Kinetic roles and conformational properties of the non-native two-disulphide intermediates in the refolding of bovine pancreatic trypsin inhibitor.

The most productive folding pathway of reduced bovine pancreatic trypsin inhibitor (BPTI) proceeds through the disulphide intermediates (30-51), (30-51, 5-14), and (30-51, 5-38); these are important kinetic intermediates in folding, even though the latter pair contain non-native disulphide bonds. Analogues of these intermediates have been prepared by protein engineering methods and their conformational properties examined by circular dichroism and 1H-nuclear magnetic resonance. The (30-51), (30-51, 5-14) and (30-51, 5-38) analogues exhibit comparable degrees of stable structure, which cannot include those portions of the polypeptide chain involving Cys5, Cys14 and Cys38. These properties are consistent with the roles of (30-51, 5-14) and (30-51, 5-38) in the folding pathway of BPTI, which demand that they exhibit a considerable degree of conformational flexibility in part of the molecule.

Antigenic structure of Coxiella burnetii. A comparison of lipopolysaccharide and protein antigens as vaccines against Q fever.

The antigenic structure of Coxiella burnetii is being investigated by identifying both external and internal cellular epitopes of the morphologic cell types. Both the phase I lipopolysaccharide (LPSI) and several surface proteins are candidates for the development of subunit multivalent vaccines. The protective efficacy of purified LPSI was demonstrated in A/J mice. The purified LPSI preparations contained residual peptides detected by amino acid analysis. Therefore, the protection afforded by LPSI may be, in part, due to the presence of peptides. The purification of proteins free of LPSI must be accomplished before the protective efficacy of proteins or peptides can be established. We have identified three proteins that are both antigenic and immunogenic, as indicated by either enzyme immunoassay, radioimmunoprecipitation, immunoblot assay, or lymphocyte transformation. A 62-kDa protein antigen encoded by the htpB gene of C. burnetii was analyzed for immunogenicity. The purified protein antigen was immunogenic, as it elicited specific antibodies and performed as recall antigen in lymphocyte stimulation assays. The antigen was not detected on the surface of phase I cells but was highly represented on the surface of phase II cells. Therefore, the protein may not be a good candidate for vaccine development. The diagnostic utility of the 62-kDa protein antigen lies in the fact that convalescent and chronic Q fever sera from human patients reacted with the antigen, whereas acute sera did not. Although the 62-kDa protein is a "common antigen," specific peptide-based diagnostic reagents may be useful in the detection of Q fever disease progression. A major surface protein (P1) of roughly 29.5 kDa was purified from the phase I Nine Mile (clone 7) strain. No LPSI was detected in the P1 preparation by three different LPSI monoclonal antibodies. Monoclonal antibodies prepared against P1 were effective in localizing the protein on the cell surface, in the cell wall, and associated with the peptidoglycan of large cells of C. burnetii. Small, pressure-resistant cells did not contain P1. Mice immunized with two 25-micrograms injections of LPSI produced antibodies against LPSI and phase I whole cells. No antibody was detected against phase II whole cells. Immunization with P1 induced antibody against the LPSI fraction and phase I and phase II whole cells. P1 was more effective than LPSI in reducing the number of infectious C. burnetii in the spleens of challenged mice. The gene encoding another protein (P2) recognized by P1 monoclonal antibodies was cloned and sequenced.(ABSTRACT TRUNCATED AT 400 WORDS)

Inactivation of Coxiella burnetii by gamma irradiation.

The gamma radiation inactivation kinetics for Coxiella burnetii at -79 degrees C were exponential. The radiation dose needed to reduce the number of infective C. burnetii by 90% varied from 0.64 to 1.2 kGy depending on the phase of the micro-organism, purity of the culture and composition of suspending menstruum. The viability of preparations containing 10(11) C. burnetii ml-1 was completely abolished by 10 kGy without diminishing antigenicity or ability to elicit a protective immune response in vaccinated mice. Immunocytochemical examinations using monoclonal antibodies and electron microscopy demonstrated that radiation doses of 20 kGy did not alter cell-wall morphology or cell-surface antigenic epitopes.

Oxidation of Manganese and Formation of Mn(3)O(4) (Hausmannite) by Spore Coats of a Marine Bacillus sp.

Isolated spore coats of a marine Bacillus species were incubated in 25 mM MnCl(2) at pH 7.5. Manganese precipitates, formed on the coat surfaces, were analyzed by transmission electron microscopy, electron diffraction, and energy-dispersive X-ray spectroscopy. Initially, an amorphous manganese oxide was observed on the coats which recrystallized to hausmannite after prolonged incubation in the MnCl(2) solution. The spore coats catalyze the oxidation of Mn(II) and have no structural influence on the final mineral phase precipitated.

