Elastase is the only human neutrophil granule protein that alone is responsible for in vitro killing of Borrelia burgdorferi.

Phagocytosis of Borrelia burgdorferi by human polymorphonuclear leukocytes triggers oxygen-dependent and -independent mechanisms of potentially cidal outcome. Nevertheless, no factor or process has yet been singled out as being borreliacidal. We have studied the B. burgdorferi-killing ability of the myeloperoxidase-H2O2-chloride system and that of primary and secondary granule components in an in vitro assay. We found that neither secondary granule acid extracts nor the chlorinating system could kill these microorganisms, while primary granule extracts were effective. The Borrelia-killing factor was purified to homogeneity and demonstrated to be elastase. Its cidal activity was found to be independent of its proteolytic activity.

Biologically active polypeptides in the venom of the jellyfish Rhopilema nomadica.

A tropical jellyfish, Rhopilema nomadica (Scyphozoa, Rhizostomeae) has recently invaded the eastern Mediterranean. Its painful stings have been the bane of bathers and fishermen from Egypt to Turkey. This paper reports on the presence of haemolytic activity and alpha-chymotrypsin-like serine protease activity in the venom of the R. nomadica nematocysts. In addition, the presence of phospholipase A2 activity, which has been described previously, is confirmed. Some properties of these activities are defined.

Purification and properties of a cytolytic toxin in venom of the jellyfish Carybdea marsupialis.

A haemolytic toxin was purified by ion-exchange chromatography and FPLC gel filtration from the nematocysts of the jellyfish Carybdea marsupialis. Sheep red cells, but not human or rabbit red cells, were susceptible to lysis by the toxin. The toxin is a protein with an apparent molecular mass of about 102-107 kDa, is heat labile, highly unstable in polar media, inactivated by reducing agents, and devoid of phospholipase activity. The experimental data speak in favour of a pore-forming mechanism of toxin action.

Physical characterization of the pore forming cytolysine from Gardnerella vaginalis.

The cytolytic toxin (CTox) produced by Gardnerella vaginalis is able to form voltage-dependent cationic channels when incorporated in lipid membranes (Moran et al. (1991) FEBS Lett. 283, 317-320). Osmotic protection experiments show that toxin incorporated in human erythrocytes forms pores between 18 A and 28 A in diameter. A hypothesis of pore formation as a primary event to produce cytolysis is proposed. The CTox activity increases when cells are depolarized by increasing the extracellular K+ concentration, probably reflecting the voltage dependent character of CTox formed channels. The cytolytic effect of the toxin was prevented by low temperatures and was a function of the extracellular Ca2+ concentration, suggesting a Ca2+ influx as part of the lytic mechanism. Binding of CTox to erythrocytes was dependent on external Ca2+ and was less temperature-dependent. Dose-response analysis suggests cooperativity of the toxin for the lytic activity, although no direct evidence of oligomerization has been found.

Voltage-dependent cationic channels formed by a cytolytic toxin produced by Gardnerella vaginalis.

A cytolytic toxin produced by G. vaginalis was incorporated in artificial membranes and giant liposomes. The toxin formed ionic channels when incorporated in lipid bilayers. The electrical properties of such channels were studied. Current records revealed a unitary conductance of 126 pS (in symmetrical 150 mM KCl). The open state probability of the cytolysin formed channels was a function of the applied membrane potential. The permeability ratio of cations to anions was estimated to be 6.5.

Monoclonal antibodies as probes to investigate the molecular changes of C5 associated with the different stability of the molecule on sheep erythrocytes and Escherichia coli 0111:B4.

The fifth C component (C5) exhibits a different stability when bound to sheep E or Escherichia coli 0111:B4, being fairly stable on the bacterial intermediate sensitized E. coli 0111:B4 coated with C components up to C5 (BAC1-5) and extremely labile on the RBC intermediate sensitized sheep E coated with C components up to C5 (EAC1-5). We examined the possibility that molecular changes of membrane-bound C5 might be responsible for the different functional behavior of the two intermediates using mAb to C5 and sensitive immunoassays to detect bound C5. The decay of EAC1-5 over 30 min of incubation at 37 degrees C was associated with a significant drop in the reactivity of bound C5 with three of four mAb used. These results contrasted with those obtained with BAC1-5, which showed unchanged reactivity with all mAb tested over the same period of incubation. The effect of mAb on the activity of C5 was then investigated in an attempt to relate the change of the reactivity pattern of EAC1-5 with the functional modification of bound C5. MAb 1.5 and 1.6 were the only antibodies that interfered with the functional activity of C5, although through a different mechanism. In particular, mAb 1.5 was active both on fluid-phase and on membrane-bound C5 and is therefore likely to interact with the binding site for the late components on C5. Conversely, mAb 1.6 was only effective on fluid-phase C5 and acted by promoting a decay of BAC1-5 similar to the spontaneous decay of EAC1-5. We suggest that the bacterial outer membrane may protect C5 from functional decay and that mAb 1.6 interferes with the stabilizing effect of the bacteria in an as yet unclear manner.

