In vitro generation of procoagulant activity by Corynebacterium parvum-stimulated mononuclear leukocytes.

Thromboembolic complications are known in cancer patients after i.v. administration of Corynebacterium parvum. We examined the ability of this organism to induce production of procoagulant activity by human blood mononuclear leukocytes in vitro. After 4 hours incubation Corynebacterium parvum was an effective stimulant for mononuclear leukocytes, behaving in the same way as the typical gram-negative bacterium Escherichia coli, whereas mononuclear cells incubated with Staphylococcus aureus were not affected. Corynebacterium parvum used was found devoid of endotoxin by the Limulus assay and was not affected by Polymyxin B, which, on the contrary, inhibited Escherichia coli-induced production of procoagulant activity. Intact Corynebacterium parvum may be required for the production of procoagulant activity and, although this specific aspect of the research will require further study, from the exposed data it is concluded that such a production could be a factor contributing to the pathogenesis of the coagulopathy following Corynebacterium parvum therapy.

Evaluation of absorption with Limulus amebocyte lysate to remove contaminating endotoxin from interferon and lymphokine preparations.

Alpha and beta human interferon (IFN) preparations and lymphokines (supernatants of PHA-stimulated blood lymphocytes) were deliberately contaminated with endotoxin (20 ng/ml) and subsequently rendered endotoxin-free by absorption with Limulus amebocyte lysate (LAL). Absorption with LAL did not appreciably affect the antiviral activity of IFN and lymphokines in 8 experiments and caused a 30-50% reduction in two. The capacity of these agents to stimulate natural killer cell activity and monocyte cytotoxicity was not consistently modified by absorption on LAL. When the chemotactic activity of lymphokine for monocytes was measured, the maximal number of monocytes induced to migrate and the maximal active lymphokine concentration were not affected by absorption with LAL. LAL-treated lymphokines, however, showed a prozone phenomenon, presumably related to the release of chemotaxis inhibitor(s) from the LAL gel.

Embryonal cardiotoxicity of the Helicobacter pylori lipopolysaccharide.

Helicobacter (H.) pylori is the causative agent of the peptic ulcer disease and a co-factor in the development of gastric malignancies. Recently, it has been maintained that chronic H. pylori infections in adults are linked to a higher risk of coronary heart diseases. In this respect, the acute toxic effects of the H. pylori lipopolysaccharide (LPS) on embryonal cardiomyocytes at different developmental stages was evaluated. White Leghorn chick embryos and smooth (S)--form NCTC 11637 strain H. pylori organisms were used. Both whole heath-killed H. pylori suspensions (3.10(6) bacteria/egg) and isolated S-LPS (500 ng/egg) or S-Lipid A (500 ng/egg) were non-lethal to 4-day embryos, becoming moderately lethal (5% to 30%) to 6- and 8-day embryos and highly lethal (> 90%) to 10- to 17-day embryos. The contractile activity of isolated atrial fragments from 10-day embryos was completely inhibited, within 5 min, following treatments with heath-killed H. pylori (3 x 10(6)/ml), or S-LPS (500 ng/ml), or S-Lipid A (500 ng/ml); the block determined by S-LPS and S-Lipid A was irreversible, while the block by bacterial suspensions was completely reversible upon withdrawal. Following a 24-hour treatment with S-LPS or S-Lipid A of single-cell cultures of cardiomyocytes (isolated from 10-day embryos) a dose-dependent cell loss was observed, as assessed by total protein dosage and direct counting of adherent cells. Propidium Iodide/Annexin V FACS-analysis confirmed the occurrence of cellular necrosis, but did not show any evidence of apoptotic processes. The release of superoxide anion radicals by cultured cardiomyocytes was as follows: S-Lipid A (25 micrograms/ml) > S-LPS (25 micrograms/ml) > heath killed H. pylori suspensions (3 x 10(6)/ml); control cultures did not release detectable amounts of superoxide anion radicals. Furthermore, cultured cardiomyocytes produced increased amounts of NO (N-monomethylarginine-inhibitable) following stimulation with S-LPS (25 micrograms/ml) or S-Lipid A (25 micrograms/ml) (but not heath killed H. pylori 3 x 10(6)/ml suspensions). Under all the above experimental conditions S-polysaccharide proved to be non-toxic. Concluding, H. pylori LPS is relatively non-toxic to the less differentiated cardiomyocytes; cardiomyocytes which are more advanced in their biochemical differentiation become highly sensitive to LPS and produce ROS and NO. ROS are probably responsible for the early toxic actions, while both ROS and NO are likely to be involved in the later degenerative/necrotic effects.

The ability of mononuclear cells to coagulate blood in response to Coxiella burnetii.

