Chlamydia pneumoniae infection induces inflammatory changes in the aortas of rabbits.

Chlamydia pneumoniae, a common human respiratory pathogen, has been associated with atherosclerosis in several seroepidemiological studies. Moreover, its presence in lesions of vessel walls has been demonstrated by culture, immunohistochemistry, PCR, and electron microscopy. In this study, we infected intranasally with C. pneumoniae New Zealand White rabbits which had been fed a normal diet. Reinfection was given 3 weeks later. Six of the nine reinfected animals showed inflammatory changes consisting of intimal thickening or fibroid plaques resembling atherosclerosis in 2 to 4 weeks after reinfection. One rabbit had calcified lesions. Immunohistochemistry for C. pneumoniae was strongly positive in the three older affected animals. No lesions were seen in the controls. The results suggest that C. pneumoniae infection is capable of inducing inflammatory atherosclerosis-like changes in the aortas of infected rabbits.

Biological characterization of Campylobacter fetus lipopolysaccharides.

Lipopolysaccharides (LPS) of three strains of Campylobacter fetus (subspp. fetus and venerealis, and serotypes A and B), a bacterium of veterinary importance but also a cause of various infections in humans, were assessed for their ability to induce mitogenicity, gelation of Limulus amebocyte lysate, lethal toxicity in mice, and pyrogenicity in rabbits. All C. fetus LPS exhibited activities lower than those of Salmonella typhimurium LPS. LPS of C. fetus subsp. fetus serotype A had the lowest activity in all assays. Since the majority of C. fetus subsp. fetus isolates from humans are serotype A, the lower biological activities of this LPS may aid the pathogenesis of such strains. The lower activities of C. fetus LPS compared with those of S. typhimurium LPS may reflect the presence of longer fatty acid chains in the lipid A of C. fetus LPS, whereas interstrain differences in C. fetus LPS bioactivities may be related to some property influenced by composition of the saccharide moiety.

Chlorhexidine susceptibilities of mutans streptococcal serotypes and ribotypes.

The susceptibilities of 379 clinical mutans streptococcal isolates to chlorhexidine (CHX) were tested by agar dilution according to the standards of the National Committee for Clinical Laboratory Standards. Isolates were obtained from saliva samples of 34 young mothers who had high or moderate salivary levels of mutans streptococci at baseline. Samples were collected on three occasions, before childbirth, when each child was 6 months old, and 1 year later. Of these isolates, 50% were inhibited at 1 microgram of CHX per ml, 90% were inhibited at 2.0 micrograms/ml, and all were inhibited at 4.0 micrograms/ml. The MICs for Streptococcus mutans isolates (serotypes c, e, and f) were lower than those for Streptococcus sobrinus isolates (serotypes d and g). In some subjects, the MICs for isolates of the same serotype were different. This phenomenon was studied by ribotyping isolates (n = 45) from selected subjects (n = 7). It was found that if there were intraindividual differences in the MICs for isolates of the same serotype, then the ribotypes of these isolates were different. In order to decrease the mutans streptococcal infection risk for children, 24 mothers (test group) brushed their teeth periodically with a gel that contained 0.3% CHX digluconate and 0.2% NaF, pH 5.8, between the second and third sampling occasions. The gel was used twice a day for the first 10 days of each month. Development of resistant strains during CHX-NaF gel use was not detected. The serotype distribution of isolates from the test group after 1 year of periodic CHX-NaF gel use did not differ from that at baseline. Periodic CHX-NaF gel brushing did not lead to lower salivary mutans streptococcal counts.

Biological activities of lipoteichoic acid and peptidoglycan-teichoic acid of Bacillus subtilis 168 (Marburg).

To evaluate the suitability of Bacillus subtilis as a production host of heterologous proteins for pharmaceutical purposes, we assessed the biological activity of this bacterium and its major cell envelope components, lipoteichoic acid (LTA) and peptidoglycan-teichoic acid complex (PG-TA) in several eukaryotic effector assays. LTA and PG-TA were found to be non-toxic for mice and guinea-pigs in a short-term toxicity assay. PG-TA was weakly pyrogenic and weakly mitogenic. Both LTA and PG-TA acted as immunologic adjuvants in mice and when injected in mice, also caused an increase in the number of granulocyte-monocyte colony-forming cells in the bone marrow probably via stimulation of production of granulocyte-macrophage colony-stimulating factor.

Effect of carbohydrates on the pharmacokinetics of human interferon-gamma.

