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.

Polymerase chain reaction detection of Bartonella henselae bacteraemia in an immunocompetent child with cat-scratch disease.

UNLABELLED: A case of Bartonella henselae bacteraemia is reported in an immunocompetent 8-year-old boy with cat-scratch disease. Serology to B. henselae, diagnosed by polymerase chain reaction, was positive. DNA was extracted from peripheral whole blood and amplified with specific primers targeting the htrA gene of B. henselae. A non-isotopic hybridization assay with a species-specific oligonucleotide probe was used to detect the amplified product. CONCLUSION: The polymerase chain reaction can be used for the rapid laboratory diagnosis of bacteraemia in cat-scratch disease.

Detection of Bartonella henselae and Afipia felis DNA by polymerase chain reaction in specimens from patients with cat scratch disease.

Polymerase chain reaction (PCR) amplification and colorimetric identification of amplicons were performed to detect Bartonella henselae and Afipia felis DNA in specimens from patients who were clinically and histologically suspected of having cat scratch disease. PCR products were revealed using 2% ethidium bromide agarose-gel electrophoresis and identified with specific probes in a commercial colorimetric hybridization assay (DEIA) (GEN-ETI-K; DiaSorin, Italy). Six paraffin-embedded lymph node biopsies from 18 patients as well as 18 samples of peripheral whole blood and 18 sera were investigated. Bartonella henselae DNA was recovered from the whole blood of four patients, and Bartonella henselae and Afipia felis DNA were detected in one patient's lymph node biopsy. This study suggests that PCR-DEIA is sufficiently sensitive to be considered feasible for the molecular diagnosis of cat scratch disease.

Prevalence of antibodies to Bartonella henselae in patients with suspected cat scratch disease (CSD) in Italy.

Cat scratch disease (CSD) is a relatively new diagnosed illness with clinical signs of self-limiting regional lymphadenopathy accompanied by symptoms of fever and malaise, to encephalopathy and neuropathy, occurring after a cat scratch or flea bite. Bartonella henselae is now accepted as the etiologic agent of CSD. From January 1994 to September 1998, 412 patients were evaluated for suspect CSD in Italy. Sera were tested for antibodies to B. henselae by a commercially available indirect immunofluorescent assay (IFA), based on B. henselae-infected Vero-cells as the antigen substrate. Of the 412 patients, 26 (6.3%) were considered positive having titers of immunoglobulin G (IgG) to B. henselae of 64 or higher. In these patients CSD was indeed confirmed by either histopathologic examination of lymph nodes biopsy or fourfold raise in antibody titers. Nevertheless, sera were tested by IFA for Afipia felis and one showed a double reactivity to B. henselae and A. felis. Finally, three sera, negative to B. henselae serology, were positive to A. felis. Three hundred and eighty-six patients received alternative diagnoses. One hundred and twenty-five serum samples from control subjects were negative by IFA for either B. henselae or A. felis. Moreover, a cross-reactivity with sera from patients affected by other diseases was not observed. Our study shows that the ascertained cases of CSD are etiologically determined by B. henselae, IFA assay is confirmed as a useful tool in the laboratory diagnosis and, over a 5 years period of study, the incidence of CSD in Italy has been low.

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.

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.

Effect of Helicobacter pylori lipopolysaccharide (LPS) and LPS derivatives on the production of tissue factor and plasminogen activator inhibitor type 2 by human blood mononuclear cells.

Different Helicobacter pylori lipopolysaccharides (LPSs) and LPS-derivatives were studied for their ability to induce the production of procoagulant activity (PCA) and plasminogen activator inhibitor type 2 (PAI-2) by human blood mononuclear leukocytes. Smooth (S)- and rough (R)-form LPSs caused a similar increase in cell-associated PCA (tissue factor) and PAI-2 antigen release. Both effects were potentiated by fetal bovine serum via a CD14-mediated mechanism. The potency of H. pylori LPSs was approximately 1000-fold lower than that of Salmonella typhimurium LPSs. When H. pylori LPS derivatives (dephosphorylated R-LPS, S-lipid A, and R-lipid A) were used, PCA and PAI-2 production were markedly reduced. R-lipid A was approximately 4-fold less efficient than S-lipid A. These findings suggest that the induction of monocyte tissue factor and PAI-2 by H. pylori LPS is influenced by the lipid A structure and modulated by the core oligosaccharide and that phosphate groups present in both regions may play an important role.

