Serological microarray for a paradoxical diagnostic of Whipple's disease.

Whipple's disease is a systemic chronic infection caused by Tropheryma whipplei. Asymptomatic people may carry T. whipplei in their digestive tract and this can be determined by PCR, making serological diagnosis useful to distinguish between carriers and patients. Putative antigenic proteins were selected by computational analysis of the T. whipplei genome, immunoproteomics studies and from literature. After expression, putative T. whipplei antigens were screened by microimmunofluorescence with sera of immunized rabbit. Selected targets were screened by microarray using sera from patients and carriers. Paradoxically, with 19 tested recombinant proteins and a glycosylated native protein of T. whipplei, a higher immune response was observed with asymptomatic carriers. In contrast, quantification of human IgA exhibited a higher reaction in patients than in carriers against 10 antigens. These results were used to design a diagnostic test with a cut-off value for each antigen. A blind test assay was performed and was able to diagnose 6/8 patients and 11/12 carriers. Among people with positive T. whipplei PCR of the stool, patients differ from carriers by having positive IgA detection and a negative IgG detection. If confirmed, this serological test will distinguish between carriers and patients in people with positive PCR of the stool.

Structural and functional studies of the metalloregulator Fur identify a promoter-binding mechanism and its role in Francisella tularensis virulence.

Francisella tularensis is a Gram-negative bacterium causing tularaemia. Classified as possible bioterrorism agent, it may be transmitted to humans via animal infection or inhalation leading to severe pneumonia. Its virulence is related to iron homeostasis involving siderophore biosynthesis directly controlled at the transcription level by the ferric uptake regulator Fur, as presented here together with the first crystal structure of the tetrameric F. tularensis Fur in the presence of its physiological cofactor, Fe2+. Through structural, biophysical, biochemical and modelling studies, we show that promoter sequences of F. tularensis containing Fur boxes enable this tetrameric protein to bind them by splitting it into two dimers. Furthermore, the critical role of F. tularensis Fur in virulence and pathogenesis is demonstrated with a fur-deleted mutant showing an attenuated virulence in macrophage-like cells and mice. Together, our study suggests that Fur is an attractive target of new antibiotics that attenuate the virulence of F. tularensis.

In vitro and in vivo evaluation of fluoroquinolone resistance associated with DNA gyrase mutations in Francisella tularensis, including in tularaemia patients with treatment failure.

Fluoroquinolones (FQs) are highly effective for treating tularaemia, a zoonosis caused by Francisella tularensis, but failures and relapses remain common in patients with treatment delay or immunocompromised status. FQ-resistant strains of F. tularensis harboring mutations in the quinolone-resistance determining region (QRDR) of gyrA and gyrB, the genes encoding subunits A and B of DNA gyrase, have been selected in vitro. Such mutants have never been isolated from humans as this microorganism is difficult to culture. In this study, the presence of FQ-resistant mutants of F. tularensis was assessed in tularaemia patients using combined culture- and PCR-based approaches. We analyzed 42 F. tularensis strains and 82 tissue samples collected from 104 tularaemia cases, including 32 (30.7%) with FQ treatment failure or relapse. Forty F. tularensis strains and 55 clinical samples were obtained before any FQ treatment, while 2 strains and 15 tissue samples were collected after treatment. FQ resistance was evaluated by the minimum inhibitory concentration (MIC) for the bacterial strains, and by newly developed PCR-based methods targeting the gyrA and gyrB QRDRs for both the bacterial strains and the clinical samples. None of the F. tularensis strains displayed an increased MIC compared with FQ-susceptible controls. Neither gyrA nor gyrB QRDR mutation was found in bacterial strains and tissue samples tested, including those from patients with FQ treatment failure or relapse. Further phenotypic and genetic resistance traits should be explored to explain the poor clinical response to FQ treatment in such tularaemia patients.

Plasma antiproteinase screen and neutrophil-mediated platelet activation. A major role played by alpha 1 antitrypsin.

