Enhancement of immunogenic activity of ribosomal preparations from Haemophilus influenzae by various adjuvants.

Ribosomes from Haemophilus influenzae type b have been reported to have immunoprotective activity in animals that can be enhanced by adjuvants. In this report we evaluated the adjuvant activity of several compounds in conjunction with ribosomes from the b and c serotypes of H. influenzae. Alhydrogel, saponin, and DPT were found to significantly enhance the immunoprotective response in mice, equalling or exceeding the activity of Freund's incomplete adjuvant. All of these adjuvants also enhanced significantly the IgM response of mice to sheep red blood cells. Ribosomes were also found to enhance this response. Among the compounds failing to provide adjuvant activity for ribosomes were poly(A:U), muramyl dipeptide, mycobacterial extract, dimethylglycine, methylated bovine serum albumin, sodium diethylthiocarbamate, and cetyltrimethylammonium bromide.

Induction of active immunity with membrane fractions from Haemophilus influenzae type b.

Using Escherichia coli strain E-1 as a model, we developed procedures for the preparation of outer- and inner-membrane-enriched fractions as structural units. These procedures could be used to prepare relatively pure inner and outer membrane fractions as determined by succinate dehydrogenase activity, ketodeoxyoctonate levels, and polyacrylamide gradient gel electrophoresis. The use of these procedures to fractionate membrane components from Haemophilus influenzae type b strains H-2 and H-E led to good separation of outer- and inner-membrane-enriched fractions as determined by succinate dehydrogenase and ketodeoxyoctonate levels but incomplete separation as determined by polyacrylamide gradient gel electrophoresis. Although there were differences between the electrophoresis profiles of outer membrane fractions of strains H-2 and H-E, immunization with outer membrane of either strain led to the induction of a high degree of immunoprotection against challenge with the H-2 strain. Protection could also be elicited with inner membrane preparations, but such protection may be due to contamination with outer membrane. Extracted membrane protein induced levels of protection that were comparable to those induced by whole membrane fractions.

Opsonization and phagocytosis of Haemophilus influenzae type B organisms by mouse polymorphonuclear leucocytes and antiribosomal serum.

Sera from rabbits immunized with ribosomes passively protect mice challenged with Haemophilus influenzae type b. The protective antibody interacted with organisms in the blood and possibly at the sites of dissemination, but not at the site of inoculation. Macrophages did not phagocytize oposonized bacteria in our system. However, immune serum enhanced phagocytosis and intracellular killing by polymorphonuclear leucocytes (PMN) by reducing viable counts by 77 to 93% and 35 to 50%, respectively. There was a strong correlation between opsonizing activity and passive protection in immune and normal serum. Inactivation of complement significantly reduced the opsonizing activity of the immune serum. A significant portion of the protection associated with the immune serum is localized in the IgM fraction. Immune serum, depleted of IgG, enhanced phagocytosis to a degree similar to intact immune serum. However, immune serum depleted of IgM, opsonized bacteria to the same degree as normal serum. Therefore, the immune component of serum responsible for protection and opsonization appears to be localized in the IgM fraction. These data indicate that protection induced by antiribosomal antibodies results from an interaction with the cell surface of H. influenzae organisms, leading to increased phagocytosis by PMN.

Kinetics of Haemophilus influenzae type B infection in normal and ribosome-immunized mice using intraperitoneal and intracerebral routes of inoculation.

The kinetics of infection was studied in normal and ribosome-immunized mice challenged with Haemophilus influenzae Type b organisms. Ribosomal preparations extracted by the differential-centrifugation and sodium-dodecyl-sulphate treatment or ammonium-sulphate-precipitation procedures were highly immunoprotective when mice were challenged by the i.p. route. After i.p. injections, organisms rapidly spread to blood, liver, lungs and brain in normal and immunized mice. However, by 24 h after injection, evidence of organism clearance could be seen in immunized mice. By 32 h organisms were cleared from blood, brain and lungs of all immunized mice and from spleens in 2 of 3 mice. However, organisms persisted in high numbers of unimmunized mice until their death by 48 h. These data indicate that i.p. injections of H. influenzae mixed with gastric mucin leads to a true infection and can be used as a model to evaluate immunoprotective activity. The kinetics of infection induced by intracerebral (i.c.) inoculation also was studied. The LD50 for this type of infection was more than 1000 times the LD50 for i.p. infection. The patterns of infection induced by i.c. challenge were similar in normal and immunized mice and immunoprotection could not be detected using this model.

Serological and biological activities of anti-Haemophilus influenzae ribosomal serum.

