Polyamine oxidase in human retroplacental serum inhibits the growth of Plasmodium falciparum.

Human retroplacental serum (RPS) containing polyamine oxidase inhibited the growth of the Camp strain of Plasmodium falciparum in vitro as assayed by the parasite's decreased incorporation of 3H-hypoxanthine. Inhibition was dose-dependent on the concentrations of serum polyamine oxidase and added polyamines. Almost complete inhibition was seen in 96-hr asynchronous cultures containing 10% RPS and in those containing 1.2% RPS plus 50 microM polyamine. Subtle morphologic changes in mature stages and decreased numbers of new rings were associated with inhibition seen in 19-hr synchronous cultures initiated at the trophozoite stage. These incubation times were longer than in previous reports showing inhibition of malaria parasites by bovine polyamine oxidase but not by human polyamine oxidase. Macrophages contain polyamine oxidase, the reaction products of which are known to be similar to those of RPS polyamine oxidase but different from those of bovine polyamine oxidase. It remains to be determined whether human polyamine oxidase, acting upon ubiquitous polyamines, contributes to host defenses against malaria.

Production and characterization of cloned T-cell hybridomas that are responsive to Rickettsia conorii antigens.

T-cell hybridomas produced by the fusion of Rickettsia conorii immune T cells to the AKR thymoma BW 5147 produced interleukin-2 when stimulated with the antigens of three different R. conorii strains. One cloned hybridoma responded only to R. conorii antigens, whereas a second and third cloned hybridoma also responded to the antigens of Rickettsia rickettsii Sheila Smith and Rickettsia sibirica 246, respectively. Antigen responses required antigen-presenting cells, and this interaction was major histocompatibility complex restricted. Fluorescence-activated cell-sorter analysis demonstrated that all three hybridomas were of the Thy-1.2+, Lyt-2- phenotype and that two of the three were L3T4+. These data demonstrated the presence of an antigenic epitope that is R. conorii species specific and other epitopes that are common to various members of the spotted fever group which can stimulate interleukin-2 production by T-cell hybridomas.

Cross-reactive lymphocyte responses and protective immunity against other spotted fever group rickettsiae in mice immunized with Rickettsia conorii.

Lymphocyte proliferation in response to antigens on spotted fever group rickettsiae was used as a method to investigate the group-specific protective immunity to rechallenge characteristic of this group of rickettsiae at the T-cell receptor level. Spleen cells from Rickettsia conorii-immune C3H/HeJ mice proliferated in response to R. rickettsii Sheila Smith, R. sibirica 246, R. australis, and all tested strains of R. conorii (Casablanca, Moroccan, and Malish). Spleen cells from these mice, however, responded poorly or not at all to antigens prepared from the Kaplan or Hartford strain of R. akari. Proliferation of immune T cells maintained as in vitro cell lines showed a similar pattern of reactivity to these antigens; however, response to R. akari was consistently demonstrable. Spleen cells from C3H/HeJ mice immunized with R. akari responded to R. akari and R. conorii antigens as well as antigens from the other spotted fever group rickettsiae. Lymphocytes obtained from lymph nodes draining foot pads infected with R. conorii or R. akari demonstrated cross-reactivity similar to that found with immune spleen cells. If immunization was accomplished with R. conorii antigen emulsified in Freund complete adjuvant, the resulting lymph node cells were able to respond to R. akari antigens. These data suggest that infection with R. conorii induces a population of T lymphocytes that recognize an antigen(s) that also is found on other spotted fever rickettsiae and that may be responsible for cross-protective immunity. This antigen probably is not a major antigen on R. akari.

Identification of strain-specific and group-reactive antigenic determinants on the Karp, Gilliam and Kato strains of Rickettsia tsutsugamushi.

