The metabolic fate of 14C-labeled immunoadjuvant peptidoglycan monomer. II. In vitro studies

Peptidoglycan monomer (GlcNAc-MurNAc-L-Ala-D-isoglutamine-meso-diaminopimelic acid-D-Ala-D-Ala), labeled with 14C both in the disaccharide and pentapeptide portions, was incubated with slices of mouse liver, kidney or spleen as well as with mouse and human blood, blood cells plasma and serum. Peptidoglycan monomer was isolated unchanged after incubations with mouse organs and blood cells. However, upon incubation with mouse or human blood, 10-50% of the peptidoglycan monomer underwent hydrolysis to the corresponding disaccharide and pentapeptide. After incubations with plasma and serum more than 90% of the [14C]peptidoglycan monomer was metabolized: about 50% of the administered radioactive dose was recovered in the disaccharide unit and about 35% in the pentapeptide part. These results suggest that in blood, plasma and serum of mouse and man, an N-acetylmuramoyl-L-alanine amidase (mucopeptide amidohydrolase, EC 3.5.1.28) exists which splits the amide bond between the lactyl carboxyl group of the muramyl residue and the amino group of the peptide moiety in the peptidoglycan molecule.     

The metabolic fate of 14C-labeled immunoadjuvant peptidoglycan monomer: II. In vitro studies

Peptidoglycan monomer (GlcNAc-MurNac-L-Ala-D-isoglutamine-meso-diaminopimelic acid-D-Ala-D-Ala), labeled with ¹⁴C both in the disaccharide and pentapeptide portions, was incubated with slices of mouse liver, kidney or spleen as well as with mouse and human blood cells, plasma and serum. Peptidoglycan monomer was isolated unchanged after incubations with mouse organs and blood cells. However, upon incubation with mouse or human blood, 10–50% of the peptidoglycan monomer underwent hydrolysis to the corresponding disaccharide and pentapeptide. After incubations with plasma and serum more than 90% of the [^14C]peptidoglycan monomer was metabolized: about 50% of the administered radioactive dose was recovered in the disaccharide unit and about 35% in the pentapeptide part. These results suggest that in blood, plasma and serum of mouse and man, an $\text{N-}\text{acetylmuramoyl-}\text{L}\text{-alanine}$ amidase (mucopeptide amidohydrolase, EC 3.5.1.28) exists which splits the amide bond between the lactyl carboxyl group of the muramyl residue and the amino group of the peptide moiety in the peptidoglycan molecule.     

In vitro phagocytosis by Limulus blood cells.

Limulus blood cells maintained in culture are able to phagocytose particles under conditions where bacterial endotoxin is absent. In the presence of endotoxin, phagocytosis is inhibited because the cells are immobile under these conditions and because the extracellular gel found in the presence of endotoxin prevents cell-particle contact. It is suggested that Limulus blood cells respond to Gram-negative organisms by the formation of an extracellular gel matrix that entraps the bacteria and handles other types of foreign particles by phagocytosis.     

The birth of immunology. III. The fate of the phagocytosis theory.

During the period of 1895-1910, immunology was preoccupied with defining the cellular (Elie Metchnikoff's phagocytosis theory) as opposed to the humoral basis of bactericidal defense. Although initial discovery of immunopathologic phenomena had been made (e.g., relating to transplantation, autoimmunity, allergy), focus on microbicidal therapy and diagnosis of infectious diseases remained the major stimuli of inquiry. The debate concerning the relative roles of phagocytes, complement, amboceptors (sensibilizing factors, antibody, antitoxins), various lysins (e.g., bacteriolysins, spermatolysins, hemolysins), agglutinins, stimulines, and then Almoth Wright's opsonins reflects the ambiguity of a scientific language being created in an era still struggling with a poorly defined experimental system, for the language, both its vocabulary (newly studied phenomena) and grammar (operational mechanisms) was yet to be codified. The joint award of the Nobel Prize to Metchnikoff and Paul Ehrlich in 1908 for their respective contributions to the "theory of immunity" appeared to proclaim a consensus, but the secret Nobel Committee reports that evaluated Metchnikoff's contributions reveal only a grudging acceptance of his position, and the award was clearly made on the basis of an apparent complementarity between the theoretical views of the humoralists and those elements of the phagocytosis theory that fit the then current discussion of immunity. In this regard, opsonins played an especially important role as both an experimental and conceptual bridge between the competing schools. What was no longer under consideration (and in fact never was explicitly debated) concerned the intellectual foundation of Metchnikoff's original concept of immunity as those activities that defined organismal identity, (developed from Metchnikoff's research in developmental biology) and which regarded host defense mechanisms as only subordinate to this primary function. Immunology in the first half of the 20th century pursued issues pertinent to chemically characterizing immune specificity and only later returned to the Metchnikovian question of how the immune identity was established. This latter venture has achieved molecular sophistication, but even such a formulation may be an inadequate answer to the Metchnikovian postulate. The theoretical discussion between cellularists and humoralists continues in new guises, for the essential debate remains unresolved.     