The antibody content in serum from rabbits immunized with ribosomes from Haemophilus influenzae type b was determined by passive hemagglutination, enzyme-linked immunosorbent assay, and complement fixation. Attempts to use passive hemagglutination to assay anti-ribosomal antibodies were unsuccessful. In the enzyme-linked immunosorbent assay tests, rabbit antiserum was allowed to react with ribosomes that adhered to microtiter plates. The enzyme-linked immunosorbent assay method detected, in two ribosome-immunized rabbits and by 3 days postimmunization, titers which rose to plateaus on days 24 to 31 and declined thereafter. With the complement fixation method, the serum from one immunized rabbit also showed a titer on day 3 and reached a plateau on days 20 to 31. Serum from the other immunized rabbit did not develop a titer until day 11; it reached a lower plateau on days 20 to 24 and then declined on days 27 to 55. Although there were no apparent differences between the two immunized rabbits by the enzyme-linked immunosorbent assay, there were differences between the complement fixation antibodies in these rabbits. Passive protection experiments were performed with these sera. Maximal passive protection was achieved when mice were challenged intraperitoneally with 100 50% lethal doses of H. influenzae and immunized intravenously 1 h later with rabbit serum collected 27 days postimmunization. Rabbit anti-ribosomal sera were evaluated for bactericidal activity. Undiluted immune sera showed bactericidal activity; however, when diluted 1:10, activity was lost. Although bactericidal activities of immune sera were correlated to passive protection activities, it is unlikely that such protection was due to bactericidal antibodies. Immune serum had opsonizing activity since it enhanced phagocytosis of H. influenzae by mouse leukocytes.

Effects of glucose and fructose loading on glycogenesis in chicks infected with avian tuberculosis.

The chick was used as a rapid metabolic model to determine the fate of ingested fructose vs. glucose in noninfected chicks and in those subjected to the stress of avian tuberculosis. The chicks were crop-loaded with either a 72% fructose or glucose solution 21 and 28 days post TB inoculation and killed 2 and 4 hr after loading. In noninfected chicks, both sugars were rapidly converted to glycogen, and there was an interaction with time and the amount of glycogen formed from each sugar. Infection depressed glycogen formation from both fructose and glucose. While the total amount of glycogen formed from glucose could be directly correlated to increased liver size in the TB chicks loaded with glucose, in the chicks loaded with fructose less glycogen was formed even though liver size was increased as a result of the TB infection. The depression in glycogen formation was not related to the severity of the infection since the TB involvement was not the same in the two experiments conducted; but in both cases chicks loaded with fructose showed a greater reduction in the capacity of the liver to synthesize glycogen.

Role of bovine serum albumin in the nutrition of Mycobacterium tuberculosis.

Bovine serum albumin promotes the growth of small inocula of Mycobacterium tuberculosis in media containing unesterified fatty acids. Albumin binds fatty acids present in concentrations toxic for the organisms. In the present study, additional roles of albumin were investigated. When present in a basal medium, fatty acid-free albumin could be utilized by M. tuberculosis as a sole source of carbon. Since albumin could not substitute for the amino acids in basal medium as a nitrogen source, it was concluded that the protein component in albumin was not utilized as a nutrient by the organisms. An ether extract of fatty acid-free albumin supported a small but significant amount of growth. Analysis of the lipids in fatty acid-free albumin by gas chromatography revealed the presence of 686 microgram of fatty acid per g of albumin. Although a small amount of growth occurred when a lipid extract of albumin was present in the medium, growth stimulation was dependent in major part on the presence of undenatured albumin in the medium. Lipids, when bound to albumin, can serve as a nontoxic source of carbon and energy.

Characterization of the immunoprotective antigen of ribosomal preparations from Haemophilus influenzae.

This investigation was designed to characterize the immunoprotective antigen of ribosomal preparations from Haemophilus influenzae. The ribosomes that elicited 80 to 90% protection contained 25% protein and 75% ribonucleic acid but did not contain any detectable hexoses. The immunodiffusion and hemagglutination inhibition tests also failed to demonstrate that the capsular material (polyribose phosphate) was in ribosomal preparations. Treatment of ribosomes with ribonuclease degraded 78% ribonucleic acid but did not affect the immunogenicity of such preparations. The proteolytic enzymes reduced the immunogenicity of ribosomes corresponding to the amount of protein degraded. The protection elicited by ribosomal protein extracted with 2-chloroethanol was comparable to that induced by intact ribosomes. In contrast, the low levels of protection observed by immunization with phenol-extracted ribonucleic acid were dependent on the amounts of contaminating protein. Finally, immunogenicity of ribosomal ribonucleic acid and protein was abrogated by treatment with proteolytic enzymes. These results clearly indicate that the protein associated with Haemophilus ribosomes is the major immunoprotective antigen.

Adoptive transfer of immunity from mice immunized with ribosomes or live yeast cells of Histoplasma capsulatum.

