Cellular fatty acid composition of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus.

Strains of Actinobacillus actinomycetemcomitans isolated from deep pockets of patients with juvenile periodontitis were analyzed for their content of cellular fatty acids. Oral Haemophilus strains, morphologically and biochemically similar to Haemophilus aphrophilus, were also examined for their content of cellular fatty acids. The extractable lipids of the actinobacilli represented approximately 10% of the cell dry weight, with the bound lipids representing 2 to 5%. The major fatty acids consisted of myristic (C14:0) and palmitic (C16:0) acids and a C16:1 acid, possibly palmitoleic acid, accounting for 21, 35, and 31% of the total extractable fatty acids, respectively. Haemophilus strains had a similar cellular fatty acid content.     

Streptococcus mutans Genes That Code for Extracellular Proteins in Escherichia coli K-12

Chromosomal DNA from Streptococcus mutans 6715 (serotype g) was cloned into Escherichia coli K-12 by using the cosmid pJC74 cloning vector and a bacteriophage λ in vitro packaging system. Rabbit antiserum against S. mutans extracellular proteins was used for immunological screening of the clone bank. Twenty-one clones produced weak to strong precipitin bands around the colonies, but only after the λ c1857 prophage was induced by being heated to lyse the E. coli cells. None of the clones expressed enzyme activity for several known S. mutans extracellular enzymes. One of these clones contained a 45-kilobase recombinant plasmid designated pYA721. An 8.5-kilobase fragment of S. mutans DNA from pYA721 was isolated and recloned into the BamHI restriction site of the plasmid vector pACYC184 to construct pYA726. pYA726 contained all, or nearly all, of the gene for a surface protein antigen (the spaA protein) of S. mutans 6715. This was deduced from immunological studies in which extracts of cells harboring pYA726 reacted with antisera against both purified 6715 spaA protein (about 210,000 daltons) and the immunologically similar antigen I/II of serotype c strains of S. mutans. In addition, the S. mutans spaA protein was found to possess at least one antigenic determinant not present on the protein specified by pYA726. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of E. coli clone extracts revealed that pYA726 produced a polypeptide with a molecular mass of about 180,000 daltons which was predominantly found in the periplasmic space of E. coli cells. Antisera to the spaA protein of S. mutans reacted with extracellular protein from representative strains of S. mutans serotypes a, c, d, e, f, and g, but not b.     

Immune complexes in early arthritis. L Detection of immune complexes before rheumatoid arthritis is definite

Fifty-three patients with early arthritis were studied longitudinally for up to 3 years. During this time, 24 developed sufficient features for definite rheumatoid arthritis (RA) to be diagnosed. The other (arthralgia patients) differed from the RA patients as, in the majority, C-reactive protein and ESR were normal and anti-nuclear antibodies or rheumatoid factors were rarely found. Moreover, in time their signs and symptoms improved or disappeared. Circulating immune complexes were detected in both groups of patients by the platelet aggregation test whereas complexes detected by abnormal Clq-binding activity were found mainly in the RA patients. Platelet-aggregating complexes were usually present in the first samples studied and disappeared in the arthralgia patients with recovery from their symptoms. In the RA patients, Clq-binding complexes appeared simultaneously or later than platelet-aggregating complexes but both tests were positive several months before RA could be diagnosed. These results suggest that immune complexes are one of the first immunological abnormalities to appear in patients with arthritis. Although the constituent antigen and antibody of complexes detected by either test are unknown, their possible nature is discussed.     

Identification of antibody epitopes in the CB-11 peptide of bovine type II collagen recognized by sera from arthritis-susceptible and -resistant rhesus monkeys.

Sera from eight rhesus monkeys that had been immunized with native bovine type II collagen were tested for antibodies to cyanogen bromide peptides (CB peptides) of type II collagen by Western blotting. The monkeys produced IgG antibodies to a number of different CB peptides, with five out of eight animals producing antibodies to the CB-11 peptide (four arthritic, one non-arthritic). Antibody epitopes on the CB-11 peptide of bovine type II collagen recognized by these sera were investigated by epitope mapping. Peptides (8-mers overlapping by seven amino acids) representing the CB-11 region were synthesised and the sera screened for binding to these peptides to determine areas of high IgG antibody binding to this region of type II collagen. The profiles obtained were not identical, though there were some epitopes that were commonly recognized. Antibodies to one epitope, also present in human type II collagen, were found only in the sera of two animals with the severest arthritis. The technique of epitope mapping has successfully identified a number of epitopes within the CB-11 peptide of type II collagen recognized by antibodies from bovine type II collagen-immunized monkeys. Studies on the relevance of responses to the identified epitopes can now be undertaken.     