Human interferon-gamma (IFN-gamma) has two N-linked glycosylation sites at positions 25 and 97 of the 143-amino-acid-long secretory form. To study the role of glycan residues in the pharmacokinetics of IFN-gamma, we produced recombinant IFN-gamma molecules lacking either one or both of the glycosylation sites (Asn mutated to Gln) by baculovirus expression in insect cells. In addition, we produced the fully glycosylated forms both in insect cells and in human leukocyte cultures. Two million units of each IFN were injected intravenously or intramuscularly into rabbits. The glycosylated IFN-gamma molecules from the insect cells were rapidly eliminated from the blood. This is probably due to the fact that their oligosaccharides are of a high mannose type that are rapidly taken up by the liver. The unglycosylated IFN-gamma persisted longer in the blood than the glycosylated recombinant forms. However, the natural IFN-gamma exhibited the longest survival in the blood. The results emphasize the importance of the carbohydrate groups in human IFN-gamma to its pharmacokinetic properties.

Frequency and stability of mono- or poly-infection by Actinobacillus actinomycetemcomitans serotypes a, b, c, d or e.

The aims of the study were to determine the prevalence of simultaneously multiple Actinobacillus actinomycetemcomitans serotypes in one individual, stability of infection by the same serotype and the occurrence of previously not described serotypes of A. actinomycetemcomitans. The serotypes of 515 clinical isolates of A. actinomycetemcomitans from 91 Finnish, Caucasian subjects, including 321 follow-up samples from 51 subjects, were determined with immunodiffusion assay. Most subjects (n = 86, 95%) were infected with one serotype only; 466 (91%) isolates from 80 subjects belonged to serotype a (25% of isolates/25 subjects), b (25% of isolates/27 subjects) or c (41% of isolates/30 subjects). Fifteen isolates from 4 subjects reacted with the antiserum raised against previously untypable clinical strain IDH 781 (serotype d) and 18 isolates from 5 subjects with the antiserum raised against strain IDH 1705 or IDH 3096 (serotype e). Sixteen (3%) isolates from 5 subjects remained untypable. The same infecting A. actinomycetemcomitans serotype(s) persisted for the 1-6 years of follow-up. In conclusion, the study indicates a rare simultaneous occurrence of multiple oral A. actinomycetemcomitans serotypes, the stability of infection by the same serotype(s) and the existence of serotypes of A. actinomycetemcomitans not previously described.

Low biological activity of Helicobacter pylori lipopolysaccharide.

Lipopolysaccharide from the gastroduodenal pathogen Helicobacter pylori was tested for its ability to induce mitogenicity in mouse spleen cells, pyrogenicity in rabbits, and toxic lethality in galactosamine-sensitized mice. Compared with those for enterobacterial lipopolysaccharide, mitogenicity and pyrogenicity were a thousand-fold lower and lethal toxicity was 500-fold lower. We suggest that the phosphorylation pattern and acylation in lipid A are responsible for the low biological activity.

Evaluation of anaesthetic potency of medetomidine-ketamine combination in rats, guinea-pigs and rabbits.

In many animal species ketamine hydrochloride is combined with xylazine, an alpha 2-adrenoceptor agonist, to achieve anaesthesia. The purpose of this study was to evaluate the anaesthetic efficacy of various dose combinations of another alpha-2-adrenoceptor agonist medetomidine and ketamine in rats, guinea pigs and rabbits. Both sexes of all three species in groups of five were used. In rats, dose combinations medetomidine/ketamine, both in mg/kg, used were 0.25/60, 1.0/60 and 0.5/70; in guinea pigs 0.5/40 and in rabbits 0.5/25 and 0.5/60. In rats and rabbits the righting reflex was lost within 2-3 min. In rats with doses 0.25/60 and 0.5/75 other reflexes disappeared, reappearing after 20-25 min and 60-70 min in males, the respective values being 120-140 min and 150-180 in females. The righting reflex reappeared after 130-135 and 160-190 min in males and 240 and greater than 300 min in females in the order above. In guinea pigs irrespective of route of ketamine administration all reflexes remained. In rabbits, a dose 0.5/25 caused disappearance of ear pinch reflex. We conclude that the medetomidine-ketamine combination can be used in rats and rabbits as an anaesthetic, and in guinea pigs only for immobilization.

Host cell-mediated selection of influenza A (H3N2) virus variant subpopulations: lack of association between antigenic and receptor-binding properties.