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).

Downregulation of human polymorphonuclear cell activities exerted by microorganisms belonging to the alpha-2 subgroup of Proteobacteria (Afipia felis and Rochalimaea henselae).

Intracellular pathogens have evolved effective mechanisms in order to survive in an intracellular environment, thus avoiding destruction by phagocytic cells. In this regard, a correlation between resistance to phagocytic killing and expression of pathogenic potency has been established. In this report, we have studied the interaction between human polymorphonuclear cells (PMN) and two gram-negative microorganisms, Afipia felis and Rochalimaea henselae, which belong to the alpha-2 subgroup of the class Proteobacteria. A. falis has been previously proposed as the causative agent of Cat Scratch Disease (CSD), but several recent lines of evidence attribute a major role to R. henselae. Of note, CSD is a syndrome characterized by a chronic lymphoadenopathy, involving macrophages and endothelial cells with a progression towards a granulomatous process and/or angiogenesis. Since members of the alpha-2 subgroup of Proteobacteria have the property to survive intracellularly, we have evaluated the effects exerted by A. felis and R. henselae on human PMN in terms of chemotaxis locomotion, degranulation and oxidative metabolism. Results will show an impairment of PMN activities as a consequence of the challenge with both microrganisms. In particular, inhibition of PMN oxidative function occurred either as result of a direct exposure to both A. felis and R. henselae or when PMN were primed by bacteria for the N-formyl-methionyl-leucyl-phenylalanine enhancement of the oxidative burst. These findings may account for the ability of A. felis and R. henselae to survive within PMN as expression of a further mechanism of pathogenic potency, influencing also the nature and the evolution of inflammatory response in the lesion sites.

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.

Rochalimaea henselae organisms possess an elevated capacity of binding to peripheral blood lymphocytes from patients with cat scratch disease.

Cat scratch disease (CSD) is a clinical condition whose aetiological agent, according to recent findings, is of bacterial origin. Two Gram-negative bacteria are invoked as causative agents of CSD, namely Afipia felis and Rochalimaea henselae. In this paper, five patients with suspected CSD were studied in terms of binding capacity of A. felis and R. henselae to their own peripheral blood lymphocytes (PBL). This parameter was correlated with serum antibody titres to both A. felis and R. henselae, as determined by an indirect fluorescence assay (IFA). Results demonstrate that in four out of five cases binding of R. henselae to PBL was higher than that observed with A. felis. In two cases serum antibody titres to both bacteria were lower or absent, while in the other two patients serum antibody titres to R. henselae were significantly high. In one case only, characterized by elevated titres of serum antibodies to A. felis, values of cytoadherence exhibited by this bacterium were similar to those observed with R. henselae. The results suggest that bacterial binding to lymphocytes may represent an additional parameter to support diagnosis of CSD.

The epidemiology of Lyme borreliosis in Italy.

Erythema migrans was described in Italy only in 1971 although Italian dermatologists were familiar with it a long time before. In 1983, the first case of Lyme borreliosis with multisystem involvement was identified. The endemic areas in Italy are the Ligurian coast, the province of Friuli Venezia Giulia, and the region surrounding the town of Bologna. In Liguria, the incidence of Lyme disease is about 17/100,000 inhabitants per year. Serosurveys of the general population and of sentinel animals were useless in determining the diffusion of Lyme borreliosis whereas evaluation of people at risk of tick bites and patients with suggestive signs or symptoms was more effective. Among the clinical manifestations of Lyme borreliosis, cutaneous involvement is four times more frequent than neuroborreliosis and arthritis is less frequent in Italy than in the USA.