Adriana Santos

Real-Time PCR Assay for Rapid and Accurate Detection of Point Mutations Conferring Resistance to Clarithromycin in Helicobacter pylori

ABSTRACT The main cause of failure of Helicobacter pylori eradication therapy is resistance to clarithromycin. The resistance is due to three point mutations in two positions on the 23S rRNA (A2142C, A2142G, and A2143G). Our aim was to develop a rapid and accurate method to detect these mutations directly on biopsy specimens. We developed a real-time PCR that included a simultaneous detection of the amplicons by hybridization of two probes labeled with LC-Red and fluorescein by using the fluorescence resonance energy transfer (FRET) technology and melting curve analysis with the LightCycler thermocycler. The assay was first applied successfully on reference strains, reference plasmids, and H. pylori-negative biopsies. Biopsies from 200 patients having failed a first eradication attempt and for whom the H. pylori strain was available were then tested with the new assay. A result was obtained in 199 cases; a single genotype was detected in 157 cases, two genotypes were detected in 41 cases, and three genotypes were detected in one case. There were, in total, seven discrepancies between the real-time PCR and the phenotypic method of determination of clarithromycin susceptibility, and in an additional four cases the two phenotypic methods were in disagreement. PCR-restriction fragment length polymorphism was applied to a sampling of biopsies, including all of the cases with multiple genotypes and all the cases with discrepant results. Finally, in four cases with discrepant results, the real-time PCR detected the resistant population at a concentration so low that it could not be detected by the phenotypic method, while in three cases other mutations could be involved. This assay had an accuracy at least as satisfactory as that of the phenotypic tests and could be performed within 2 h, allowing it to be used before the administration of therapy in the case of a first H. pylori eradication.

CCL2 and CCL5 mediate leukocyte adhesion in experimental autoimmune encephalomyelitis—an intravital microscopy study

Experimental autoimmune encephalomyelitis (EAE) models multiple sclerosis (MS) and is characterized by marked mononuclear cell influx in the brain. Several studies have demonstrated a role for chemokines during EAE. It remains to be determined whether these mediators modulate EAE primarily by mediating leukocyte influx into the CNS or by modifying lymphocyte activation and/or trafficking into lymphoid organs. After induction of EAE with MOG(35-55), leukocyte recruitment peaked on day 14 and correlated with symptom onset, TNF-alpha production and production of CCL2 and CCL5. Levels of CXCL-10 and CCL3 were not different from control animals. Using intravital microscopy, we demonstrated that leukocyte rolling and adhesion also peaked at day 14. Treatment with anti-CCL2 or anti-CCL5 antibodies just prior to the intravital microscopy prevented leukocyte adhesion, but not rolling. Our data suggest that induction of leukocyte adhesion to the brain microvasculature is an important mechanism by which CCL2 and CCL5 participate in the pathophysiology of EAE.

Transmission of Helicobacter pylori infection in families of preschool-aged children from Minas Gerais, Brazil.

We evaluated the role of the family in the transmission of Helicobacter pylori infection in preschool‐aged children from a rural district in the State of Minas Gerais, Brazil. Sixty‐six families (66 index children, 63 mothers, 60 fathers and 134 siblings), defined as at least one parent living in the same household with at least one offspring up to 8 years old, were studied. Odds ratios (OR) and 95% confidence intervals (CI) were estimated by logistic regression controlling for age, gender, number of children in household and H. pylori status of the father, mother and siblings. The prevalence of the infection was 69.7% (469 of 673) and it increased with age (P < 0.001). Positive mothers were a strong and independent risk factor for infection (OR 22.70; 95% CI 2.31–223.21). Positive siblings were also positively associated with infection (OR 1.81; 95% CI 1.01–3.30).

babA2- and cagA-positive Helicobacter pylori strains are associated with duodenal ulcer and gastric carcinoma in Brazil.

ABSTRACT The babA2 and cagA genes were investigated in 208 Brazilian Helicobacter pylori strains. A strong association between babA2 and duodenal ulcer or gastric carcinoma was observed, even after adjusting for confounding factors, such as age, gender, and cagA status. cagA-positive strains were also independently associated with H. pylori-related diseases.

New Pathogenicity Marker Found in the Plasticity Region of the Helicobacter pylori Genome

ABSTRACT Comparison of gastric carcinoma and gastritis isolates showed the presence of genes, probably carcinoma associated (JHP947 and JHP940), that are situated in a Helicobacter pylori genome region (45 kb in J99 and 68 kb in 26695) called the “plasticity region.” This region presents a great variability of DNA sequences. We investigated, by PCR, the presence of the JHP940 and JHP947 genes, as well as the presence of a third gene which seems to be associated with gastritis (HP986), on H. pylori strains isolated from 200 Brazilian patients, 79 of whom had gastric carcinomas and 53 of whom had duodenal ulcers, to confirm this association. Gastritis isolates (n = 68) were included as a control. We also evaluated if these genes were related to the virulence-associated cagA genotype. The present methodology did not permit definitive conclusions to be reached regarding the association between the JHP940 gene and gastric carcinoma or between the HP986 gene and gastritis. However, we showed that the JHP947 gene might be implicated in the development of both duodenal ulcer and gastric carcinoma. The presence of the JHP947 gene was associated with the cagA-positive genotype. The JHP947 gene is a novel virulence marker candidate of H. pylori.