Identification and partial characterization of a cytolytic toxin produced by Gardnerella vaginalis.

Generation and release into the culture medium of a cytolytic toxin by Gardnerella vaginalis has been demonstrated. Addition of starch and of the nonionic detergent Tween 80 to the culture medium was essential to recover cytolytic activity. A protein with an apparent molecular mass of 61 to 63 kDa was purified from the culture supernatants showing lytic activity towards erythrocytes and nucleated cells, such as human endothelial cells and human neutrophils. The protein had marked selectivity for human erythrocytes, while erythrocytes from other species were not lysed or were lysed at much higher concentrations of the protein than those needed for human erythrocytes. The cytolytic activity was remarkably unstable in polar media, but was stabilized by nonionic detergents, by binding, or by insertion into the target cell membrane, suggesting its amphiphilic nature.

Bactericidal-activities of human polymorphonuclear leukocyte proteins against Escherichia coli O111:B4 coated with C5 or C8.

The postnuclear supernatant of disrupted polymorphonuclear leukocytes exhibited bactericidal activity on Escherichia coli O111:B4 coated with immunoglobulin M antibodies and C5 or C8 but not on C3- or C7-coated bacteria. To characterize this antimicrobial activity further, granules obtained from the postnuclear supernatant were extracted with sodium acetate (pH 4) and the soluble extract was subsequently fractionated through carboxymethyl cellulose and Sephacryl S-200. Over 90% of the activity present in the starting material was recovered in the soluble granule extract. Kinetic and dose-response analyses of the bacterial activity of the polymorphonuclear leukocyte extract on BAC1-5 and BAC1-8 revealed different susceptibilities to killing of these two bacterial intermediates; they also differed for their susceptibilities to killing at 37 degrees C and at room temperature. The suggestion raised by these data, that BAC1-5 and BAC1-8 could be killed by different bactericidal factors, was confirmed by the findings that separate fractions of the soluble granule extract obtained by carboxymethyl cellulose and Sephacryl S-200 chromatography exhibited specific activity on either BAC1-5 or BAC1-8, whereas other fractions were active on both intermediates.

Kinetics of assembly and decay of complement components on Escherichia coli O111:B4 preparation of stable intermediates.

The preparation of bacterial intermediates bearing complement components at various steps of the complement sequence was investigated by suspending immunoglobulin M-opsonized Escherichia coli O111:B4 cells in complement-deficient sera at different temperatures and ionic strengths. The optimal conditions for the formation of the intermediates at Tmax were found to be an ionic strength of 0.091 mu and a temperature of 37 degrees C, except for BAC142, which could be formed equally well at room temperature. In contrast to all the other intermediates, which, once formed at Tmax, were stable in the presence of the whole serum, BAC142 decayed with a half-life of 10 min due to the lability of bound C2. Washing with a buffer of either 0.091 or 0.046 mu did not affect the bacterial intermediates, with the exception of BAC1-3 formed either in the presence of C5-deficient serum or with purified C3 added to BAC142. All the intermediates were found to be stable after incubation in 0.091-mu buffer for 30 min at 37 degrees C.

A new method for assessment of serum-induced damage to E. coli.

A simple and rapid spectrophotometric assay for the kinetic evaluation of serum-induced damage to E. coli is described, based on changes in the optical density (OD) of a bacterial suspension. Exposure of antibody-coated E. coli to human absorbed serum results in a diphasic response, namely an increase in OD, which reaches a maximum at about 17 min and is followed by a progressive decrease in OD until a minimum value is reached after 45 min. The increase and the decrease in OD are related to bacterial death and bacterial lysis, respectively.

Incidence of myeloperoxidase deficiency in an area of northern Italy: histochemical, biochemical and functional studies.

Forty-five subjects with a complete deficiency of myeloperoxidase were identified in an area of the region Friuli-Venezia Giulia in north-eastern Italy using the Hemalog D system as the screening technique. Histochemical and biochemical tests performed on the leucocytes of some of these subjects confirmed the defects shown by the Hemalog D system. The defect was of genetic origin in seven subjects. The genetic origin could be suspected in another eight subjects since more than two affected members were present in a given family. Eosinophil peroxidase, which is present in MPO deficient subjects, interfered with the guaiacol assay of MPO, and in several cases masked the genetic transmission. An assay was developed using o-dianisidine as the electron donor which considerably reduced the interference by EPO. With this assay an autosomal recessive pattern of inheritance was found. The MPO deficient leucocytes had a higher respiratory burst than control cells and an impaired bactericidal activity, at early post-phagocytic periods, which became comparable to that of control cells at later stages. Particle ingestion by the MPO-deficient cells was normal.

Moculating effect of the late-acting components of the complement system on the bactericidal activity of human polymorphonuclear leukocytes on E. coli 0111:B4.