Upon activation, human polymorphonuclear neutrophils (PMN) release two serine proteinases, cathepsin G (Cat.G) and elastase (HLE), which in turn synergize to activate nearby platelets. We looked for the inhibitory effect of plasma and the involvement of alpha 1 antichymotrypsin (alpha 1 ACT) and alpha 1 antitrypsin (alpha 1 AT), on this cell-to-cell cooperation. It was observed that inhibition by plasma of PMN-mediated platelet activation was rather correlated with an effect on HLE (r = 0.95) than on Cat.G (r = 0.65) enzymatic activity. Purified alpha 1 AT suppressed in a concentration-dependent manner HLE activity present in the supernatant of activated PMN. When HLE was fully blocked, alpha 1 AT started to inhibit Cat.G activity. By contrast and as expected, purified alpha 1 ACT inhibited only Cat.G activity. Using specific blocking polyclonal antibodies against alpha 1 AT and alpha 1 ACT, it was demonstrated that the inhibitory effect of plasma vs. HLE was entirely mediated by alpha 1 AT. By contrast, blockade of Cat.G activity was only partly due to plasma alpha 1 ACT and around 50% was attributable to alpha 1 AT. When plasma from patients with an acute inflammatory state was used in place of plasma from normal subjects, the inhibitory effect was more pronounced, while plasma depleted in alpha 1 AT and alpha 1 ACT was less effective. These data indicate a predominant role of alpha 1 AT in the inhibition by plasma of the PMN-mediated platelet activation.

Preliminary transcriptional analysis of spoT gene family and of membrane proteins in Rickettsia conorii and Rickettsia felis.

Rickettsiae survival implicates adaptation to different environmental conditions. We hypothesized that multiple copies of genes in bacteria with reduced genomes might account for such a process. Transcription of spoT and sca paralogs was thus analyzed in R. conorii and R. felis.

Rickettsia felis, from culture to genome sequencing.

Rickettsia felis has been recently cultured in XTC2 cells. This allows production of enough bacteria to create a genomic bank and to sequence it. The chromosome of R. felis is longer than that of previously sequenced rickettsiae and it possess 2 plasmids. Microscopically, this bacterium exhibits two forms of pili: one resembles a conjugative pilus and another forms hair-like projections that may play a role in pathogenicity. R. felis also exhibits several copies of ankyrin-repeat genes and tetratricopeptide encoding gene that are specifically linked to pathogenic host-associated bacteria. It also contains toxin-antitoxin system encoding genes that are extremely rare in intracellular bacteria and may be linked to plasmid maintenance.

All-trans retinoic acid rapidly decreases cathepsin G synthesis and mRNA expression in acute promyelocytic leukemia.

The cells from patients with acute promyelocytic leukemia (AML M3) undergo terminal differentiation when treated with all-trans retinoic acid (ATRA). We have analyzed the expression of the mRNA for cathepsin G, a promyelocyte stage-specific transcript, in the leukemia and in retinoic acid responsive cell lines. We showed that the transcript is perpetually synthesized in patients' cells and that it rapidly disappears when the cells are treated with ATRA. In ATRA-sensitive (HL-60, NB4) cell lines and an ATRA-resistant (HL-60R) cell line we have shown that this process is dependent on proteins synthesized during the first 6h of ATRA-triggered differentiation and may involve both pre- and post-transcriptional mechanisms. A corresponding decrease in cathepsin G protein synthesis then follows. These findings indicate that the maturation arrest in AML M3 results in cells that may constitutively continue to produce proteins whose production is temporally confined during normal hemopoiesis. This would explain the elevated plasma-free serine protease activity we have demonstrated in this disease, and has implications for both the coagulopathy and the 'retinoic acid syndrome' in AML M3.

Inhibition of neutrophil-endothelial cell adhension by a neutrophil product, cathepsin G.

In the present study we investigated the modulation of the polymorphonuclear neutrophil (PMN)-endothelial cell adhesion process by the two main proteinases released from activated PMN during their adhesion to endothelium. Our results showed that, in contrast with elastase, cathepsin G was a powerful inhibitor of PAIN adhesion to interleukin-1 (IL-1)-treated human umbilical vein endothelial cells. This inhibitory effect was linked to the enzymatic activity of the proteinase and was selectively directed against PMN. Because the viability and the reactivity of PMN were not modified by cathepsin G, we looked for a possible effect on adhesion molecules. L-selectin was not cleaved by cathepsin G, whereas it was by chymotrypsin, a closely related proteinase. Cathepsin G blocked PMN adhesion to activated endothelial cells, but also to serum- or fibrinogen-coated plates, three adhesion processes mediated by CD11b/CD18. However, by FACScan analysis or by immunoprecipitation, we failed to find evidence of modifications of CD11b/CD18 expression. Although the precise molecular target(s) of cathepsin G remain(s) to be defined, these data indicate that this proteinase, which is known as an inflammatory mediator, can also be considered as a potential down-regulator of adhesion reactions involved in the inflammatory process.