Endocarditis is a rather frequent complication of Q fever caused by Coxiella burnetii. We examined the ability of phase I (virulent) or phase II (avirulent) C. burnetii to coagulate blood in the presence of human blood mononuclear cells in vitro. After incubation for 4 h, virulent phase I C. burnetii was an effective stimulant for mononuclear cells. Since this interaction is a potent trigger of blood coagulation through the extrinsic pathway, it could be responsible for the local deposition of fibrin on the surface of infected valves and the development of large vegetations in cases of endocarditis complicating Q fever.

Aeromonas cytotonic enterotoxin cross reactive with cholera toxin.

Isolation by affinity chromatography from crude culture filtrate of Aeromonas sobria of protein that cross reacted with cholera toxin (CT) revealed a toxin that produced fluid accumulation in rat ileal loops and in infant mice and caused rounding of Y1 adrenal cells. All these activities were neutralised by antiserum to CT. There was no haemolytic or cytotoxic activity associated with this CT-cross reactive cytotonic enterotoxin. CT-cross reactive material detected in enzyme linked immunosorbent assay (ELISA) was produced by 25% of Aeromonas isolates from faeces of children with or without diarrhoea-26% of A. sobria, 20.0% of A. hydrophila and 24% of A. caviae tested gave positive ELISA results. Most strains that produced this cytotonic enterotoxin but no cytotoxic enterotoxin were isolated from children without diarrhoea. Toxin preparations from Aeromonas spp. that completely inhibited adenosine-5'-diphosphate-induced platelet aggregation, an effect related to elevation of intracellular cAMP, were, with one exception, cross reactive with CT in ELISA.

In vitro production of tumor necrosis factor-alpha, interleukin-6 and interleukin-8 from normal human peripheral blood mononuclear cells stimulated by Rhodococcus equi.

The capability of heat-killed Rhodococcus equi organisms to induce in vitro release of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-8 from normal human mononuclear cells as well as the secretion kinetics of these inflammatory cytokines over a 48 h period were evaluated. Results show that normal human mononuclear cells are efficiently triggered to secrete TNF-alpha, IL-6 and IL-8 following R. equi stimulation according to a different kinetics. In particular, release of IL-B was already maximally expressed after 2 h of stimulation, while TNF-alpha amounts progressively increased in a time-dependent fashion. Finally, IL-6 secretion reached peak levels as soon as 18 h of incubation. Taken together, these data point out that monocyte-derived cytokines may play an important role in the immunological control of R. equi infection in immunocompetent people.

Production of procoagulant activity, tissue factor-like, by human mononuclear cells and its role in the pathogenesis of Legionella prosthetic valve endocarditis.

The production of procoagulant activity, tissue factor-like, by human mononuclear phagocytes stimulated by Legionella microorganisms is here hypothesized as a key moment in the pathogenesis of Legionella prosthetic valve endocarditis.

The permeability of dialytic membranes to endotoxins: clinical and experimental findings.

This study of 20 endotoxemic patients submitted to 70 hemodialyses (HD) found a reduction of the pre-HD limulus amebocyte lysate (LAL) positivity in 50 HD (71%), without appreciable differences in terms of effectiveness between cuprophan and AN 69 membranes. To define the mechanisms responsible for the reduction in LAL positivity during HD, the membranes were used in two in vitro studies, the first of which showed that the LAL positivity of blood containing lipopolysaccharide (LPS), submitted to hemofiltration (HF) for 300 min, remained unchanged and the ultrafiltrate remained constantly LAL negative. These results suggest that the reduction in LAL positivity observed in HD in vivo, an expression of reduced endotoxemia, cannot be attributed either to the filtration of the LPS as such or to its fragmentation following blood-membrane interaction into theoretically less filtrable molecules or to mechanisms of LPS adsorption on the membrane. The in vivo reduction of LAL positivity is more likely due to removal of the filtrable endotoxin fragments already released in the body, like lipid A, the biologically active component of LPS, known to react to LAL. This hypothesis was borne out by the second in vitro study, where the LAL positivity of blood containing lipid A, treated by HF for 80 min, gradually decreased, and dialytic permeability to lipid A was confirmed by the appearance of LAL positivity in the ultrafiltrate.

Cat-scratch disease in Italy: a serological approach.

The results of studies on the serologic responses to Afipia felis and Rochalimaea henselae in suspected patients for Cat Scratch Disease (CSD) are illustrated. This preliminary study performed using Indirect Immunofluorescence Assay, proved negative for A. felis and R. henselae in some patients and positive in others; in a few instances the test was positive for both organisms. Additional microbiological and serological studies are needed to clarify the exact role of these microorganisms in causing CSD.

Clinical epidemiological survey of Legionella pneumophila infections in Italy.