Hybridoma antibodies (Hab) were prepared against the Karp, Gilliam and Kato strains of Rickettsia tsutsugamushi and were examined for homologous and heterologous reactivity using an indirect immunofluorescence assay. Strain-specific Hab demonstrated homologous IFA titers ranging from 1/320 to 1/1,280 and did not react (less than 1/10) with the heterologous strains. The cross-reactive Hab generally reacted equally with all three strains in the scrub typhus group; however, there were some Hab that reacted with only one of the two heterologous strains tested. The Hab also were examined in enzyme-linked immunosorbent assays with scrub typhus antigens eluted from SDS-polyacrylamide gels. Most Hab reacted with either one or several of the six eluted antigens detected with a polyclonal immune serum. It was also observed that strain-specific and cross-reactive Hab sometimes reacted with the same antigen, suggesting the existence of multiple antigenic determinants in one electrophoretic peak. The data suggest that strain-specific Hab can be used in the indirect immunofluorescence assay to identify isolates of R. tsutsugamushi without the cross-reactions usually observed with polyclonal antisera, and that they are useful probes for detection and analysis of rickettsial antigens.

Susceptibility of inbred mice to rickettsiae of the spotted fever group.

A mouse strain susceptible to lethal infection with Rickettsia conorii was required for testing vaccine efficacy and for studying the immunology and pathogenesis of infection. Among 20 strains of inbred mice inoculated intraperitoneally with the Malish strain of R. conorii, the C3H/HeJ mouse strain was the most susceptible, with a 50% lethal dose of approximately 10 PFU. Infection of all mouse strains resulted in a measurable antibody response; the highest titers correlated with the greatest degree of rickettsial replication as measured by plaque assay of infected spleen homogenates. Inoculation of C3H/HeJ mice with 5.0 log10 organisms of strain Malish by the subcutaneous route did not result in lethal infection. The Casablanca and Moroccan strains of R. conorii were not lethal for C3H/HeJ mice and, in addition, produced plaques in L-929 cells morphologically distinct from those produced by the Malish strain. The only other spotted fever group rickettsia tested which produced a lethal infection in C3H/HeJ mice was Rickettsia sibirica. Sublethal infection with any of the spotted fever rickettsiae tested protected against lethal infection with R. conorii. These data established a lethal challenge system for examining the protective efficacy of spotted fever immunogens and presented evidence of biological variation among strains of R. conorii.

Role of T-lymphocytes in production of antibody to antigens of Rickettsia tsutsugamushi and other Rickettsia species.

The requirement of thymus-dependent lymphocytes for antibody production to Rickettsia tsutsugamushi, Rickettsia akari, Rickettsia conorii, and Rickettsia typhi was investigated by comparing antibody production in athymic (nu/nu) or thymus-bearing BALB/c mice. Athymic BALB/c mice produced antibody after infection with R. akari, R. conorii, and R. typhi as measured by indirect fluorescent antibody titration or radioimmunoassay. Antibody production in these mice was a great or greater than in the thymus-bearing mice and demonstrated similar kinetics. In contrast, athymic BALB/c mice infected either intraperitoneally or subcutaneously with the Gilliam strain of R. tsutsugamushi failed to produce demonstrable antibody. The requirement of thymus-dependent lymphocytes for antibody production to R. tsutsugamushi was further suggested by the demonstration of antibody production after transfer of immune thymus-dependent lymphocytes to athymic mice and the demonstration of R. tsutsugamushi-specific T helper cells in immune thymus-bearing mice. The antibody produced in athymic mice after infection with R. akari, R. conorii, and R. typhi was predominantly immunoglobulin M, based on isotype-specific radioimmunoassays and sucrose gradient fractionation. Furthermore, the antibody produced by athymic mice in response to R. akari infection reacted with a carbohydrate-containing outer membrane component.

Antigens of scrub typhus rickettsiae: separation by polyacrylamide gel electrophoresis and identification by enzyme-linked immunosorbent assay.