During the outbreak of influenza due to A (H3N3) viruses in Finland in 1985/6 virus pairs were isolated from the same clinical specimens in embryonated hens' eggs (CE) and in canine kidney cell cultures (MDCK). Some of these isolates, the E and M pairs, were distinguished by their reactions in haemagglutination inhibition (HI) tests carried out using polyclonal antisera, and by receptor-binding properties, as evidenced by differences in their elution activity from erythrocytes. Passage of the E- and M-virus isolates in the foreign host affected their serological characteristics, but the E virus did not convert to an M-like virus and the M virus did not convert to an E-like virus. Returning the viruses to grow in the host used for their isolation changed the serological reactions so that they were once more close to the reactions of the original isolates. This contrasts with the changes in receptor-binding properties. Rapid elution from hen erythrocytes, which has been described as a property of viruses binding to the SA alpha 2,3Gal sequence in preference to SA alpha 2,6Gal, characterized the virus passages grown solely in MDCK cell cultures. Cultivation of the M virus in CE, at any stage of its passage history, made the virus irreversibly incapable of elution. The M virus was more sensitive than the E virus to HI antibodies against heterologous viruses of the H3N2 subtype, and, when used as an antigen in HI serology, it more frequently (90% vs. 69%; P less than 0.01) detected diagnostic antibody responses in patients infected with viruses of this subtype in 1985/6. Use of antigens with a different passage history in HI serology provided evidence that this superiority, which may be due to the ability of the virus to pick out anamnestic antibody responses, is unrelated to the receptor-binding peculiarity of the M virus under consideration. The results support the concept that the host cell can select a diversity of virus variant subpopulations from a single clinical specimen during isolation and subsequent cultivation procedures. Moreover, the MDCK-grown influenza viruses may correspond better than the egg-grown isolates to the natural epidemic viruses.

Lipopolysaccharide-induced non-specific resistance to systemic Escherichia coli infection in mice.

A high degree of non-specific resistance to a lethal systemic Escherichia coli infection was induced in mice by pretreatment with a small dose (less than 5 micrograms/mouse) of the homologous lipopolysaccharide (LPS) or with heterologous rough-type LPS from E. coli K-12. The route of LPS administration, intraperitoneally or subcutaneously, did not influence the development of resistance, suggesting that a systemic cell activation was responsible for the effect. The enhanced elimination of bacteria was similar to that in mice recovering from a sublethal E. coli infection. In the LPS-treated mice, elimination of the challenge bacteria from the peritoneal cavity and the blood started 3-4 h after challenge whereas, in controls, the bacterial numbers continued to increase until the mice died. The detoxified LPS derivative, monophosphoryl lipid A (MPL), also increased the survival of mice infected with E. coli O18:K1. However, the dose of MPL required for optimal infection resistance was 100-fold greater than that of native, E. coli K-12 LPS, corresponding to the 100-fold reduced toxicity of MPL for mice and rabbits in lethality and pyrogenicity assays.

Egg-grown and tissue-culture-grown variants of influenza A (H3N2) virus with special attention to their use as antigens in seroepidemiology.

A field strain of influenza A (H3N2) virus isolated in embryonated eggs during the 1984-5 influenza outbreak (A/Finland/13/85E) was compared in an antigenic analysis with virus from the same clinical specimen isolated in MDCK cell cultures (A/Finland/13/85M). The M-virus appeared to be more sensitive to haemagglutination-inhibiting antibodies against heterologous viruses than did the E-virus. The results of propagation and plaque purification experiments support the hypothesis that a single clinical specimen may consist of distinct antigenic variant subpopulations promoted selectively by the host during isolation procedures. Receptor-binding properties are discussed as a possible explanation for this selectivity. A set of 471 paired sera consisting of pre-epidemic and post-epidemic specimens taken from the same subjects in 1984-5 was studied for haemagglutination-inhibiting antibodies to six influenza A (H3N2) virus strains, including the E-virus and the M-virus from A/Finland/13/85. Of the antigens used, the M-virus detected significant antibody increases more frequently than did the E-virus (10.0 v. 5.9%). The superiority of the M-virus may rest primarily in its ability to pick out anamnestic antibody responses. Irrespective of this cross-reactivity, pre-epidemic antibody to the M-virus was fairly well associated with protection. In the set of sera (230 specimens) collected in summer 1985 to represent different age groups, the antibody status against the M-virus was significantly better than the status against the E-virus. The results suggest that, at least in some instances, antibody to MDCK-grown virus is a more accurate indicator of the immune status of a community than antibodies to egg-grown virus variants.

Antigenic analysis of intraepidemic variants of influenza A (H3N2) viruses by hyperimmune rat antisera.

Hyperimmune rat antisera prepared against 5 recent antigenic variants of influenza A (H3N2) viruses were studied for haemagglutination inhibiting (HI) antibodies to the homologous and the heterologous viruses. The ratios of homologous to heterologous reactions varied from one animal to another in immunizations with each of the immunogens. Some antisera exhibited a ratio high enough to allow differentiation of the epidemic variants and demonstration of an intraepidemic heterogeneity of field strains isolated during the outbreak of 1985/86. The variation of cross-reactions of polyclonal antisera may reflect differences in the range of specificities of anti-haemagglutinin antibodies produced by individual animals. The significance of this finding in the classification of influenza A (H3N2) viruses is discussed. Lack of nonspecific inhibitors interfering with the HI test is an additional advantage of hyperimmune rat antisera in typing influenza A and B virus isolates.

Effect of bacterial lipopolysaccharide on serum lipids and on the development of aortic atherosclerosis in rabbits.

