Maximizing plasmid stability and production of released proteins in Yersinia enterocolitica.

Virulent serotypes of Yersinia enterocolitica carry a plasmid (pYV) encoding a family of proteins that are released into the medium and whose expression is temperature and calcium regulated. The plasmid is easily lost from cells during their growth in the laboratory. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with a monoclonal antibody (3.2C) that is specific for a 25-kDa released protein to show that 32 degrees C is the lowest temperature at which plasmid-encoded proteins are expressed in quantity. The highest calcium concentration allowing full expression of these proteins was 445 to 545 microM at 32 degrees C. Calcium concentrations of 745 microM and above at 37 degrees C completely prevented the loss of pYV during multiple subcultures, while at 32 degrees C, calcium concentrations of 245 microM and greater were sufficient to stabilize the plasmid. Growth of Y. enterocolitica at pH 5.5 was slower than at neutral pH values, but it also resulted in greatly increased stability of pYV. These studies showed that bacterial growth, retention of pYV, and expression of plasmid-encoded proteins may be maximized at 32 degrees C with 445 microM calcium and that pYV stability is enhanced by growth at low pH. These observations suggest new approaches for isolation of plasmid-bearing virulent strains of Y. enterocolitica from samples contaminated with this organism and also may improve our understanding of pYV retention in vivo.

Monoclonal antibody to a major outer membrane protein of feline Chlamydia psittaci: antibody specificity and anti-idiotype antibody production.

A murine monoclonal antibody, F1-8, was developed against the purified major outer membrane protein (MOMP) of Chlamydia psittaci feline pneumonitis (FPn). F1-8 showed a serotype-specific activity against intact Fpn elementary bodies in a micro-immunofluorescence assay. In immunoblot, F1-8 reacted only with the Fpn MOMP but did not react with the MOMPs from other strains of C. psittaci and C. trachomatis. F1-8 neutralized Fpn infectivity in L929 cell culture in a dose-dependent and complement independent fashion. These results suggested that the monoclonal antibody (mAb) binds with an epitope on the MOMP region that is exposed at the cell surface and plays an important role in FPn infection. Polyclonal anti-idiotype (anti-Id) antibodies to mAb F1-8 were elicited by F1-8 coupled to keyhole limpet hemocyanin. These anti-Id antibodies inhibited F1-8 binding to FPn MOMP.

An antibody adsorption technique facilitates antigen selection for development of serotype-specific monoclonal antibodies to Yersinia enterocolitica.

A simple procedure is described to aid in the selection of antigens from gram-negative organisms that are most likely to exhibit serotype-specific epitopes when used as immunogens in the mouse. Several such proteins of Yersinia enterocolitica serotype O:8 were identified by immunoblotting through their binding to polyclonal antibodies that remained in immune serum after the serum had been adsorbed with common antigens prepared from a variety of closely related serotypes and Gram-negative organisms. The selected proteins were purified electrophoretically and used to immunize mice to obtain immune spleen cells for cell fusions. Serotype-specific monoclonal antibodies (MAbs) were successfully developed to a 36-kDa outer membrane protein resembling the porin OmpF. Additional MAbs also were produced that cross-reacted with other serotypes. Only cross-reactive epitopes were detected on a 34-kDa protein. The MAbs were used to show that the relative expression of the 36-kDa protein varied severalfold according to the medium used to grow the cells. These MAbs are promising agents for serological identification of Y. enterocolitica serotype O:8. The procedure should be useful in many situations where extensive cross-reactivities impede generation of highly specific MAbs.

A rapid method for detection of Yersinia enterocolitica serotype O:3 in pig feces using monoclonal antibodies.

A simple colony immunoblotting method using monoclonal antibodies (MAbs) was developed to detect Y. enterocolitica serotype O:3 in pig feces. One of the MAbs studied was able to detect single colonies of the organism in the presence of calculated 3.1 x 10(8) heterologous organisms in pig feces. The MAb was found to be specific for the lipopolysaccharide (LPS) O-antigens of Y. enterocolitica serotype O:3. No significant cross-reactivity was found against a variety of closely related serotypes and Gram-negative organisms. The MAb could also be used in a slide agglutination test and an indirect fluorescence antibody assay for rapid identification of Y. enterocolitica serotype O:3.

Monoclonal antibodies to Chlamydia psittaci guinea pig inclusion conjunctivitis (GPIC) strain.