In vitro modulation of fish phagocytic cells by beta-endorphin

The activation of rainbow trout, Oncorhynchus mykiss, and carp, Cyprinus carpio, phagocytic cells by synthetic chum salmon, O. keta, beta-endorphin was analysed in vitro. Rainbow trout head kidney leukocytes were cultured in RPMI 1640 medium containing 1, 10, 50 or 100 ng ml-1 of chum salmon beta-endorphin and the production of superoxide anion was measured via the reduction of nitroblue tetrazolium (NBT) in vitro. Macrophages incubated with 10 ng ml-1 up to 100 ng ml-1 of beta-endorphin showed an increase in their production of superoxide anion in comparison with control macrophages which were cultured without hormone. beta-endorphin also increased the production of superoxide anion in phagocytic cells prepared from kidney of carp. This stimulation was inhibited by naloxone. Phagocytic cells treated with beta-endorphin also displayed increased phagocytic activity and phagocytic index. These results showed that beta-endorphin in lower vertebrates activates the function of phagocytic cells in vitro.     

Resistance to phagocytosis by group A streptococci: failure of deposited complement opsonins to interact with cellular receptors

The previous finding that phagocytosis-resistant M+ group A streptococci bear quantities of C3 which are sufficient for phagocytosis of their M- derivatives was investigated at two levels. It was first established that the C3 associated with M+ streptococci was not able to promote adherence to cells bearing the complement receptors CR1 and CR3 under conditions in which M- streptococci readily attached. The molecular form of C3 bound to M+ and M- streptococci was then defined by adding 125I-C3 to serum used for opsonization. C3 eluted from the bacteria by chaotropic and hydrolytic agents was analyzed by SDS-PAGE, and revealed that both cell types bound the opsonic forms of C3, C3b, and iC3b. Furthermore, approximately 80% of the C3b and iC3b associated with both cell types was covalently bound to a surface component, although most of the C3 bound to M+ streptococci was detergent-extractable, whereas greater than 50% of that bound to M- streptococci was not. These findings demonstrate that the M+ surface is interfering with the receptor binding of deposited C3b and iC3b, and that this contributes to resistance to phagocytosis by these organisms.     

In vitro studies of age-associated diseases.

We have carried out studies on cultured human fibroblasts in an attempt to trace the origins of age-dependent disorders to the cellular and molecular levels. Three interrelated areas are discussed. First, skin donors with diabetes mellitus (a disease complex that features inappropriate hyperglycemia) produce cultured fibroblasts with a moderate reduction in growth capacity, while two inherited disorders of inappropriate hyperglycemia and premature aging, progeria and Werner syndrome, yield fibroblast cultures with more severely impaired growth capacity. Second, there is a decreased response of progeria level and donor age; evidence is presented that this defective hormone responsiveness in aging cells may reside at the hormone receptor on the surface membrane, the cyclic AMP system, the intracellular enzymatic machinery, or all of these sites. Third, tissue factor, a procoagulant that activates the extrinsic clotting mechanism, is more abundant in cells from the premature aging syndromes of progeria and Werner syndrome. Fibroblast aging in vitro may help to explain various concomitants of normal aging and diabetes mellitus including cell dropout, impairment of hormone responsiveness, and increased atherothrombosis.     

In vitro adhesion of tufted oral streptococci toBacterionema matruchotii

The adhesive interaction between the ectosymbionts of the corn cob configuration (CCC), a naturally occurring bacterial consortium in human dental plaque, was studied. In vitro association was produced in mixed cultures consisting ofBacterionema matruchotii and streptococci resemblingStreptococcus sanguis previously isolated from human CCC. Phase-contrast, selective immunofluorescence, and scanning electron microscopy showed that in this aggregative system, a tuftlike polar structure typical of the coccal epibiont mediated binding to the core filament. This aggregative model provides evidence that a specialized structure on the cell surface of epiphytic organisms may participate in interbacterial adherence.