Biomedical subjects
Publications and source records attributed to F Galdiero.
Female mice were maintained on a lipid-rich diet. After 33 days, the mice showed a decrease in the cell population bearing MHC class II molecules, and in the production of interleukin-1 (IL-1). After 60 days, we reported a further decrease of the cell population with MHC class II molecules and the secretion IL-1.
The fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) in the outer membrane bilayer of two mutant strains of Salmonella thyphimurium, i.e., SH 5014 and SH 6261, at different temperatures was analyzed in terms of continuous Lorentzian lifetime distributions. The results were compared with those obtained for the free fluorophore in an isotropic nonviscous solvent. The incorporation of DPH in the outer membrane fragments resulted in a broadening of the lifetime distribution which was attributed to the microenvironmental heterogeneity of the membrane bilayer for the extrinsic fluorophore. The differences observed between the two types of membrane bilayers were interpreted in terms of a different molecular organization and, to a lesser extent, in terms of a different fluidity. The comparison between the DPH lifetime distributions obtained using two different excitation wavelengths, i.e., 280 and 350 nm, suggested that the structural organization of the membrane domains, which are richest in proteins, is almost identical in the two examined mutant strains. This observation indicates that the different susceptibility of the two mutant strains toward phagocytosis and complement-mediated lytic action may depend on the molecular organization and dynamics of the lipid regions far from those containing proteins.
Our study considered the possibility of modifying the functional response of human neutrophils, of mouse lymphocytes and macrophages treated with phospholipids having different polar groups, different isomerisms with saturated and unsaturated fatty acids from C12 to C20 carbon atoms. The results are as follows. a) Most of the phospholipids containing fatty acids from C12 to C20 cause inhibition of the blastogenic capacity of the polyclonal activators tested. b) The phospholipids tested cause a decrease in adherence of polymorphonuclear leukocytes with the exception of the phosphatidyl-choline containing saturated and unsaturated fatty acids. c) A decrease in polymorphonuclear leukocytes migrational capacity almost always occurs. d) The cells treated with L-phosphatidyl-ethanolamine having fatty acids from C14 to C17 show an increase in chemiluminescence; those treated with phosphatidyl-choline and L-phosphatidyl-glycerol show a decrease of the chemiluminescence; L-phosphatidic acid and L-phosphatidyl-ethanolamine having Microbial fatty acids (FAs) at C16 cause a decrease in the formation of phagolisosomes in the macrophages tested.
Salmonella typhimurium and Escherichia coli K12 mutants of R chemotype, with varying contents of major proteins, were studied with respect to serum-mediated killing. The mutants demonstrated a different susceptibility to serum lytic action. These results were related to phospholipid and fatty acid content, as well as different physico-chemical surface properties, such as outer membrane fluidity. Tests were carried out on all parameters considered in the literature to demonstrate the resistance to complement. Our results showed that in sensitive strains such as Salmonella strains SH6261, SH6378, SH5551, SH6017 and E. coli PC0479 tests taken alone were not sufficient to explain the resistance to complement. Therefore, complement susceptibility is probably determined by many factors influencing the microheterogeneity of the membrane system.
The inflammatory activity of porins purified from Salmonella typhimurium has been investigated. Porins (0.3 to 30 micrograms) injected into the rat paw induced a dose-related edema that was not due to lipopolysaccharide contamination and did not appear to be dependent on the activation of the complement system. The edema induced by 30 micrograms of porins was comparable to that caused by 1 mg of carrageenin and was inhibited by indomethacin (5 mg/kg) and dexamethasone (0.1 mg/kg). Porins (1 to 10 micrograms/ml) induced a concentration-related release of histamine from rat peritoneal cells. These results are discussed in the light of the possible pathogenic role of porins in infections.
The effect of SV-IV, one of the major proteins secreted from the rat seminal vesicle epithelium, on phagocytosis and chemotaxis of human polymorphonuclear leukocytes (PMNs) has been studied. Various cytological, biochemical, metabolic, and physical correlates of both biological activities have been found to be markedly reduced by the presence in the medium of micromolar concentrations of protein SV-IV. Moreover, the Scatchard analysis of the labeled SV-IV binding to PMN cell surface has demonstrated that such binding is specific. The binding sites contain only saturable components, completely displaceable by unlabeled SV-IV. The number of the specific sites has been calculated to be 87,000/cell, with a Kd of 1.72 X 10(-7) M. The molecular mechanism of the inhibitory effect is discussed along with the possible biological and clinical implications of the experimental findings.
Female mice were maintained on lipid diet for 20 days. The nonspecific and immunological defense capability was determined by in vitro and in vivo methods. It was found that mice held mostly on a lipid diet demonstrate an all-round lowered response. Following 20 days of lipid diet the splenocytes exhibit: (1) an inversed lipid-protein ratio; (2) an inability to respond to sheep erythrocytes; (3) a reduction in [3H] thymidine incorporation in splenocytes stimulated with lipopolysaccharide (LPS) or with concanavalin A; (4) a reduction in the number of cells bearing surface immunoglobulins in splenocytes stimulated with LPS; (5) an inhibition of phagocytosis and intracellular killing in macrophages; (6) a lowering in granulocyte chemotaxis and adherence capacity; (7) a higher mortality to LPS after loading with galactosamine; and (8) a lowered complement activity even following LPS activation.