A clinical epidemiological survey of Legionella pneumophila infections occurring in Italy between 1 December 1985 and 31 May 1986 was carried out to evaluate the incidence of sporadic, epidemic and nosocomial L. pneumophila pneumonia. A total of 355 cases of pneumonia were studied of which 11.5% were due to Gram positive bacteria, 11% were due to Gram negative bacteria, 7.9% were due to Mycoplasma pneumoniae, 4.5% were due to L. pneumophila and 8.5% were due to sundry aetiological agents. The remainder (45.6%) could not be diagnosed accurately. In addition, the anti L. pneumophila antibody titres were assessed. The results are discussed in terms of the occurrence of the disease in Italy and regarding the importance of considering the possibility of legionellosic aetiology when diagnosing pneumonia.

Inhibition of neutrophil functions by scrapie prion protein: description of some inhibitory properties.

The effect of scrapie prion protein (PrP) either in the native or in the denatured form was studied on in vitro responses of human neutrophils. Incubation of neutrophils with native PrP caused an inhibition of their aggregation induced by cytochalasin B. Moreover, the denatured form was in itself a strong aggregation inducer. When evaluating the effect on generation of neutrophil superoxide anion (O2) we found that neutrophils released O2 in response to the denatured from only but the native form was ineffective. Similarly, neutrophil discharge of beta-glucuronidase which represents the azurophilic granule marker was stimulated in a dose-dependent form by the denatured PrP 27-30 whereas the native form was almost completely devoid of any activity. These results indicate that several aspects of neutrophil function can be altered by the native form of prion protein PrP 27-30. This might be responsible for the impaired phagocytic cell activity explaining, at least in part, the absence of any inflammatory reaction during scrapie infection.

Chlamydia pneumoniae infection in Italian patients.

Of 91 adult patients with respiratory tract infections, 13 (14%) had serological evidence of recent infection with Chlamydia pneumoniae. The clinical picture was consistent with asthmatic bronchitis in three patients, whilst exacerbations of chronic obstructive pulmonary disease were present in five subjects and, pneumonia was diagnosed in the remaining five. Our findings provide evidence of an aetiological association between C. pneumoniae and respiratory infections in our region (Bari, South Italy).

[Enterobacteria and endotoxins in abdominal surgical pathology. Critical review and clinico-experimental studies]. / Enterobatteri ed endotossine nella patologia chirurgica addominale. Rassegna sintetico-critica e contributo clinico-sperimentale

A detailed critical review on the role of endotoxins from gram-negative bacteria in the pathogenesis of most of the intestinal experimental models of surgical interest is presented. Data of an investigation on 10 cases of acute appendicitis and 2 cases of intestinal occlusion associated with toxaemia are then presented: in all these cases the Limulus test significatively revealed the presence of endotoxin in the blood and in the peritoneal fluid, also in absence of bacteria in the blood. The implications of these results and the use of the test in clinical practice are discussed.

[Permeability of dialysis membranes for endotoxins. Clinical and experimental results]. / Permeabilita dialyzacních membrán pro endotoxiny. Klinické a experimentální výsledky.

In the presented investigation in 20 endotoxaemic patients 70 haemodialyses were performed. It was revealed that the prehaemodialyzation LAL positivity in 50 haemodialyses (71%) declined, while no differences were observed in the effectiveness of cuprophan and AN 69 membranes. Research in vitro revealed that LAL positivity of blood which contains endotoxins and is subjected for 300 minutes to haemofiltration remains unaltered; in ultrafiltrates LAL is permanently negative. This is due to a mechanism of endotoxin fragmentation as a result of interaction of blood and membrane. Moreover, the LAL positivity of blood containing lipid A declines gradually when subjected to 80 minutes haemofiltration; the dialyzation permeability for lipid A is then proved by the fact that LAL positivity appears in the ultrafiltrate. From the submitted results ensues that reduction of LAL positivity by haemodialysis occurs as a result of elimination of filtrable endotoxin fragments (lipid A) which are released in the body.

Role of lipopolysaccharide and related cytokines in Helicobacter pylori infection.

Helicobacter pylori is a gram-negative bacterium which accounts for the development of chronic gastritis and peptic ulcer in man. In this review, emphasis has been laid on the role of lipopolysaccharide (LPS) of the H. pylori cellular wall in the pathogenesis of gastroduodenal disease. H. pylori LPS exhibits a reduced endotoxic potency in terms of pyrogenicity, lethality, toxicity, mitogenicity, cytokine (CK) release and chromogenic limulus amebocyte lysate assay. This low biological activity of the LPS could be ascribed to the underacylation and underphosphorylation pattern of the lipid A backbone. However, also LPS core structures seem to contribute to the biological activity of the molecule. Despite this low immunological potential, an array of proinflammatory CKs are produced both in vitro and in vivo following stimulation of mucosal cells with H. pylori organisms. It is likely that LPS plays a major role in triggering interleukin (IL)-8, IL-1 and tumor necrosis factor-alpha production from both epithelial cells and macrophages. Finally, the lower immune response elicited by H. pylori LPS in comparison with other enterobacterial LPS may represent an escape mechanism from the host immunosurveillance exerted by this bacterium, thus allowing its survival and persistence in the gastric niche.