Multiplexed whole bacterial antigen microarray, a new format for the automation of serodiagnosis: the culture-negative endocarditis paradigm.

The serological diagnosis of blood culture-negative endocarditis due to Coxiella burnetii, Bartonella spp., Brucella melitensis and Legionella pneumophila is based on a manual immunofluorescence assay (IFA), which is taken to be the reference method. The automated IFA InoDiag multiplexed antigenic microarray, which includes a slide with all the above bacteria and four internal controls, an incubator, a fluorescent reader and software with an algorithm of interpretation for infectious endocarditis (IE) was evaluated. A single serum dilution at 1/128 was used. Eleven patients with Bartonella spp. IE and ten with C. burnetii IE, diagnosed using the modified Duke criteria, as well as one patient with B. melitensis infection and three patients with L. pneumophila IE were tested. In total, 236 sera were used as negative controls, with the reference method. The results of IgG detection were: C. burnetii phase I, 'sensitivity (Se) = 88% and specificity (Sp) = 99%', and C. burnetii phase II, Se = 88% and Sp = 99%; for Bartonella henselae, Se = 100% and Sp = 100%; for Bartonella quintana, Se = 78% and Sp = 96%; for B. melitensis, Se = 100% and Sp = 99%; and for L. pneumophila, Se = 100% and Sp = 99%. With the algorithm interpretation, the negative and positive predictive values of the test 'were 100% for the diagnosis of IE caused by the four bacteria tested. These results were confirmed by two other assays, one using triplicate testing and one blind testing performed by another centre. This multiplexed test is therefore a valuable tool for the rapid diagnosis of blood-culture negative IE.

Activation of protein tyrosine kinases by Coxiella burnetii: role in actin cytoskeleton reorganization and bacterial phagocytosis.

Coxiella burnetii, the agent of Q fever, is an obligate intracellular microorganism that grows in monocytes/macrophages. The internalization of virulent organisms by monocytes is lower than that of avirulent variants and is associated with actin cytoskeleton reorganization. We studied the activation of protein tyrosine kinases (PTKs) by C. burnetii in THP-1 monocytes. Virulent organisms induced early PTK activation and the tyrosine phosphorylation of several endogenous substrates, including Hck and Lyn, two Src-related kinases. PTK activation reflects C. burnetii virulence since avirulent variants were unable to stimulate PTK. We also investigated the role of PTK activation in C. burnetii-stimulated F-actin reorganization. Tyrosine-phosphorylated proteins were colocalized with F-actin inside cell protrusions induced by C. burnetii, and PTK activity was increased in Triton X-100-insoluble fractions. In addition, lavendustin A, a PTK inhibitor, and PP1, a Src kinase inhibitor, prevented C. burnetii-induced cell protrusions and F-actin reorganization. We finally assessed the role of PTK activation in bacterial phagocytosis. Pretreatment of THP-1 cells with lavendustin A and PP1 upregulated the uptake of virulent C. burnetii but had no effect on the phagocytosis of avirulent organisms. Thus, it is likely that PTK activation by C. burnetii negatively regulates bacterial uptake by interfering with cytoskeleton organization.

[Noise and communal dining facilities]. / Rumore e ristorazione collettiva.

Noise is a sound which is unwanted, either because of its effect on humans, its effect on fatigue or multifunctions of physical equipment, or its interference with the perception or detection of other sounds. It is a part of environmental pollution which can, in certain circumstances, reach worrying levels for the population (130 dB cause pain). Unsuitable exposure to noise for even short periods of time is responsible for symptomology involving the hearing organs (hypoacusis) and other parts of the body such as the cardiovascular, muscular and digestive systems via the connection between the central and the autonomous nervous systems. Noise in communal eating areas can be classed as coming from 3 sources: 1) operation of cooking machinery; 2) banging of pans and equipment; 3) voices of both staff and diners. The intensity of noise on these premises varies generally between 60 and 80 dB (discomfort threshold). The Regulations governing this subject are D.Lgs n.277 of 15/8/1991 regarding the protection of employees, D.P.C.M. of 1/3/1991 which establishes the maximum levels of noise both in the home and outside, and the more recent D.P.C.M. of 21/5/1999 referring to noise in public places, which includes restaurants. To contain the exposure to noise in public eating places, we believe that action should be taken at legal levels with stricter limits than the recently passed level of 105 dB, in the building planning departments and also with technological intervention in order to reduce the effects that noise has on the auditive and extra-auditive organs and thus limit possible sublimal messages which certainly do not benefit the psycho-physical well-being of the diners.

Subversion of monocyte functions by coxiella burnetii: impairment of the cross-talk between alphavbeta3 integrin and CR3.