Animal models in Q fever: pathological responses of inbred mice to phase I Coxiella burnetii.

The susceptibility of inbred strains of mice to infection by phase I Coxiella burnetii, the aetiological agent of Q fever, was investigated by evaluating morbidity, mortality, antibody production and in vitro proliferative responses of splenic lymphocytes. Among the 47 strains of mice tested for morbidity and mortality to C. burnetii infection, 33 were resistant, 10 were of intermediate sensitivity, and four were sensitive. A/J mice exhibited the highest mortality, and surviving mice of this strain yielded high concentrations of viable rickettsiae from essentially all organs for more than 3 weeks after inoculation. However, A/J mice developed a protective immune response after vaccination with inactivated C. burnetii cells. Induction of gross pathological responses and antibody production were similar in sensitive mice (strain A/J) and resistant mice (strain C57BL/6J). The LD50 of phase I C. burnetii for A/J mice was about 1000-fold lower than that for the more resistant C57BL/6J mice. Mice of both strains developed antibody titres against phase I cells, phase II cells, and phase I lipopolysaccharide after the injection of one or more viable phase I organisms of C. burnetii; five or more rickettsiae caused splenomegaly that was almost proportional to the infecting dose. Suppression of in vitro proliferative responses of splenic lymphocytes to concanavalin A, a T-cell mitogen, was apparent after infection of sensitive A/J mice with as few as one to five phase I micro-organisms. However, suppression of proliferation of splenic lymphocytes from resistant C57BL/6J mice required 10(7) phase I C. burnetii.

Delayed-type hypersensitivity responses induced by bovine colostral components.

Transfer factor-type substances obtained from leukocytic cells and fluid portions of bovine colostrum caused effective passive transfer of delayed-type hypersensitivity responses across species barriers. Passive transfer of Brucella abortus sensitivity was obtained with equal regularity when using components derived from peripheral blood and colostrum of dams sensitized at 3 and 9 months of age. Colostral feedings to calves caused the passive transfer of delayed-type hypersensitivity as early as 2 days after parturition. The findings indicated that colostral components were important in the process of cell-mediated immunity.

Glucan-induced enhancement of host resistance to selected infectious diseases.

We conducted studies with mice, rats, and monkeys which demonstrated the ability of glucan to induce either nonspecific or specific enhancement of host resistance to infectious diseases. Intravenous pretreatment of mice with glucan significantly enhanced the survival of mice challenged with either Venezuelan equine encephalomyelitis (VEE) virus or Rift Valley fever virus. Pretreatment was beneficial when initiated 3 days before challenge with VEE virus and 7 days before challenge with Rift Valley fever virus. Treatment of mice after VEE challenge did not increase their survival compared with controls. Glucan pretreatment of rats provided increased resistance to both intraperitoneal and low-dose aerosol challenges with virulent Francisella tularensis when the glucan was given intravenously, but not when it was administered intranasally. In contrast, intranasal glucan pretreatment enhanced the survival of mice when they were challenged by aerosol with Pseudomonas pseudomallei, whereas intravenous glucan pretreatment did not increase survival. mice given glucan combined with a marginally immunogenic dose of VEE vaccine were more resistant to homologous virus challenge than were mice given either Freund complete adjuvant plus vaccine or vaccine alone. Similarly, both primary and secondary VEE antibody titers in cynomolgus monkeys given glucan with VEE vaccine were significantly greater than titers in vaccine controls.

Experimental Coxiella burnetii infection of guinea pigs and mice.

The susceptibility of five inbred strains of mice, designated DBA/1J, DBA/2J, C57BL/6J, Balf/CJ, and AKR/J, as well as outbred Hartley and Moen-Chase guinea pigs to infection with Coxiella burnetii by several routes was studied. The DBA/2J mice were more susceptible to infection and had higher mortality rates than other strains of mice. Guinea pigs were more susceptible to infection than mice. Lesions observed in the infected animals were similar to those previously described in man and experimentally infected animals.

Activity of amantadine, rimantadine, and ribavirin against swine influenza in mice and squirrel monkeys.