Monoclonal antibodies to a strain of Chlamydia psittaci isolated from guinea pig inclusion conjunctivitis (GPIC) were developed. Only five of the 15 hybridomas isolated produced antibodies specific for the GPIC strain, while seven others produced antibodies which cross reacted with other strains and another species. Strain-specific and species-specific monoclonal antibodies were isotyped as IgG2a and IgG3, respectively. It appears that the GPIC strain has at least two epitopes, one of which is specific for the strain and the other common to the species. These monoclonal reagents may be used to immunotype GPIC agents, better than available methods and may be of potential use in the development of vaccines against chlamydial infections.

Distinguishing between ovine abortion and ovine arthritis Chlamydia psittaci isolates with specific monoclonal antibodies.

Monoclonal antibodies to Chlamydia psittaci were prepared by both in vivo and in vitro immunization methods, using an abortion strain of C psittaci as the immunizing antigen. Seven of the 8 monoclonal antibodies produced were genus-specific by the enzyme-linked immunosorbent assay and immunofluorescence test. The genus-specific antibodies were reactive with a protease-resistant, periodate-sensitive antigen of less than 14 kilodaltons. The remaining monoclonal antibody, 10D7, was specific for ovine abortion strains of C psittaci and nonreactive with 2 strains isolated from the joints of lambs with polyarthritis. The type-specific antigen was protease sensitive, but could not be detected in the immunoblot assay.

Monoclonal antibodies specific for Corynebacterium sepedonicum, the causative agent of potato ring rot.

BALB/c mice were immunized with Corynebacterium sepedonicum, and spleen cells from the immunized animals were fused with cells of the mouse myeloma line P3-X63-Ag8.653. Several hybridoma cell cultures were selected for further study. Monoclonal CS-B-5 was specific for C. sepedonicum and did not react significantly with other closely related phytopathogenic corynebacteria in an enzyme-linked immunosorbent assay (ELISA). As few as 10(3) organisms could be detected. This approach should prove useful for developing improved diagnostic procedures for a number of bacterial plant pathogens.

Amphotericin B in liposomes: a novel therapy for histoplasmosis.

Incorporation of amphotericin B into liposomes significantly altered its toxicity, tissue distribution, and efficacy. Compared with intravenously administered amphotericin B-desoxycholate, liposome-amphotericin B showed a reduced acute toxicity and a maximal tolerable dose 9 times greater than amphotericin B-desoxycholate. Liposome-amphotericin B also produced higher tissue and lower serum concentrations than amphotericin B-desoxycholate, and was significantly more effective in prolonging survival of mice infected with Histoplasma capsulatum.

Treatment of murine cryptococcosis with liposome-associated amphotericin B.

Liposomes were prepared to incorporate large amounts of amphotericin B. BALB/c mice were challenged with Cryptococcus neoformans and given liposome-associated amphotericin B (AMBL) or amphotericin B-deoxycholate (AMBD) intravenously. Mice that were treated with AMBL survived longer and had lower tissue counts of cryptococci than mice treated with AMBD or untreated control mice. The reduced acute toxicity of AMBL permitted much larger doses of amphotericin B to be given than were possible with AMBD. AMBL is a novel vehicle of administration that reduces toxicity and concentrates the drug in the appropriate target organs.

Marked stimulation of lymphocyte-mediated attack on tumor cells by target-directed liposomes containing immune RNA.

A marked stimulation of normal guinea pig lymphocytes was obtained by incubating them with liposomes that contained both antibody to lymphocytes to provide "homing" of the vesicles and immune RNA isolated from guinea pigs immunized with syngeneic line 10 hepatocarcinoma cells. Tumor cell cytotoxicity was monitored by a 51Cr release assay. This target cell delivery of immune RNA by liposomes produced a dose-dependent stimulation up to 12 times that achieved by in vitro methods with naked immune RNA.

A comparison of negatively and positively charged liposomes containing entrapped polyinosinic-polycytidylic acid for interferon induction in mice.