Several intracellular pathogens exploit macrophages as a niche for survival and replication. The success of this strategy requires the subversion or the avoidance of microbicidal functions of macrophages. Coxiella burnetii, the agent of Q fever, is a strictly intracellular bacterium that multiplies in myeloid cells. The survival of C. burnetii may depend on the selective use of macrophage receptors. Virulent C. burnetii organisms were poorly internalized but survived successfully in human monocytes, whereas avirulent variants were efficiently phagocytosed but were also rapidly eliminated. The uptake of avirulent organisms was mediated by leukocyte response integrin (alphavbeta3 integrin) and CR3 (alphaMbeta2 integrin), as demonstrated by using specific Abs and RGD sequence-containing peptides. The phagocytic efficiency of CR3 depends on its activation via alphavbeta3 integrin and integrin-associated protein. Indeed, CR3-mediated phagocytosis of avirulent C. burnetii was abrogated in macrophages from integrin-associated protein-/- mice. In contrast, the internalization of virulent C. burnetii organisms involved the engagement of alphavbeta3 integrin but not that of CR3. The pretreatment of monocytes with virulent C. burnetii organisms prevented the CR3-mediated phagocytosis of zymosan particles and CR3 activation assessed by the expression of the 24 neo-epitope. We conclude that the virulence of C. burnetii is associated with the engagement of alphavbeta3 integrin and the impairment of CR3 activity, which probably results from uncoupling alphavbeta3 integrin from integrin-associated protein. This study describes a strategy not previously reported of phagocytosis modulation by intracellular pathogens.

Coxiella burnetii induces reorganization of the actin cytoskeleton in human monocytes.

Coxiella burnetii, an obligate intracellular bacterium which survives in myeloid cells, causes Q fever in humans. We previously demonstrated that virulent C. burnetii organisms are poorly internalized by monocytes compared to avirulent variants. We hypothesized that a differential mobilization of the actin cytoskeleton may account for this distinct phagocytic behavior. Scanning electron microscopy demonstrated that virulent C. burnetii stimulated profound and polymorphic changes in the morphology of THP-1 monocytes, consisting of membrane protrusions and polarized projections. These changes were transient, requiring 5 min to reach their maximum extent and vanishing after 60 min of incubation. In contrast, avirulent variants of C. burnetii did not induce any significant changes in cell morphology. The distribution of filamentous actin (F-actin) was then studied with a specific probe, bodipy phallacidin. Virulent C. burnetii induced a profound and transient reorganization of F-actin, accompanied by an increase in the F-actin content of THP-1 cells. F-actin was colocalized with myosin in cell protrusions, suggesting that actin polymerization and the tension of actin-myosin filaments play a role in C. burnetii-induced morphological changes. In addition, contact between the cell and the bacterium seems to be necessary to induce cytoskeleton reorganization. Bacterial supernatants did not stimulate actin remodeling, and virulent C. burnetii organisms were found in close apposition with F-actin protrusions. The manipulation of the actin cytoskeleton by C. burnetii may therefore play a critical role in the internalization strategy of this bacterium.

Effect of cytotoxic necrotizing factor-1 on actin cytoskeleton in human monocytes: role in the regulation of integrin-dependent phagocytosis.

Cytotoxic necrotizing factor-1 (CNF1) is isolated from pathogenic strains of Escherichia coli and catalyzes the activation of Rho GTPases by the deamidation of a glutamine residue. This toxin induces stress fiber formation, cell spreading, and membrane folding and promotes phagocytosis competence in epithelial cells. We show that CNF1 induces morphologic changes in monocytic cells: polarized-like shape in THP-1 cells, lamellipodia, and cell spreading in adherent monocytes. CNF1 also increased filamentous actin (F-actin) content in a time- and dose-dependent manner. In addition, the toxin profoundly reorganized the actin cytoskeleton: redistribution of F-actin in polarized deformations of THP-1 cells and disorganization of microfilament network in monocytes. We also studied the effects of CNF1 on phagocytosis. It markedly impaired the ingestion of unopsonized zymosan involving CR type 3. However, CNF1 had no effect on the uptake of iC3b-coated zymosan or IgG-mediated phagocytosis of SRBC. In addition, CNF1 induced clustering of CR3 and Fc gammaRII (CD32) but selectively impaired the colocalization of CR3 with F-actin. It is likely that CNF1-induced reorganization of actin cytoskeleton down-modulates integrin activation-dependent phagocytosis by preventing the codistribution of CR3 with F-actin. CNF1 may control some features of integrin-dependent phagocytosis in myeloid cells through its action on Rho GTP binding proteins and cytoskeletal organization.