Proteolysis of thrombospondin during cathepsin-G-induced platelet aggregation: functional role of the 165-kDa carboxy-terminal fragment.

The serine-proteinase cathepsin G (CG) is a potent agonist of platelet aggregation inducing the release and surface expression of alpha-granule adhesive proteins such as fibrinogen (Fg) and thrombospondin-1 (TSP-1). Because Fg and TSP-1 are potential substrates for the enzymatic activity of CG, we investigated the fate of these proteins during CG-induced platelet aggregation using an immunoblot technique. Only a small proportion of secreted Fg was proteolyzed by CG and platelet aggregation was efficiently inhibited by anti-fibrinogen Fab fragments. In contrast, TSP-1 was extensively proteolyzed on aggregated platelets releasing in the milieu a fragment with Mr approximately 28 000, corresponding to the amino-terminal heparin-binding domain (HBD). Several antibodies, directed against the cell-associated carboxy-terminal TSP-1f fragment (Mr approximately 165000) impaired the formation of stable macroaggregates, indicating that this fragment may contribute to platelet aggregation in the absence of the HBD.

Protective effect of platelet activating factor antagonists on cultured endothelial cell lysis induced by elastase or activated neutrophils.

1. The mechanism(s) responsible for injury of endothelial cells induced by human leukocyte elastase (HLE) was investigated in an immortalized venous human endothelial cell line (IVEC). 2. First, the proteinase concentrations and incubation delays necessary to trigger a significant IVEC cytotoxicity were determined by chromium assays. Thus, exposure of IVEC for 6 h to 10 micrograms ml-1 HLE resulted in 22 +/- 2.8% lysis and 36.4 +/- 5.4% detachment (mean +/- s.e. mean; n = 4; P < 0.05). 3. WEB 2086, a specific platelet-activating factor (PAF) receptor antagonist, induced a significant concentration-dependent decrease of such a lysis (39.6 +/- 7.7% protection at 100 microM; n = 4). This potential role for PAF was confirmed with two other antagonists of this lipid mediator, i.e., BN 52021 and RP 48740. 4. Finally, we demonstrated that pretreatment of IVEC with WEB 2086 protected significantly against cell lysis induced by stimulated human neutrophils, an experimental model in which HLE participates.

Interference of anti-inflammatory and anti-asthmatic drugs with neutrophil-mediated platelet activation: singularity of azelastine.

1. The capacity of various drugs (acetylsalicylic acid (ASA), ketoprofen, diclofenac, piroxicam, BW 755C, BW A4C, nedocromil sodium and azelastine) to inhibit human polymorphonuclear neutrophil (PMN)-mediated platelet activation was investigated. In this model, stimulated PMN release cathepsin G (Cat G), a serine proteinase which, in turn, induces platelet activation. 2. Among the different tested drugs, azelastine (100 microM for 1 min) was the only one able to prevent platelet aggregation. The cyclo-oxygenase inhibitors were all inactive, although used at effective concentrations as judged by inhibition of thromboxane B2 (TxB2) formation. Inhibition of platelet aggregation by azelastine was concentration-dependent, the range of active concentrations being of 20-70 microM. Release from platelets of 5-hydroxytryptamine was also inhibited at 30 microM and above, but never reached 100%. 3. The inhibition by azelastine is due to an effect on both cells. Indeed, beta-glucuronidase release from activated PMN and platelet activation by purified Cat G were both affected. 4. However, used at high concentrations (greater than 100 microM) azelastine was toxic since it released significant amounts of lactate dehydrogenase (LDH) from PMN and platelets. 5. These results show the capacity of azelastine, an anti-allergic and anti-asthmatic compound, to inhibit the cell-to-cell communication between PMN and platelets, an effect which may be relevant for its therapeutic efficacy or for a new application in diseases in which PMN and platelets are involved.