The effect of complement (C) components on the intracellular killing of E. coli 0111:B4 by human PMN was studied. Various intermediate bacteria were prepared by opsonizing IgM-coated 0111:B4 with yeast cell-treated human serum (BAC1-3), a C6-deficient human serum (BAC1-5), a C8-deficient human serum (BAC1-7), a C8-deficient human serum, and with partially purified C8 (BAC1-8). All these bacterial preparations were phagocytosed by human PMN, but only BAC1-8 and, to a lesser extent, BAC1-5 were killed. Similar results were obtained when the 400 x G postnuclear supernatant (PNS) of PMN homogenate was used instead of intact leukocytes. The C9 nature of the killing factor in the PMN homogenate was ruled out by its inability to lyse EAC1-8 and by the finding that the killing of BAC1-8 by the PMN factor was not inhibited by the antiserum against human C9. The anti-C5 and anti-C8 antisera were unable to inhibit the killing by the PNS of BAC1-5 and BAC1-8, respectively, suggesting that bound C5 and C8 do not provide a binding site for the killing factor.

Effect of antibodies and complement on the interaction between Escherichia coli 0111:B4 and polymorphonuclear leukocytes.

The interaction of Escherichia coli 0111:B4 with polymorphonuclear leukocytes in the presence of specific antibodies and complement was studied. This strain, which is resistant to phagocytosis by polymorphonuclear leukocytes, may be ingested and killed by the phagocytes in the presence of both antibodies and fresh serum. The ineffectiveness of fresh serum to promote ingestion of E. coli 0111:B4 by the phagocytes in the absence of antibodies reflects the inability of this strain to activate the complement system through the alternative pathway. Investigation of the mechanisms of the bacterial killing by polymorphonuclear leukocytes showed that both antibodies and complement were required for the oxygen-independent bactericidal system, whereas they were not needed for the oxygen-dependent system.

[Pathogenicity of E. coli: biological aspects and problems related to laboratory diagnosis (author's transl)]. / Patogenicità di Escherichia coli: aspetti biologici e problemi diagnostici di laboratorio.

The biological properties which may contribute to the pathogenicity of E. coli are reviewed in this article. Specifically the following topics are discussed in detail: 1) adhesion to the intestinal epithelial cells, 2) production of enterotoxins, 3) invasiveness of and ability to multiply within the epithelial cells, 4) insensitivity to complement lysis or inability to activate the alternative pathway of complement, 5) resistance to phagocytic killing. The various techniques which might be of potential usefulness in the clinical laboratory to test the parameters of the pathogenicity of E. coli are briefly outlined. The dissociation between the definition of pathogenicity, as established on the basis of the results of E. coli serotyping, and the criteria of pathogenicity listed above is brought to the attention of the reader. As specificity regards the toxinogenicity, what emerged from a survey of the literature, was that only one of the so called "enteropathogenic" strains of E. coli, according to the serotype classification, was found to produce an enterotoxin.

Correlation between the susceptibility of E. coli to phagocytosis and their ability to invade HeLa cells in vitro.

The susceptibility of several strains of E. coli to phagocytic killing by polymorphonuclear lencocytes and the ability of the same strains to invade HeLa cells were studied. It was found that only the strains resistant to killing by leucocytes were able to penetrate and multiply within HeLa cells.

Influence of E. COLI POLysaccharide on the interaction of E. coli K+ and K- with polymorphonuclear leukocytes in vitro.

Both a K+ strain and a K- strain of E. coli withstand phagocytic killing by polymorphonuclear leukocytes and do not stimulate the respiratory burst, that accompanies phagocytosis in these cells, as compared with E. coli J 53 which are extensively and rapidly killed and stimulate the respiration of leukocytes. Both K+ and K- E. coli become readily phagocytosable after removal of their polysaccharide by heat treatment and are able to stimulate oxygen consumption by PMN. Heat treated E. coli J 53 stimulate the oxygen uptake of PMN less than live E. coli J 53. The polysaccharide extracted from either K+ or K- E. coli inhibits both phagocytosis of and the respiration stimulated by all the three heat-treated strains used. Instead the polysaccharide extracted from the phagocytosable strain J 53 stimulates the oxygen consumption of PMN exposed to the heat treated K- strain or heat treated J 53 itself.

Correlation between phagocytic activity and metabolic response of polymorphonuclear leukocytes toward different strains of Escherichia coli.

The bactericidal activity, the phagocytic capacity, and the metabolic stimulation of polymorphonuclear leukocytes challenged with different strains of Escherichia coli were studied. It was found that only two strains out of 10 tested stimulated the oxygen consumption and carbohydrate metabolism of leukocytes and were readily killed by the phagocytes. The lack of killing of the other eight strains was shown to be due to absent or poor phagocytosis rather than to resistance to intracellular killing. Evidence was presented that the surface K antigen plays an important role in conferring antiphagocytic properties to some strains of E. coli. It was suggested that K antigen acts by interfering with the early step of the phagocytic process, that is, the attachment step.