This investigation was designed to compare the role of lymphoid cells and immune serum in protective immunity induced by immunization with ribosomes or live yeast cells of Histoplasma capsulatum. Spleen cells, peritoneal cells, and serum from C3H mice immunized with Histoplasma ribosomes or live cells were transferred intravenously to separate groups of syngeneic recipients. All recipients along with a set of immunized and control mice were challenged intravenously with 4 x 10(6) yeast cells of H. capsulatum, and protection was assessed. Immunization with ribosomes or live cells provided 90 to 100% protection. Mice receiving filtered spleen cells or peritoneal cells from donors immunnized with live cells showed 90 to 100% protection; 80 to 90% protection was observed for mice receiving cells from ribosome-immunized donors. In contrast, no evidence of protection was seen in mice receiving serum from either live-cell- or ribosome-immunized mice. Peritoneal cells were far more efficient than spleen cells in adoptive transfer of immunity. The adoptive immunity in recipients persisted for at least 3 weeks after transfer, the longest period tested in the present study. These results indicate that the immunity elicited by immunization with Histoplasma ribosomes or live cells is mediated by a cellular mechanism.

Immunoprotective activity of ribosomes from Haemophilus influenzae.

Immunization with ribosomal preparations from Haemophilus influenzae type b elicited protective immunity in mice. Ribosomes from disrupted cells where isolated by differential centrifugation using sodium dodecyl sulfate. The washed ribosomes contained 25% protein and 75% ribonucleic acid and sedimented as a single peak on sucrose density gradient analysis with a sedimentation coefficient of 67S, using Escherichia coli ribosomes as a 70S marker. Immunodiffusion tests with antipolyribose phosphate serum showed that the ribosomes were free from capsular material. Mice immunized subcutaneously with ribosomes, with or without adjuvant, were challenged intraperitoneally with 100 to 1,000 mean lethal doses of H. influenzae type b suspended in gastric mucin. Significant protection was induced by ribosomes and was compared to that obtained after sublethal infection with live cells. The protection was greatly enhanced after incorporation of ribosomes into adjuvants. Maximum protection (90 to 95%) was observed at 1 to 2 weeks after immunization. Ribosomes from a nonencapsulated strain of H. influenzae were as immunogenic as those from the encapsulated strain, demonstrating that the capsular material is not responsible for immunogenicity of Haemophilus ribosomes.

Ribonucleic acid synthesis in normal and immune macrophages after antigenic stimulus.

Macrophage ribonucleic acid (RNA) synthesis is an important metabolic process intimately related to the function of these cells. Mouse peritoneal macrophage RNA was extracted with phenol in the presence of bentonite and electrophoresed on composite agarose-polyacrylamide gels. The pulse-chase technique was used to follow the precursor relationships in macrophage ribosomal RNA (rRNA) maturation. The rRNA species at 18S and 28S appeared at 15 and 45 min, respectively, after RNA synthesis was halted. Their appearance corresponded closely to decreases in the rRNA precursors at 45S, 36S, and 34S. Studies of RNA methylation aided in confirming the identity of these ribosomal species. Unmethylated RNA species appeared as messenger RNA between 5S and 15S, and at about 55S probably represented heterodisperse nuclear RNA. When normal macrophages were incubated with heat-killed Salmonella enteritidis, an acceleration in the maturation of RNA was observed. The accelerated maturation was indicated by the earlier appearance of 28S rRNA and the more rapid development of an equilibrium state, where further labeling did not change the RNA profile. In macrophage RNA from mice immunized with S. enteritidis, rRNA species appeared rapidly but did not accumulate to the same extent as observed for normal macrophages. Precursor rRNA and other RNA species developed as usual, suggesting specific degradation of mature rRNA. Such rRNA wastage could indicate a mechanism controlling ribosome assembly in the non-proliferating activated macrophage. The pattern of RNA synthesis in immune macrophages was essentially unchanged by the presence of heat-killed S. enteritidis in vitro.

Comparative chemotherapeutic activity of amphotericin B and amphotericine B methy ester.

The comparative efficacy of amphotericin B and amphotericin B methyl ester (AME) against experimental histoplasmosis, blastomycosis, cryptococcosis, and candidosis in mice was assessed by determining the effect of daily intraperitoneal therapy on 21-day survival and persistence of organisms in internal organs. AME, like amphotericin B, was effective against each of the experimental infections, but the efficacy was lower than the parent compound. For Histoplasma and Blastomyces infections the mean effective dose (ED(50)) of amphotericin B was 0.3 mg/kg, whereas the corresponding values for AME, respectively, were 2.4 and 2.8 mg/kg. For Cryptococcus infection the ED(50) for amphotericin B was 0.2 mg/kg compared with 2.0 mg/kg for AME. The ED(50) of amphotericin B for Candida infection was lower than 0.05 mg/kg and the value of AME was between 0.5 to 0.05 mg/kg. The colony counts from internal organs of the surviving animals after the therapeutic regimens were compatible with the data on survival.

