Breast gross cystic disease fluid analysis. I. Isolation and radioimmunoassay for a major component protein.

Human breast gross cystic disease (GCD) fluid was analyzed by sodium dodecyl sulfate-acrylamide gel electrophoresis, and four major proteins (GCDFP-70), GCDFP-44, GCDFP-24, and GCDFP-15) were identified. By fractionation techniques, these proteins were separated from one another. The GCDFP-70 was immunologically identical to human albumin and was present in GCD fluid at approximately a 100-fold lower concentration than in plasma. The GCDFP-44 was immunologically identical to human plasma Zn-alpha2-glycoprotein; however, it was present in GCD fluid at an approximately 50-fold higher concentration than in plasma. The GCDFP-24 was the major component protein of GCD fluid. It had progesterone binding activity, and immunologically it was identical to a component of human plasma; however, antisera that identified 30 separate components of plasma failed to identify the GCDFP-24 as one of these plasma proteins. The GCDFP-24 concentration in GCD fluid was approximately 100-fold higher than the plasma analog. The GCDFP-15 component was immunologically distinct from any plasma components, as judged by Ouchterlony analysis. It was, however, immunologically identical with a component of both human milk and saliva. As revealed by radioimmunoassay, plasma levels in normal subjects were 7-85 ng/ml. In patients with metastatic breast carcinoma, markedly plasma levels (150-30,000 ng/ml) of this protein were detected. Short-term tissue cultures of breast carcinoma explants released this protein into the culture medium.

Paramagnetic spin label interactions with the envelope of a group A arbovirus. Lipid organization.

Electron paramagnetic resonance observations were made on nitroxide spin- labeled molecules which were bound to the TC-83 vaccine strain of Venezuelan equine-encephalomyelitis virus. Paramagnetic resonance parameters derived from the observations and their dependence on sample temperature were similar but not identical to those which have been reported for these labels dissolved in lipid bilayer membranes of mammalian and bacterial origin. The data has a mechanical rigidity substantially greater than that of bilayers in cellular membranes. A model is presented which assumes the location of the lipid bilayer outside the nucleoprotein capsid and inside a spherical layer of envelope proteins. The model is in accord with Harrison's X-ray diffraction results for Sindbis virus. The model is discussed in terms of its implications with respects to the role played by lipid in viral maturation and infectivity.

A microprecipitation test for rapid detection and identification of Venezuelan, eastern and western equine encephalomyelitis viruses.

The development of a new diagnostic procedure for the identification of Venezvelan, eastern and western equine encephalomyelitis (VEE, EEE, WEE) viruses is described. The procedure utilizes virus precipitation with reference fluorescein-conjugated gamma globulin, followed by cellulose acetate electrophoresis. Clinical specimens containing varying concentrations of virus yielded, in primary duck embryo cell culture, sufficient virus for detection within 22 to 44 hours. Identification of VEE, EEE and WEE virus in specimens was accomplished by microprecipitation within this time. In contrast to conventional identification methods, our procedure eliminates the cost of utilizing laboratory animals and considerably reduces the time required for virus identification.

Changes in blood serum constituents and hematologic values in Macaca mulatta with Rocky Mountain spotted fever.

Forty-seven male Macaca mulatta, 3 to 4 kg weight, were inoculated intravenously or subcutaneously with various doses of yolk sac-grown Rickettsia rickettsii. Thirty-four macaques became febrile and exhibited signs of infection ranging from transient illness with a few days of fever to severe illness with subsequent death. The rash appeared more frequently in the macaques inoculated subcutaneously. Febrile macaques that survived had leukocytosis, with concomitant neutrophilia. Febrile macaques that died had, in addition, marked terminal leukopenia and thrombocytopenia. Packed cell volume of all febrile macaques decreased. In almost all of the febrile macaques, there were increased serum urea nitrogen, glutamic-oxaloacetic transaminase, and lactate dehydrogenase and decreased total serum protein and amylase concentrations. A few febrile macaques had increased bilirubin values and decreased sodium, chloride, phosphorus, and alkaline phosphatase concentrations. Changes did not occur in serum glucose, potassium, calcium, and glutamic-pyruvic transaminase values. The experimental form of Rocky Mountain spotted fever in the macaque provides a subhuman primate model for studying the pathophysiology of this disease.