The effect of repeated intravenous administration of bacterial lipopolysaccharide (LPS) on serum lipids and on aortic atherosclerosis was studied in rabbits on basal diet and on hypercholesterolemic diets containing 0.15-1.0% cholesterol. LPS (10 or 100 ng/kg body weight) was administered 3 times per week for 3 or 6 weeks. No difference was observed in serum lipid levels or in aortic atherosclerosis between LPS- and saline-treated animals. These observations do not support the hypothesis that LPS has an effect on the progression of atherosclerosis.

Intraepidemic heterogeneity of influenza A (H3N2) viruses in 1985: antigenic analysis and sensitivity to non-specific inhibitors.

During the influenza outbreak of 1984-85 22 strains of H3N2 viruses were isolated in Finland. An intra-epidemic heterogeneity was demonstrated in an antigenic analysis by haemagglutination inhibition test with antisera produced in rats. The strains could be classified into three groups which corresponded to the following reference strains: group I: A/Hong Kong/1/84, A/Hong Kong/3/84; group II: A/Philippines/2/82; group III: A/Caen/1/84. Seven of the isolates were entirely insensitive to gamma-inhibitors of guinea-pig sera, which is in contrast to the small number of these viruses found among H3N2 strains isolated in the 1970s. The insensitive strains could not be isolated until the second or third passage through the eggs, whereas about half of the sensitive and intermediate strains were already isolated during the first passage. Conversions in reactivity with gamma-inhibitors could be detected only from an intermediate or an insensitive virus to a sensitive virus when several strains were passed serially in ovo and in MDCK cultures. The findings suggest that the gamma-inhibitor-insensitive strains corresponded well to the viruses of the human host or arose from dimorphic virus populations under an arbitrary selection of terminal dilution conditions prevailing during isolation in eggs. The insensitive strains did not differ substantially from the sensitive viruses in their ability to agglutinate erythrocytes of different laboratory animals or in their disagglutination patterns. On the other hand, propagation of viruses in MDCK cultures had an effect on these properties. The results are discussed with respect to Q phase variants and receptor binding properties.

Effect of bacterial lipopolysaccharide on serum high density lipoprotein cholesterol in rabbits.

The effect of bacterial lipopolysaccharide (LPS) on serum lipids was examined in rabbits. LPS was prepared from the smooth Salmonella typhimurium LT2 strain and given intravenously at a dose of 100 ng/kg b.wt. There were no significant changes in serum triglyceride or cholesterol levels in 1-3 days after the administration of LPS. There was, however, a decrease in serum high density lipoprotein (HDL) cholesterol, which was greatest after 2 days (P less than 0.001). Simultaneously, the HDL/total cholesterol ratio decreased (P less than 0.005).

Circulating interferon in rabbits and monkeys after administration of human gamma interferon by different routes.

Rabbits and rhesus monkeys were injected with 3 X 10(5) units of human gamma interferon (IFN) prepared in human leukocyte suspensions. Circulating IFN was detected up to 4 h after intravenous administration. Intramuscular injection maintained a relatively stable serum IFN level of about 50 units/ml for 7 to 9 h. The results in both species were similar. Little or no circulating IFN was detected after subcutaneous injection of 3 X 10(5) units, but 1.5 X 10(6) units maintained about 50 units per ml of serum for 30 h. Pharmacokinetically, human gamma IFN resembled human alpha interferons rather than human beta IFN.

Toxicity studies with human leukocyte interferon in newborn rabbits.

Daily subcutaneous injections of 5 to 10 million units of partially purified human leukocyte interferon were given to newborn rabbits for 2 weeks or 1 month. The control groups received mock interferon, saline or nothing. The interferon treatment had no overt effect on the development of the animals during the period of treatment. The rabbits treated with interferon had leukocytosis, splenomegaly and prolonged postnatal extramedullary hematopoiesis in the liver and spleen. Certain immune responses were also demonstrated in the rabbits treated with interferon and mock interferon preparations. Platelet counts and the serum-ASAT, -ALAT, -LD and alkaline phosphatase values were normal.

Pharmacokinetics of human leukocyte interferon.

Most of the human leukocyte interferon or rabbit serum interferon administered intravenously to rabbits was detectabble in their plasma 1 min after injection. An increase in the dose did not affect the early clearance of human interferon but did prolong its persistence in the serum. Repeated intramuscular injections had no effect on the kinetics of circulating interferon. The purity of the preparation influenced the rate at which interferon entered the blood after intramuscular or subcutaneous injection but did not affect clearance from the blood after intravenous injection. The circulating interferons obtained after intravenous or intramuscular administration cleared at similar rates. During the first 1-2 hr after intravenous or intramuscular injection into rats, the interferon levels were lower in lymph than in serum; after 2 hr lymph and serum samples contained similar amounts of interferon.