The protein SV-IV, one of the major secretory proteins produced by the rat seminal vesicle epithelium, has been found to possess a marked ability to inhibit in vitro the phagocytic properties of activated peritoneal rat macrophages, by a mechanism that apparently involves phagocytes and target cells. Although SV-IV is a substrate for transglutaminase (TGase), an enzyme secreted by activated macrophages, TGase does not seem to play any significant role either in the binding of the protein to the cells participating in the phagocytic process or in the inhibition of macrophage phagocytosis by SV-IV. The significance of the findings in relation to the reproductive process and their possible clinical implications are discussed.
The effect of Salmonella typhimurium porins on human polymorphonuclear leukocytes (PMNs) was studied. Labeled porins were shown to bind to the PMNs, and could be completely displaced by unlabeled porins. The binding caused modifications of membrane integrity and of the physico-chemical characteristics of the PMN surface, e.g. decreased oxidative burst, decreased hydrophobicity and altered cell morphology. The porins acted as both chemotaxins and chemotaxinogens. When PMNs were preincubated with porins their migration in the presence of commonly used chemoattractants (serum activated by zymosan or N-formyl-L-methionyl-L-leucyl-L-phenylalanine) was inhibited.
The adherence of polymorphonuclear neutrophils was examined in 16 children affected by enteritis, pneumonia, hepatitis and infectious mononucleosis. The results were compared with those obtained in 30 healthy adult volunteers and in 15 healthy children of the same age. Adhesiveness was significantly higher in adults than in healthy children, and significantly higher in healthy children than in children with viral infection. In 7 patients tested one month after regression of the disorder, PMN adhesiveness had returned to normal.
A study was conducted on the granulocytic phagocytosis of bacterial aggregates obtained under ideal environmental conditions. For the strains studied, aggregation was favored by low salt concentrations, low pH and temperatures between 30 degrees C and 40 degrees C. Our results show that the phagocytic capacity of granulocytes depends on the type and size of these aggregates. Those formed by a smaller number of cells are more easily phagocytized than the larger ones.
This study examines the action of the cell wall components of enterobacteria on the vitality of human spermatozoa. Lipopolysaccharides extracted from Escherichia coli K12 killed about 80% of the spermatozoa at a concentration of 50 micrograms/ml. Porins extracted from E. coli, Proteus mirabilis and Salmonella typhimurium killed between 80% and 100% of the spermatozoa at a concentration of 50 micrograms/ml. Muramic acid and N-acetylmuramic acid caused about 60% mortality at a concentration of 50 micrograms/ml. The possibility that the products of cellular lysis in the course of gram-negative infections cause temporary sterility is discussed.
Escherichia coli K12 suspended in different media showed a loss of phospholipids. Mg2+ and Ca2+ 0.01 M prevented phospholipid loss and stabilized Escherichia coli K12 for bactericidal and bacteriolytic assays.
The adherence of bacterial cells to valvular prostheses has been studied. Bacteria were selected on the basis of their surface features (fimbriae, hydrophobicity and specific receptors). It was found that only strains having fimbriae and high cell surface hydrophobicity adhered to bioprostheses, while they did not adhere to metallic prostheses to any significant extent. Adherence to bioprostheses depended on the exposure time and it was affected by the saline concentration of the suspension medium. Furthermore, different bacterial binding capacity was observed for bioprostheses from different companies.
The peritoneal macrophages from mice on a lipidic diet have shown an increase of surface hydrophobicity of cytoplasmatic membrane. This fact is correlated with a decrease of the phagocytic index and with an impairment of Salmonella typhimurium.
Major outer membrane proteins of Eikenella corrodens, an organism frequently isolated from patients with periodontal disease, were tested for some biological activities. Mouse peritoneal macrophages, exposed at low concentrations of the above-mentioned proteins (between 0.05 and 5 micrograms/ml), showed evident and marked morphological modifications consisting of increases in the size and vacuolation of the cells. Higher concentrations showed a toxic effect. Low concentrations resulted in a selective release of lysosomal enzymes without any significant release of lactatedehydrogenase, and cytoplasmic marker; while concentrations of 25-50 micrograms/ml, which were toxic in trypan-blue exclusion test, increased LDH release. Eikenella corrodens major proteins increased the platelet aggregation of ADP and thrombin. The residual complement activity of serum samples incubated with various amounts of proteins at 37 degrees C for 30 minutes appeared strongly reduced with respect to controls, thus showing a consumption of the complement components. These results suggested that Eikenella corrodens major proteins may play a role in the development of periodontal lesions.
Porins interact with macrophage membranes and inhibit their phagocyting activity. We have tested the porin effect on a biologically relevant membrane-bound enzymic activity, the adenylate cyclase system, which appears to be stimulated both in the presence of Mn2+ and Mg2+ or Mg2+ + Gpp(NH)p. Moreover, for mice macrophages incubated in the presence of porins, there is an increase in the intracellular cAMP content after 5 min of incubation, with a maximum after 15 min of incubation. The results shown suggest that the porin effects on the adenylate cyclase can represent the molecular basis of the porin-dependent inhibition of the macrophages phagocytosis. Our point of view, which proposes a cAMP role in inhibiting the phagocyting activity in macrophages, is supported also by the results of the experiments carried out in the presence of both dibutyryl-cAMP or aminophylline. The phagocyting activity is inhibited in all cases and independently of the bacteria to be phagocyted.
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