Studies on Macaca mulatta infected with Rocky Mountain spotted fever.

Acid-base alterations and changes in other selected serum constituents (free fatty acids, triglycerides, cholesterol, copper, cortisol, alpha1-acid glycoprotein, haptoglobin, and albumin) were measured during a study of Rocky Mountain spotted fever in 16 male rhesus macaques. Blood samples were taken from nonanesthetized macaques conditioned to repeated handling. Arterial pH increased and PCO2 decreased during the febrile period. Free fatty acids, triglycerides, copper, cortisol, alpha1-acid glycoprotein, and haptoglobin increased, whereas albumin decreased during the disease. Significant changes were not observed in arterial PO2. Cholesterol remained unchanged. The increase in arterial pH and decrease in PaCO2 indicated that respiratory alkalosis was present in macaques acutely affected with Rocky Mountain spotted fever.

Scanning and transmission electron microscopy of Rickettsia rickettsii propagated in cell culture.

Scanning electron microscopy utilizing critical-point drying and transmission electron microscopy employing air-dried agar pseudoreplicas and critical-point dried carbon replicas were used to study the surface of Rickettsia rickettsii propagated in cell culture.

Preparation and testing of vaccines prepared from the envelopes of Venezuelan, eastern, and western equine encephalomyelitis viruses.

Envelope components were separated from Venezuelan, Eastern, and Western equine encephalomyelitis viruses after treatment of the virions with detergent. Vaccines prepared from the envelope component were capable of stimulating mice to produce humoral antibodies. Protective efficacy studies were performed using mono-, di-, and trivalent vaccine combinations. These elicited varying degrees of homologous protection, and Eastern and Venezuelan equine encephalomyelitis envelope products appeared to confer protection to mice challenged with Western equine encephalomyelitis virus.

Comparative analyses of members of the Venezuelan equine encephalomyelitis virus complex.

Polyacrylamide gel electrophoretic examination of viruses selected from the Venezuelan equine encephalomyelitis (VEE) complex revealed distinct strain to strain differences in profiles of the two virion envelope proteins. The core protein was identical in all viruses tested. We detected five electrophoretic patterns into which the virus strains could be classified and these were designated alpha (alpha), beta (beta), gamma (gamma), delta (delta), and episolon (episolon). Isolates representing variant E of subtype I exhibited a profile characterized by only one apparent envelope band. The epizootic subtypes I-A, I-B, I-C and the sylvatic subtype II viruses contained at least two envelope proteins which differed in molecular weight according to virus strain but which were not necessarily specific for antigenic variety. These results generally, though not uniformly, support the serologic classification of the VEE virus complex and suggested that the usefulness of the classification scheme could be complemented by the inclusion of biochemical criteria.

Preparation of Rocky Mountain spotted fever vaccine suitable for human immunization.

Rocky Mountain spotted fever vaccine was produced from rickettsiae grown in chicken embryo cells in roller bottle cultures. The rickettsiae were concentrated and purified by passage through a sucrose gradient and inactivated with formalin. This vaccine satisfactorily passed preinactivation and final container testing and is believed to be superior to the presently available yolk sac vaccine.

Separation, isolation, and immunological studies of the structural proteins of Venezuelan equine encephalomyelitis virus.

Three viral proteins were separated from the TC-83 strain of Venezuelan equine encephalomyelitis virus by discontinuous polyacrylamide gel electrophoresis after disruption with sodium dodecyl sulfate and beta-2-mercaptoethanol. These proteins were inoculated into rabbits and the resultant antisera were tested for immunological activity by gel precipitation, plaque reduction neutralization, hemagglutination inhibition (HI), complement fixation, fluorescence microscopy, and mouse protection studies. All proteins were capable of stimulating precipitating antibody in rabbits, but the largest protein (VP 1), which is contained in the envelope, stimulated the production of detectable neutralizing and HI antibody against the intact virion. The other two proteins yielded little or no neutralizing or HI antibody.

Plaque formation by strains of spotted fever rickettsiae in monolayer cultures of various cell types.

Some strains of spotted fever rickettsiae could be distinguished by their ability or inability to form plaques in monolayer cultures of various mammalian and avian cell types.

Immune responses to Rickettsia akari infection in congenitally athymic nude mice.