Intravenous injection of negatively and positively charged liposomes containing entrapped poly(I)-poly(C) induced a vigorous interferon response in mice with serum titers of interferon reaching twenty times those observed with comparable dosages of free poly(I)-poly(C). The response did not persist over an extended time period as observed earlier for enhanced interferon production stimulated by positively charged liposomes containing the inducer. Both negatively and positively charged liposomes containing [14C]poly(I)-poly(C) were taken up chiefly by the liver when given intravenously. Negatively charged particles were concentrated somewhat preferentially by the spleen (7--9% of the dose compared to 4--6%). Less radioactivity was found in liver and spleen when negatively charged particles were given intraperitoneally than was the case when positively charged particles were injected by this route. Free [14C]poly(I)-poly(C) was extensively metabolized to low molecular weight materials within four hours of injection, while encapsulation of the polymer provided protection against in vivo degradation. When both preferential localization and protection were considered, from three to five times as much high molecular weight E114C]poly(I)-poly(C) was recovered from liver at four hours after intravenous injection when the compound was given in encapsulated form compared to free polymer. Similarly, for spleen, seven times and three times as much polymeric [14C]poly(I)-poly(C) was recovered following injection of negatively charged liposomes and positively charged liposomes respectively compared to free [14C]poly(I)-poly(C). At 48 h after an intravenous injection of positively charged liposomes, as much as four percent of the dose remained in high molecular weight form in the liver and one percent in the spleen. Following intraperitoneal injections, polymeric [14C]poly(I)-poly(C) recovered from the liver never exceeded 4.3% of the dose, showing that most of the radioactivity in the liver consisted of metabolites. These results suggest that elevated and prolonged production of interferon in animals treated with encapsulated inducer results from a combination of factors including preferential tissue location and protection of the inducer from hydrolytic cleavage.

The interaction of cationic liposomes containing entrapped horseradish peroxidase with cells in culture.

Cationic liposomes composed of sphingomyelin, cholesterol, and stearylamine were prepared with horseradish peroxidase trapped inside. Stable particles were formed in which 10-12% of the enzymic activity appeared to be located at, or near, the outer surface of the liposome. Adsorption and uptake of liposomes by HeLa cells were followed cytochemically by electron microscopy and quantitated by enzyme assay and by the distribution and fate of particles labeled with [(14)C]cholesterol and [(125)I]horseradish peroxidase. The particles were adsorbed by HeLa cells at least 300 times as efficiently as was free horseradish peroxidase. Many of the particles remained at the cell surface, but numerous membrane-bound cytoplasmic inclusions were observed to contain peroxidase-staining material. In addition, many areas of the cell membrane gave a positive staining reaction. It was concluded that many particles (presumably the larger ones) did not gain access to the interior of the cells, many were phagocytized, and some enzyme was transferred to the cell membrane, perhaps as a result of fusion of the liposomal membrane with the cell membrane.

Interferon induction by poly (I): poly (C) enclosed in phospholipid particles.

Liposomes were prepared with phospholipids (sphingomyelin, lecithin, and phosphatidylethanolamine) in combination with cholesterol and charged lipids (dicetyl phosphate and stearylamine) and contained either poly(I):poly(C) or poly(I). Neutral and positively charged liposomes attached much better to L-929 cells in tissue culture than did negatively charged particles. Liposomes were toxic to L cells at relatively low concentrations, making the determination of antiviral activity induced by particles containing poly(I):poly(C) difficult to measure by the plaque reduction assay. When injected into mice, all of the liposomes containing poly(I):poly(C), except phosphatidylethanolamine liposomes, greatly potentiated and extended the serum interferon response of poly(I):poly(C). Lecithin and sphingomyelin liposomes given intravenously were ten times more effective than free poly(I):poly(C) in stimulating production of serum interferon. Sphingomyelin liposomes containing [(14)C]poly(I):poly(C) were 88% cleared from the bloodstream of mice by 3 min after intravenous injection. Most of the radioactivity (70%) was captured by the liver and remained there for at least 4 h. By 2 h, 7% of the radioactivity could be found in the spleen. Five percent of the radioactivity was found in the lungs at 30 min, with decreasing amounts thereafter. Small amounts of radioactivity were found in the muscle and kidneys. The spleen was shown to contain appreciable levels of interferon at 4 h, and low levels were found in the liver. Radioactivity accumulated slowly in the liver following an intraperitoneal injection of sphingomyelin liposomes containing [(14)C]poly(I):poly(C). By 4 h, 26% of the dose was recovered from the liver and 4.9% from the spleen, with small amounts in the lung, kidney, and omentum.