Antigens of plaque-purified Rickettsia tsutsugamushi strains Gilliam, Karp, and Kato were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were analyzed by an enzyme-linked immunosorbent assay. Six antigens were identified in each of the three prototype strains; in strain Gilliam, these antigens were located in the cell envelope fraction of the organisms. Reactivity of these isolated antigens with homologous or heterologous immune sera indicated that different macromolecules existed in all three strains, although they exhibited very similar mobilities during electrophoresis. Antigens of strain Gilliam reacted equally well with antibodies directed against Gilliam, Karp, or Kato rickettsiae. However, strains Karp and Kato each had two distinct antigens which did not react with heterologous antisera. R. tsutsugamushi antigens retained immunogenicity after electrophoresis, and antisera raised against them reacted with intact organisms and exhibited specificity in reactions with isolated antigens.

Detection of typhus antibodies by latex agglutination.

A latex test for assay of antibodies to endemic and epidemic typhus rickettsiae is simple, group-specific, sensitive, and reproducible. Cross-reactivity within the typhus group was extensive.

Surface proteins of typhus and spotted fever group rickettsiae.

Six proteins, previously established as major constituents of intact organisms, were identified in cell envelopes obtained from intrinsically radiolabeled Rickettsia prowazekii. Extrinsic radioiodination of intact organisms conducted at 0.5 micronM iodide indicated that protein 4 was the most peripheral, although protein 1 also had reactive groups exposed on the surface of the organisms. A 10-fold increase in iodide concentration resulted in labeling of protein 2, and at 50 micronM iodide, all six major proteins were radiolabeled. Similar selective labeling was not achieved with R. conorii. Analysis of both typhus and spotted fever group organisms radiolabeled with galactose suggested that carbohydrate was associated with proteins 1, 3, and 4. Typhus soluble antigen included all major proteins except protein 2, which remained attached to particulate rickettsiae after ether extraction. Protein 4 appeared to be prominent in the surface topography of R. prowazekii, was a component of soluble antigen and may have an important role in rickettsiae-host interactions.

Rickettsial indirect hemagglutination test: isolation of erythrocyte-sensitizing substance.

The erythrocyte-sensitizing substances (ESS) of Rickettsia prowazekii and R. conorii were characterized by biological and chemical criteria. ESS could be derived from either soluble or particulate complement-fixing antigens obtained by ether extraction of rickettsiae. The soluble complement-fixing antigen exhibited two peaks of serological activity in potassium tartrate density gradients. The particulate complement-fixing antigen coincided with the more dense peak but was distinguishable by its sedimentation in rate-zonal sucrose gradients. ESS was obtained from each of the complement-fixing-reactive gradient peaks by extraction with hot alkali and was quantified by a modified indirect hemagglutination test. These ESS preparations sedimented similarly in potassium tartrate gradients and were shown to contain protein and carbohydrate, both by colorimetric tests and by incorporation of radioactive precursors. The serological activity of ESS was unaffected by trypsin, but both antigenicity and erythrocyte-binding capacity were reduced after exposure to sodium metaperiodate. Highly purified ESS was rapidly inactivated by potassium tartrate and required stabilization with bovine plasma albumin.

Proteins of typhus and spotted fever group rickettsiae.

Purified radioactive rickettsiae were obtained from irradiated and cycloheximide-inhibited L cells, and their proteins were analyzed by polyacrylamide gel electrophoresis. Rickettsial species could be distinguished by comparing the relative mobilities of constituent proteins after migration of two differentially labeled preparations in a single gel. Distinct differences were observed in gel patterns of rickettsiae from the typhus and spotted fever groups, as well as with different species within a group. Rickettsial organisms causing murine and epidemic typhus were clearly distinguished, as were the causative agentsof boutonneuse fever and rickettsialpox. The use of both internal and external molecular weight standards allowed molecular weight estimates for 19 proteins from both Rickettsia prowazekii and Rickettsia conorii. A flexible system for designating rickettsial proteins is proposed that lends itself to modification as more detailed analysis progresses.