Athymic BALB/c nude mice and euthymic BALB/c mice were infected with Rickettsia akari by the intraperitoneal route. The rickettsialpox infection was terminated in euthymic mice with only two intraperitoneal injections of the antibiotic oxytetracycline, whereas prolonged treatment was necessary to terminate the infection in athymic mice. Both athymic and euthymic mice produced specific antibody, but athymic mice were still susceptible to reinfection. Killed R. akari served as a protective immunogen in euthymic, but no in athymic, mice. When spleen cells from convalescent euthymic mice were transferred to syngeneic athymic mice, recipients showed protection against challenge. This suggests that a T-cell-dependent step is generally necessary to terminate the rickettsialpox infection.

Comparison of three rocky mountain spotted fever vaccines.

Growth of Rocky Mountain spotted fever (RMSF) rickettsiae in duck embryo cell (DEC) cultures and chicken embryo cell (CEC) cultures was evaluated. Experimental lots of duck embryo cell- and chicken embryo cell-grown Rocky Mountain spotted fever vaccines and a commercial lot of yolk sac-grown vaccine were compared for protective efficacy in rhesus monkeys. Incidence and magnitude of antibody response, febrile response, and rickettsemia, as well as incidence of fatalities, suggested that both cell culture-derived vaccines were more immunogenic than the yolk sac-grown vaccine.

Effect of vaccination schedule on immune response of Macaca mulatta to cell culture-grown Rocky Mountain spotted fever vaccine.

The effect of vaccination schedule on the immune response of Macaca mulatta to formalin-inactivated chicken embryo cell culture (CEC)-grown Rickettsia rickettsii vaccine was studied. Schedules consisted of inoculation on day 1 only, on days 1 and 15, on days 1 and 30, on days 1, 8, and 15, or on days 1, 15, and 45. Humoral antibody measured by microagglutination and indirect immunofluorescence and resistance to challenge with 10(4) plaque-forming units of yolk sac-grown R. rickettsii were assessed. Seroconversion was noted in all monkeys after the first dose of vaccine. A second dose administered 8 or 15 days after the primary infection, or a third given 7 or 30 days after the second, produced no long-term effect on antibody titer. Only monkeys given two doses of vaccine at a 30-day interval showed an increase in antibody titer during the period before challenge. Vaccination with one, two, or three doses of CEC vaccine prevented development of rash and rickettsemia after challenge. The two-dose schedules appeared to induce the highest degree of resistance to challenge, as indicated by unaltered hematological parameters and body temperature in monkeys. The one- and three-dose schedules were somewhat less effective, in that some challenged monkeys within each group displayed febrile and leukocyte responses associated with Rocky Mountain spotted fever infection. Our data suggest that administration of two doses of CEC vaccine at 15- or 30-day intervals is the immunization schedule of choice.

Evaluation of a killed Rocky Mountain spotted fever vaccine in cynomolgus monkeys.

A nonhuman primate model of Rocky Mountain spotted fever infection was developed in cynomolgus monkeys (Macaca fascicularis) infected by the subcutaneous route or by aerosol. Clinical responses, hematology and serum chemistry values, and pathological findings were similar to those found in humans ill with Rocky Mountain spotted fever. The clinical model was then used to test the efficacy of a killed Rocky Mountain spotted fever vaccine grown in chicken embryo cells. Monkeys were immunized with varying dilutions of the vaccine with a two-dose schedule and then challenged at 2 months with virulent Rickettsia rickettsii by the subcutaneous route or by aerosol. The undiluted vaccine totally protected monkeys against both challenges, even at extremely high doses.

Suppression of cellular immune responses in guinea pigs infected with spotted fever group rickettsiae.

Using a guinea pig model, we demonstrated that infections with pathogenic species of spotted fever group rickettsiae transiently and nonspecifically suppress established cellular immune responses as measured by in vitro lymphocyte transformation and in vivo delayed cutaneous hypersensitivity responses to unrelated, nonrickettsial antigens. The correlation of the duration of this immunosuppression with the virulence of the infecting rickettsial species suggests that this suppression is a pathological effect of the rickettsial infection. Although we did not specifically study the mechanism of this suppression, it is not associated with either lymphocytopenia or leukocytosis.