Amantadine, rimantadine, and ribavirin given orally, either prophylactically or therapeutically, reduced mortality and increased the survival time of 3-week-old mice infected with the type A/New Jersey/8/76 (swine) strain of influenza virus. In addition, amantadine and rimantadine, administered therapeutically, increased the rate of virus clearance from lungs of infected mice. Administration of amantadine either before or after virus challenge ameliorated the illness in squirrel monkeys; when administered therapeutically, it appeared to eliminate virus shedding from infected monkeys within hours after therapy was initiated.

Evaluation of commercially prepared vaccines for experimentally induced type A/New Jersey/8/76 influenza virus infections in mice and squirrel monkeys.

Mice and squirrel monkeys were vaccinated and subsequently challenged at selected times to evaluate the immunoprophylactic value of vaccines against influenza virus type A/New Jersey/76. Mice were challenged with virulent, homologous virus either 17 or 60 days after vaccination with 80 chick cell-agglutinating (CCA) units of whole-virus vaccine. Vaccinated mice showed minimal lesions and virus in lung tissue and had lower lung weights than unvaccinated controls. These mice had titers of hemagglutination-inhibiting (HAI) antibody in serum of greater than 1:400, but only traces of antibody were found in lung washes. Vaccinated squirrel monkeys had significantly less illness than unvaccinated controls when challenged with virulent virus 30 days after intramuscular immunization with 200 CCA units of whole virus or 400 CCA units of split virus given either once or twice (at 30-day intervals). Equal protection was observed in all monkeys despite the absence of serum HAI antibody in some monkeys after vaccination. Anamnestic reactions were observed only in monkeys vaccinated with whole virus. The possible roles of various immune factors and antibody to neuraminidase are discussed.

Use of a photodensitometric technique to quantify microscopic lung lesions in mice: Antiviral activity against swine influenza virus.

During studies of swine influenza virus (A/NJ/76) infection, a technique was devised to quantify the pulmonary lesions in infected mice treated at different time intervals with antiviral chemotherapeutic agents. The technique is based on the premise that as the severity of microscopic change increases, the optical density of lung sections also increases because of edema and increased cell numbers in infected lungs. Seven days after intranasal instillation of the virus, mice were killed and the lungs were perfused with 2% glutaraldehyde at constant pressure. Lungs were processed in a routine manner, sectioned at standard levels, and stained with hematoxylin and eosin. By using standard photomicrography equipment, multiple optical density measurements were made of lung sections in a carefully controlled systematic manner, and a mean optical density was determined for each lung. The optical density of lungs of mice treated before and after infection with amantadine, rimantadine, or ribavirin was significantly reduced compared with that of the lungs of infected, untreated controls. If treatment was delayed until 15 h after infection, amantadine and ribavirin were effective in reducing pulmonary optical density, but rimantadine was without effect. These findings correlated well with mean lung weight of each group; however, the sensitivity of the optical density technique was greater. Subjective scoring of microscopic lesions revealed differences only between infected and uninfected controls. The densitometric method offers promise as a reliable means of objectively quantifying the pulmonary response to a variety of infectious, toxic, and therapeutic agents.

Immunoglobulin-bearing cells in lungs of mice infected with influenza virus.

In sequential studies of cellular infiltrates from influenza-infected mouse lungs, increased populations of lymphocytes with surface immunoglobulins were observed, and immunoglobulin A-bearing cells exhibited the greatest relative increase.

Responses of mice immunized with influenza virus by serosol and parenteral routes.

Antibody levels in sera and respiratory secretions and resistance to respiratory infections were examined in mice given live infuenza virus in small-particle (2 mum) aerosols, large-particle (10 mum) aerosols, intraperitoneally, and subcutaneously. After parenteral administration antibody was found primarily in the serum, but small amounts were recovered in bronchoalveolar washings after 2 to 3 weeks. Specific antibody was present in both sera and bronchoalveolar washings from mice given virus in small-particle aerosols to achieve virus dissemination throughout the respiratory tract. Immunoglobulin A, immunoglobulin G, and trace amounts of immunoglobulin M, all specific for the infecting virus, were detected in bronchoalveolar washings of small-particle aerosol-infected mice. Virus administration in large-particle aerosols (for primary virus localization in upper respiratory tract) at doses greater than those required to initiate infection with small-particle aerosols failed to stimulate production of antibody in sera or bronchoalveolar washings. Small-particle aerosol-immunized mice were resistant to subsequent challenge with 10(2.0) respiratory median lethal doses of virulent virus, whereas large-particle aerosol-immunized mice were not protected. Parenteral immunization modified the course of the disease in challenged mice and reduce mortality rates but did not prevent reinfection of the respiratory tract.