Inhibition by recombinant SLPI and half-SLPI (Asn55-Ala107) of elastase and cathepsin G activities: consequence for neutrophil-platelet cooperation.

1. The capacity of recombinant human secretory leukocyte proteinase inhibitor (SLPI) to inhibit human leukocyte elastase (HLE) and cathepsin G (Cat G) was investigated and compared with a recombinant truncated form (carboxyl-terminal domain, Asn55-Ala107) called 1/2 SLPI. 2. Both compounds were efficient when tested against enzymatic activities of purified HLE and Cat G indicating that the HLE- and Cat G-inhibitory sites were preserved in the truncated form. SLPI and 1/2 SLPI also affected platelet activation induced by 0.2 microM Cat G (IC50 = 112 +/- 13 nM for SLPI and 280 +/- 12 nM for 1/2 SLPI). 3. The effects of SLPI and 1/2 SLPI were then tested against polymorphonuclear neutrophil (PMN)-mediated platelet activation, a cell-to-cell interaction mediated by HLE and Cat G released from PMN. In this experimental system, addition of SLPI or 1/2 SLPI before N-formyl-Met-Leu-Phe (fMLP) led to the inhibition of the resulting platelet activation. As was the case for Cat G enzymatic activity and Cat G-induced platelet activation, SLPI was more efficient than 1/2 SLPI (IC50 = 676 +/- 69 nM vs 1121 +/- 150 nM). 4. The ratio of the IC50 against PMN-mediated platelet activation compared to purified Cat G-mediated platelet activation was 6.03 for SLPI and 4.32 for 1/2 SLPI. This difference may be due to the smaller size of the truncated form which could allow this molecule to diffuse more easily between PMN and platelets. 5. In conclusion, 1/2 SLPI could be a promising candidate in the treatment of pathological states linked to inflammation in which participation of HLE and Cat G has been evoked.

Modulation by superoxide anions of neutrophil-mediated platelet activation.

When polymorphonuclear neutrophil-platelet suspensions were stimulated by 0.5 microM N-formyl-Met-Leu-Phe in the presence of 40 U/mL of superoxide dismutase, a significant reduction of platelet secretion was observed (51.4 +/- 6.3% vs 62.4 +/- 4.6% for control; mean +/- SEM; N = 6; P < 0.01). This was due to the superoxide anion scavenging property of superoxide dismutase since neutrophil degranulation, cathepsin G and elastase enzymatic activities (the two main mediators of this cell-to-cell interaction) and platelet reactivity were not affected. Involvement of superoxide anions was confirmed using leukotriene B4, a neutrophil agonist which induces degranulation with minimal superoxide anion production. Indeed, serotonin release induced by this agonist was unchanged whether superoxide dismutase was added or not.

Determination of the rpoB gene sequences of Bartonella henselae and Bartonella quintana for phylogenic analysis.

Using the Genome Walker procedure, which allows PCR amplification of genomic DNA using a single gene-specific primer and direct automated sequencing methodology, we obtained the nucleotide sequence of the RNA polymerase beta subunit (rpoB) from Bartonella henselae and Bartonella quintana. A phylogenetic tree constructed from these data and other rpoB sequences available in GenBank is, in part, consistent with those previously derived from 16S rRNA gene sequences and confirms the position of Bartonella within the alpha subdivision of Proteobacteria. In fact, this analysis showed that rpoB data are similar to 16S rRNA data for the alpha, beta and gamma subdivisions of Proteobacteria. In contrast, concerning other bacteria included in our study, the topologies of phylogenetic trees were different. Based on the bootstrap values derived from rpoB phylogenic analysis, we believe that this molecule should contribute to better understanding the evolutionary process.

From genes to proteins: in vitro expression of rickettsial proteins.