An engineered monomer of CCL2 has anti-inflammatory properties emphasizing the importance of oligomerization for chemokine activity in vivo

We demonstrated recently that P8A‐CCL2, a monomeric variant of the chemokine CCL2/MCP‐1, is unable to induce cellular recruitment in vivo, despite full activity in vitro. Here, we show that this variant is able to inhibit CCL2 and thioglycollate‐mediated recruitment of leukocytes into the peritoneal cavity and recruitment of cells into lungs of OVA‐sensitized mice. This anti‐inflammatory activity translated into a reduction of clinical score in the more complex inflammatory model of murine experimental autoimmune encephalomyelitis. Several hypotheses for the mechanism of action of P8A‐CCL2 were tested. Plasma exposure following s.c. injection is similar for P8A‐CCL2 and wild‐type (WT) CCL2, ruling out the hypothesis that P8A‐CCL2 disrupts the chemokine gradient through systemic exposure. P8A‐CCL2 and WT induce CCR2 internalization in vitro and in vivo; CCR2 then recycles to the cell surface, but the cells remain refractory to chemotaxis in vitro for several hours. Although the response to P8A‐CCL2 is similar to WT, this finding is novel and suggests that despite the presence of the receptor on the cell surface, coupling to the signaling machinery is retarded. In contrast to CCL2, P8A‐CCL2 does not oligomerize on glycosaminoglycans (GAGs). However, it retains the ability to bind GAGs and displaces endogenous JE (murine MCP‐1) from endothelial surfaces. Intravital microscopy studies indicate that P8A‐CCL2 prevents leukocyte adhesion, while CCL2 has no effect, and this phenomenon may be related to the mechanism. These results suggest that oligomerization‐deficient chemokines can exhibit anti‐inflammatory properties in vivo and may represent new therapeutic modalities.

Helicobacter pylori Primary Resistance to Metronidazole and Clarithromycin in Brazil

ABSTRACT Helicobacter pylori resistance to metronidazole was detected in 107 (52.97%) of 202 strains. Twenty (9.85%) strains, 18 of them harboring 23S ribosomal DNA mutations, were resistant to clarithromycin. Metronidazole resistance was associated with female gender. Resistance to metronidazole and resistance to clarithromycin were associated. Increasing clarithromycin resistance rates were observed over time.

PCR-Restriction Fragment Length Polymorphism Can Also Detect Point Mutation A2142C in the 23S rRNA Gene, Associated with Helicobacter pylori Resistance to Clarithromycin

Clarithromycin resistance, the major cause of Helicobacter pylori treatment failure ([2][1], [7][2]), is attributed to point mutations within the peptidyltransferase-encoding region in domain V of the 23S rRNA gene ([10][3], [15][4]). Three distinct point mutations have been found to be associated

Factors Associated with Helicobacter pylori Infection by a cagA-Positive Strain in Children

Although infection with a cagA-positive Helicobacter pylori strain is considered a risk factor for the development of duodenal peptic ulcer in adults, this association has not been demonstrated in children. The presence of cagA was investigated by polymerase chain reaction in H. pylori strains isolated from 27 children with duodenal ulcer and 53 without duodenal ulcer. All patients (100%) with duodenal ulcer and 33 (62.3%) without ulcer were colonized by a cagA-positive strain (P=.00007). A cagA-positive status was also associated with a more marked macroscopic gastritis, with a greater inflammatory infiltrate of both mononuclear and polymorphonuclear cells in the antral and oxyntic gastric mucosae and degenerative and regenerative changes of the gastric mucosa. Increased cagA positivity was also associated with increased age, but no association between cagA-positive status and sex was observed.

Kinin B2 receptor regulates chemokines CCL2 and CCL5 expression and modulates leukocyte recruitment and pathology in experimental autoimmune encephalomyelitis (EAE) in mice.

BackgroundKinins are important mediators of inflammation and act through stimulation of two receptor subtypes, B1 and B2. Leukocyte infiltration contributes to the pathogenesis of autoimmune inflammation in the central nervous system (CNS), occurring not only in multiple sclerosis (MS) but also in experimental autoimmune encephalomyelitis (EAE). We have previously shown that the chemokines CCL2 and CCL5 play an important role in the adhesion of leukocytes to the brain microcirculation in EAE. The aim of the present study was to evaluate the relevance of B2 receptors to leukocyte-endothelium interactions in the cerebral microcirculation, and its participation in CNS inflammation in the experimental model of myelin-oligodendrocyte-glycoprotein (MOG)35–55-induced EAE in mice.MethodsIn order to evaluate the role of B2 receptor in the cerebral microvasculature we used wild-type (WT) and kinin B2 receptor knockout (B2-/-) mice subjected to MOG35–55-induced EAE. Intravital microscopy was used to investigate leukocyte recruitment on pial matter vessels in B2-/- and WT EAE mice. Histological documentation of inflammatory infiltrates in brain and spinal cords was correlated with intravital findings. The expression of CCL5 and CCL2 in cerebral tissue was assessed by ELISA.ResultsClinical parameters of disease were reduced in B2-/- mice in comparison to wild type EAE mice. At day 14 after EAE induction, there was a significant decrease in the number of adherent leukocytes, a reduction of cerebral CCL5 and CCL2 expressions, and smaller inflammatory and degenerative changes in B2-/- mice when compared to WT.ConclusionOur results suggest that B2 receptors have two major effects in the control of EAE severity: (i) B2 regulates the expression of chemokines, including CCL2 and CCL5, and (ii) B2 modulates leukocyte recruitment and inflammatory lesions in the CNS.