Isolation and characterization of the outer membrane and lipopolysaccharide from Eikenella corrodens

The chemical composition of the outer membrane fractions (OMFs) of Eikenella corrodens strains 23834 and 470 as well as the strain 23834 lipopolysaccharide (LPS) was determined. The OMFs were obtained by Triton X-100 treatment of the heavier membrane fraction from sucrose density centrifugation of the total membrane fraction. The resulting OMFs of strains 23834 and 470, free of cytoplasmic membrane components, were found to contain 69.6 and 75.0% (wt/wt) protein, 4.8 and 9.2% lipid, 4.6 and 4.7% carbohydrate, and 2.0 and 4.6% muramic acid, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis both OMFs contained one major peptide determined to be 33,500 daltons for the strain 23834 OMF, and 37,500 daltons for the strain 470 OMF. Analysis of the OMF fatty acids revealed hexadecanoic, hexadecenoic, octadecenoic, and lesser amounts of octadecanoic acids. Transmission electron microscopic examination of the OMFs revealed typical large sheets of membrane. Structures (10 nm in diameter) resembling pores were also evident. The E. corrodens LPS was found to be composed of 34.5% (wt/wt) carbohydrate and 25.0% lipid A. Only minute amounts of 2-keto-3-deoxyoctonate and heptose could be detected. Fatty acid analysis revealed primarily octadecanoic and hexadecanoic acids, with lesser amounts of octadecenoic acid. No hydroxy fatty acids were detected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed the E. corrodens LPS to resemble other smooth-type LPSs. Transmission electron microscopic examination revealed a vesicle-like morphology. The E. corrodens LPS appears not to be a "classical," i.e., enteric, type of LPS.     

Cigarette smoke and phagocyte function: effect of chronic exposure in vivo and acute exposure in vitro.

Phagocytic function was studied in mice chronically exposed to cigarette smoke, and the effects of in vitro exposure to cigarette smoke on macrophage activity were also assessed. Cultures of radiolabeled Pseudomonas aeruginosa were employed to investigate phagocyte activity in vivo and in vitro. Mice were exposed on weekdays to fresh cigarette smoke for periods up to 37 weeks and the bactericidal and clearance activity of their lungs was measured. Both pulmonary clearance and bactericidal activity was impaired. The clearance of intravenously injected bacteria from the blood of smoke-exposed mice occurred at the same rate as in control mice, but the accumulation of radiolabel by the liver was decreased. In addition, the rate of elimination of radiolabel from the liver was less than the controls. Macrophages exposed to cigarette smoke in vitro initially had a depressed phagocytic rate, but if phagocytosis over a prolonged period was measured it was eventually enhanced over the rate of control macrophages. The vapor phase of cigarette smoke could also transiently inhibit and then enhance the phagocytic activity.     

Migration of human lymphocytes. I. A model using the mouse as host.

The distribution of radioactivity after the intravenous injection of 51Cr-labelled human lymphocytes has been examined in normal mice, irradiated mice, mice treated with anti-platelet antiserum and in mice treated with colloidal carbon. Pre-treatment with carbon and anti-platelet antiserum appears to protect the human lymphocytes from uptake by the host's reticuloendothelial system (RES). Comparison of tissue radioactivity in carbon-treated mice after the injection of viable human lymphocytes with that found after the injection of dead cells and soluble or insoluble cell debris showed that radioactivity recovered in the spleen and lymph nodes is primarily due to the migration of viable lymphocytes into these tissues. Thus the measurement of radioactivity in lymph nodes of carbon-treated mice after the injection of 51Cr-labelled human lymphocytes can be used as a model of these lymphocytes' ability to migrate into the lymph nodes during recirculation and to study factors influencing this migration.     

Drug development strategies for asthma: in search of a new paradigm

The spiraling costs of asthma treatment seem set to continue rising, given the equivocal performance of the latest generation of specific anti-inflammatory drugs in trials in adult asthmatics. We argue that the continuation of this trend is inevitable unless there is a substantial realignment of entrenched drug development policy in the pharmaceutical industry and a parallel shift in licensing policy by regulatory authorities to encourage the development of drugs capable of halting the progression from acute to chronic asthma when the disease first manifests in childhood. The theoretical framework for such an approach, including proof-of-principle data from studies in children with early-stage disease and a range of candidate drugs, already exists. What is needed is informed debate on the risks versus potential benefits of this approach.