumin in sensitized Brown-Norway rats also produced a marked "late-phase" eosinophil-rich influx of inflammatory cells into the lungs. The lung eosinophilia in the rat was prevented by two structurally unrelated leukotriene B4 (LTB4) antagonists, U-75302 and Ly255283. These data implicate LTB4 as a mediator of allergen-induced bronchopulmonary eosinophilia. Leukotriene B4 antagonists may provide leads for the development of compounds which inhibit the chronic airway inflammation associated with asthma in man.

Biochemical manifestations of oxygen toxicity in the newborn lamb.

The purpose of this project was to study the role of lipid peroxidation in oxygen-induced lung injury in the newborn lamb. It was our hypothesis that injury to the microvascular bed of the lung by oxygen would coincide with a burst of peroxidative activity and would be accompanied by an increased rate of excretion of ethane and pentane in expired gas. We measured vascular pressures, the rate of lung lymph flow and concentrations of ethane and pentane in exhaled gas in 10 newborn lambs that breathed greater than 95% oxygen continuously. Our marker for oxygen-induced lung injury was an increase in the permeability of the microvascular bed of the lung to protein (an increase in the rate of lung lymph flow accompanied by an increase in the protein concentration in lymph). Although all 10 lambs demonstrated an abrupt increase in microvascular permeability to protein within 48 to 96 h of exposure to greater than 95% oxygen, the rates of ethane and pentane excretion remained unchanged throughout the entire experimental period. Lung tissue concentrations of glutathione decreased by 40% in the oxygen-exposed lambs and the concentrations of glutathione disulfide increased 85% relative to air-breathing controls. Activities of glutathione reductase and superoxide dismutase were lower in the lungs of the oxygen-exposed lambs than in controls, whereas the activities of glutathione peroxidase and catalase were not changed. We conclude that, in the lamb, changes in the rates of excretion of ethane and pentane do not correlate with the timing of injury to the microvascular bed of the lung.

Somatotropin antibody formation in cows treated with a recombinant bovine somatotropin over two lactations.

Blood plasma from cows treated with somidobove, a form of recombinant bovine somatotropin, was assayed for development of antibodies against the protein. Forty-three Holstein cows, selected from an animal safety study, were monitored. Cows were divided into four groups and treated with placebo, 960, 2880, or 4800 mg somidobove per dose at 28-d intervals during two successive lactation periods. Blood plasma was collected at intervals prior to and during the lactations, and levels of IgG antibody reactive with somidobove were determined in an enzyme-linked immunosorbent assay. Virtually all of the cows treated with somidobove developed low levels (less than 40 micrograms/ml) of antibody against somidobove. One or two cows from each group responded with some-what higher levels, ranging from 40 to 200 micrograms/ml. Responses generally increased during the first 3 mo of treatment, then decreased, and remained constant with continued treatment. There was no sign of a memory response within or among the lactation periods, and no adverse health effects or decreases in lactational performance were associated with antibody production.

Protein crystal growth in microgravity.

The crystals of most proteins or other biological macromolecules are poorly ordered and diffract to lower resolutions than those observed for most crystals of simple organic and inorganic compounds. Crystallization in the microgravity environment of space may improve crystal quality by eliminating convection effects near growing crystal surfaces. A series of 11 different protein crystal growth experiments was performed on U.S. space shuttle flight STS-26 in September 1988. The microgravity-grown crystals of gamma-interferon D1, porcine elastase, and isocitrate lyase are larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions than the best crystals of these proteins grown on Earth.

Ethical use of Yellow Pages listings by physicians.

A recent article in the New England Journal of Medicine examined the percentage of physicians listed under specialty headings in a Yellow Pages publication who were board certified. The authors presented findings that many of the physicians listed in the Yellow Pages, particularly under the heading of "Plastic Surgery," were not board certified. The source data employed in that article were obtained and reexamined, and it was found that the assumptions used were inaccurate and the methodology flawed. Almost every physician listed under "Plastic Surgery" in the Yellow Pages is board certified. We discuss the conclusions of the original article and its recommendations for control of Yellow Pages listings. We believe that these recommendations are not in the public's best interests and may be illegal as well.

Acute myocardial infarction temporally related to cocaine use. Clinical, angiographic, and pathophysiologic observations.

Ischemic chest pain syndromes and myocardial infarction occurred within minutes to hours of cocaine use in nine persons ages 23 to 39 years. Five developed symptoms after taking cocaine intranasally; three, after intravenous use; and one, after smoking cocaine. Four were habitual users and five were recreational users; eight also smoked cigarettes heavily. Ischemic syndromes recurred in five who continued to use cocaine. Coronary arteriography showed an abnormal infarct-related vessel (more than 50% stenosis, total occlusion, or intraluminal thrombus) in seven patients. The noninfarct-related vessels were normal in eight patients. The left anterior descending coronary artery and the anteroapical left-ventricular wall were involved in all patients. After three patients had successful thrombolysis of the obstructed infarct-related vessel, angiography showed a normal underlying vessel.

The control of experimental Escherichia coli diarrhoea in calves by means of bacteriophages.