Comparative in vitro antifungal activity of amphotericin B and amphotericin B methyl ester.

The in vitro antifungal activity of amphotericin B methyl ester (AME), a water-soluble derivative of amphotericin B, was compared to that of the parent compound against a variety of pathogenic and potentially pathogenic fungi. AME has a significant antifungal activity, but the activity of AME was slightly lower than that of amphotericin B. Among the yeast-like organisms, only the yeast cells of Sporothrix schenckii were more resistant than others to both antibiotics, with a minimal fungicidal concentration of 5 to 10 mug/ml. The yeast cells of other fungi were killed at concentrations of 1 mug or less of either antibiotic per ml. The filamentous forms of S. schenckii and Oidiodendron kalrai were more resistant than the filamentous forms of other dimorphic fungi to both drugs. The minimal fungicidal concentration for S. schenckii was 10 mug/ml and for O. kalrai, 50 mug/ml. The dermatophytes, phycomycetes, and dematacious and other potentially pathogenic fungi were inhibited fairly well by both drugs, but up to 50 mug/ml was required for fungicidal action. The water solubility and wide spectrum of antifungal activity of AME warrant evaluation of its chemotherapeutic activity against experimental fungal infections.

Biological activity of heated diphtheria toxin.

Diphtheria toxin splits into two fragments when heated at 100 C for 10 min in a phosphate buffer. The separated fragments have molecular weights of 24,000 and 39,000, respectively. These molecular weights are similar to those of the A and B fragments found in diphtheria toxin preparations after thiol reduction. Since the separation of toxin into fragments is not complete, it is likely that only nicked toxin molecules having a cleaved peptide bond are split by heating. When toxin is suspended in phosphate buffer at pH 6.4, the B-like fragment precipitates, but at pH 7.8 it does not. Heated toxin is unable to intoxicate sensitive cells or cause a necrodermal response in animals. Fragment A produced by heating is active in inhibiting cell-free protein synthesis. It is able to intoxicate both HeLa and L cells when the uptake of the fragment is facilitated by addition of polyornithine to the cultures. Fragment B produced by heating is involved with binding to the cell surface. It is able to delay the action of toxin on KB cell cultures preincubated with fragment B.

Quantitation of the bioenergetics of a tuberculosis infection in chicks.

Interaction of an avian tuberculosis infection with a known metabolizable energy yield of dietary corn oil in chicks was used to quantitate total host energy expenditure necessitated by the infectious process. Three trials in which two doses of inoculum were used resulted in mild and severe involvements. Trial 1 (mild) indicated that 6% and trials 2 and 3 (severe) that 96 and 93% of the energy supplied by known quantities of corn oil were utilized by the tuberculosis process. In the birds given the low level of inoculum, the degree of tuberculosis involvement, as measured by increased liver size, was correlated with increased total quantities of hepatic ribonucleic acid, monoglycerides, free fatty acids, free cholesterol, and glucose. All of these effects were observed prior to manifestations of clinical symptoms or failure of the chicks to consume all food offered.

Site in cell-free protein synthesis sensitive to diphtheria toxin.

The effects of diphtheria toxin on cell-free protein synthesis in a bacterial system, and preparations obtained from animals that were sensitive and resistant to toxin were examined. In the presence of nicotinamide adenine dinucleotide (NAD), toxin inhibited the incorporation of amino acids by endogenous and synthetic polynucleotides in both rat liver and guinea pig liver cell-free systems that were exposed to 6 Lf units per ml of toxin. A cell-free system derived from Streptococcus faecalis was resistant to high concentrations of toxin. Dialyzed toxin-antitoxin floccules that are formed in the presence of NAD and the 105,000 x g supernatant fluid from rat liver contain NAD. Such floccules are also active in protein synthesis in the absence of added transferase I or II. An operational model presents the view that the intoxication complex is formed at the ribosomal level and occurs in two steps. First, the toxin molecule binds to transferase II and alters its stereospecific relationship to transferase I, but it does not result in an inactive complex. Second, the stereospecific alteration in transferase I, but it does not result in an inactive complex. Second, the stereospecific alteration in transferase II caused by the binding of diphtheria toxin allows NAD to bridge between transferase I and II, which then results in an inactivated complex. The sensitivity of the cell-free system derived from the normally resistant rat implies that in some cells the cell membrane serves as a permeability barrier to the toxin molecule. The resistance of bacterial cell-free protein synthesizing systems to diphtheria toxin may reflect basic differences between transferase enzymes from bacterial and mammalian sources.