The availability of the complete genome sequences of several organisms allows the comparative analysis of genomes, a branch of bioinformatics known as genomics. With this approach, much can be learned about the biology of organisms that are difficult to culture, even when few, if any, of their proteins have been isolated and studied directly. We have focused our interest on Rickettsia conorii, an obligate intracellular bacterium responsible for Mediterranean spotted fever, a disease endemic in southern Europe. While bioinformatic annotation of the complete genome of this bacteria has allowed identification of 1,374 genes, a large number of them remain functionally uncharacterized. The final goal of many experiments in molecular biology is to use biological systems to synthesize the protein encoded by the gene being studied. Because three-dimensional structures are more resilient to evolution and change than amino acid sequences, structure determination of some open reading frames should also exhibit structural similarity to previously described protein families. We have thus initiated a systematic expression and structure determination program for the proteins encoded by rickettsial genes of interest. We have cloned different genes of R. conorii by recombinational cloning (GATEWAY), Invitrogen) a method that uses in vitro site-specific recombination to accomplish a directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency. The constructions in p-Dest17 yielded several clones able to express recombinant proteins with a C-terminal histidine tag. Expression of corresponding proteins was then performed using a cell-free protein expression system (Rapid Translation System, RTS, Roche Diagnostics). The recombinational cloning approach coupled to RTS provides an approach to rapid optimization of protein expression and is very useful to express rickettsial proteins. Moreover, this system is able to overcome some of the limitations encountered with rickettsial proteins highly toxic for E. coli or insect cells.

Inhibition by human leukocyte elastase of neutrophil-mediated platelet activation.

When human polymorphonuclear neutrophils and platelets were incubated with human leukocyte elastase before N-formyl-Met-Leu-Phe (FMLP) challenge, a time- and concentration-dependent inhibition of the resulting platelet activation was observed. Thus, when the mixed cell suspension was preincubated for 6 min with 1 microM elastase before stimulation of neutrophils with 0.5 microM FMLP, resulting aggregations and serotonin releases were respectively only 4.4 +/- 4.1% (n = 4) and 1.6 +/- 2.4% (n = 4) as compared to 41.6 +/- 5.2% (n = 9) and 71.3 +/- 16.0 (n = 9) for controls. A direct inhibitory action of elastase on neutrophil activation was ruled out, as well as a breakdown of cathepsin G, a mediator involved in neutrophil-mediated platelet activation. In fact, we demonstrated that the target for the inhibitory effect of elastase in such a cell-to-cell cooperation system was the platelet. This phenomenon is likely to play a role under in vivo conditions in pathologies in which a significant granulocytic proteolytic activity has been detected in the plasma.

Activation and damage of cultured airway epithelial cells by human elastase and cathepsin G.

Accumulation of polymorphonuclear neutrophils (PMN) and epithelium damage have often been described during airway inflammation. We studied the effects of two PMN-derived proteinases, namely elastase and cathepsin G, on guinea-pig tracheal epithelial cells in culture. Both proteinases activated tracheal epithelial cells in terms of prostaglandin (PG) E2 production. A concentration- and time-dependent effect was observed with 10 micrograms/ml and 6 h as the optimal conditions for both enzymes. Optical microscopic studies confirmed an effect on tracheal epithelial cells as intercellular gaps were observed upon incubation of the monolayers with proteinases. A small cytotoxic effect was observed after 1 h incubation but remained stable up to 6 h. This cytotoxic effect, more pronounced with elastase than with cathepsin G, was dissociated from PGE2 formation.

Synergism between interleukin-8 and tumor necrosis factor-alpha for neutrophil-mediated platelet activation.

We previously showed that two polymorphonuclear neutrophil (PMN)-derived proteinases, namely cathepsin G (Cat. G) and elastase (HLE), acting in synergy activated nearby platelets in vitro. This cellular interaction could result in a pathology such as the adult respiratory distress syndrome (ARDS). Since elevated levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) were detected in these patients and therefore could be involved in this disease, we looked for their effects in the PMN-platelet cooperation system. Addition of IL-8 to mixed suspensions of PMN and platelets induced weak but significant platelet aggregations. Upon preincubation with TNF-alpha, aggregations triggered by IL-8 were significantly increased. The targets of these cytokines were not the platelets but the PMNs. This was shown by following beta-glucuronidase release and more interestingly by measuring the enzymatic activities of Cat. G and HLE in the supernatant. Inhibition of the platelet response upon addition of a serine proteinase inhibitor, eglin C, clearly demonstrated the involvement of these two enzymes. Taken together, these results constitute an additional argument for the role of the PMN-platelet interaction in ARDS.