Seven phages highly active in vitro and in vivo against one or other of seven bovine enteropathogenic strains of Escherichia coli belonging to six different serotypes were isolated from sewage. Severe experimentally induced E. coli diarrhoea in calves could be cured by a single dose of 10(5) phage organisms. It could be prevented by doses as low as 10(2), by spraying the litter in the calf rooms with aqueous phage suspensions or simply by keeping the calves in uncleaned rooms previously occupied by calves whose E. coli infections had been treated with phage. Microbiological examinations of calves used in these experiments revealed that the phage organisms multiplied rapidly and profusely after gaining entry to the E. coli-infected small intestine, quickly reducing the E. coli to numbers that were virtually harmless. The only phage-resistant E. coli that emerged in the studies on calves infected with one or other of the seven E. coli strains were K-. These organisms were much less virulent than the K+ organisms from which they were derived and did not present a serious problem in calves given adequate amounts of colostrum. Infections produced by oral inoculation of a mixture of six strains of the E. coli could be controlled by administration of a pool of the six phages that were active against them but, in general, the control was less complete than that observed in the single-strain infections. K+ phage-resistant bacteria emerged in some of the calves used in these mixed infections and they were as virulent as their parent organisms; evidence from in vitro studies suggested that they might have arisen by genetic transfer between organisms of the different infecting strains. Infections produced by these K+ mutants and their parents could be controlled by the use of mutant phages derived from phages that were active on their parents. During the experiments with mixed E. coli infection, an extraneous phage active against one of the six E. coli strains suddenly appeared in calves kept in the same rooms. Microbiological examinations revealed that this phage was effectively controlling the multiplication of organisms of that particular strain of E. coli in the small intestines of the calves.

Factors influencing the survival and multiplication of bacteriophages in calves and in their environment.

Seven phages were fairly susceptible in vitro to the lethal effect of acidified whey, more so than the enteropathogenic Escherichia coli strains on which they were active. The low acidity that prevailed in the abomasum contents of calves shortly after a milk feed had little harmful effect on orally administered organisms of these phages; they flooded into the small intestine. The high acidity that prevailed later was lethal to orally administered phage organisms; few entered the small intestine. The lethal effect could be counteracted by giving CaCO3 in the feed. Low concentrations of phage-neutralizing antibodies were found in some serum samples from human beings, cattle and pigs. Antibodies to one of the seven phages were common in the human samples and antibodies to another, phage B44/1, were common in the cattle and pig samples and in bovine colostrum. Phage B44/1 antibodies in a sample of colostral whey were destroyed at pH 3.25 or less. Giving colostrum containing phage B44/1 antibodies with CaCO3 to a calf greatly reduced the numbers of orally administered phage B44/1 organisms in its alimentary tract. Antibodies to another phage were induced in the serum of a calf suffering from E. coli diarrhoea by treating it with that phage. The phages were as susceptible as the E. coli strains to the lethal action of formaldehyde and sodium hypochlorite. In contrast to the E. coli strains, they were almost completely resistant to phenol and chloroxylenol. The in vitro virulence of 21 phages varied according to the temperature at which tests were performed.(ABSTRACT TRUNCATED AT 250 WORDS)

Two toxin-converting phages from Escherichia coli O157:H7 strain 933 encode antigenically distinct toxins with similar biologic activities.

Escherichia coli O157:H7 strain 933 contains two distinct toxin-converting phages (933J and 933W). The biologic activities and antigenic relationship between the toxins produced by 933J and 933W lysogens of E. coli K-12, as well as the homology of the genes that encode the two toxins, were examined in this study. The 933J and 933W toxins, like Shiga toxin produced by Shigella dysenteriae type 1, were cytotoxic for the same cell lines, caused paralysis and death in mice, and caused fluid accumulation in rabbit ileal segments. The cytotoxic activity of 933J toxin for HeLa cells was neutralized by anti-Shiga toxin, whereas the activity of 933W toxin was not neutralized by this antiserum. In contrast, an antiserum prepared against E. coli K-12(933W) neutralized 933W toxin but not 933J toxin or Shiga toxin. For E. coli 933, most of the cell-associated cytotoxin was neutralized by anti-Shiga toxin, whereas most of the extracellular cytotoxin was neutralized by anti-933W toxin. However, a mixture of these antisera indicated the presence of both toxins in cell lysates and culture supernatants. Among 50 elevated cytotoxin-producing strains of E. coli, we identified 11 strains isolated from cases of diarrhea, hemorrhagic colitis, or hemolytic uremic syndrome that produced cell-associated cytotoxins which were neutralized by the 933W antitoxin. Southern hybridization studies showed that the cloned toxin structural genes from phage 933J hybridized with DNA from phage 933W under conditions estimated to allow no more than 26% base-pair mismatch. These findings indicate that E. coli produces two genetically related but antigenically distinct cytotoxins with similar biologic activities which we propose to name Shiga-like toxins I and II. Strains of E. coli that produce elevated levels of Shiga-like toxin I or Shiga-like toxin II, or both, have been associated with the clinical syndromes of diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome.

Infectious bronchitis immunity: its study in chickens experimentally infected with mixtures of infectious bronchitis virus and Escherichia coli.

The live infectious bronchitis (IB) vaccine, H120, protected chickens against intranasal challenge with a mixture of Escherichia coli strains (E. coli Pool) and IB virus (IBV) strains of the same (Massachusetts) serotype as H120; it usually also protected against challenge with the E. coli Pool and IBV strains of other serological types. When these challenge strains were themselves used as vaccines they usually protected against challenge with a mixture of the E. coli Pool and an IBV strain of the Massachusetts serotype (VF69-149) or an IBV strain not of the Massachusetts serotype (HVI-116). Poor protection, when observed, was most common in those experiments involving a minority of the IBV strains that had been incriminated in recent outbreaks of disease in vaccinated flocks of chickens. Much lower concentrations of IBV strain VF69-149 and E. coli O18 were found in the nose, trachea and spleen of H120-vaccinated chickens killed at different times after they were given a mixture of these organisms than were found in these sites in similarly challenged unvaccinated chickens. Some protection against challenge with IBV and the E. coli Pool was also observed in chickens vaccinated with an inactivated IBV strain; it was much less effective than that obtained following vaccination with the corresponding live IBV strain.