Comparison of genotyping of Helicobacter pylori cagA and vacA virulence genes from gastric biopsies and stool specimens

Background. We compared results of genotyping of Helicobacter pylori cagA and vacA virulence genes in DNA from gastric biopsies, both paraffin‐embedded and frozen, and from stool samples, in order to evaluate the comparative sensitivity of the stool assay. Methods. Genomic DNA from paraffin‐embedded biopsies, unfixed frozen biopsies, and stool samples of the same 20 patients was amplified for the cagA gene, an empty site (which provides a positive signal for cagA negative strains) and for the s and m alleles of the vacA gene. Composite genotypes were determined by combining data from analysis of all three materials. Results. Analysis of none of the materials taken singly showed all of the genotypes revealed by all three materials taken together, probably because of sampling error. Analysis of paraffin biopsies revealed 83.5%, that of frozen biopsies revealed 74.7% and that of stools revealed 75.9% of the genotypes. There was no significant difference in the percentage of the H. pylori genotypes identified from the three materials. Analysis of combinations of frozen biopsies and stools revealed 89.9% of the composite genotypes, and that of paraffin biopsies and stools revealed 96.2% of the composite genotypes. Evidence of multiple genotypes was found in 10 of 20 (50%) of the cases. Conclusions. Any one of the investigated biological materials can be used for detection of cagA and vacA genes, but no single assay provided a complete genotype. The use of a combination of two materials may generate a more accurate representation of H. pylori genotypes in each individual.     

Protein extraction by reverse micelles: a study of the factors affecting the forward and backward transfer of alpha-chymotrypsin and its activity

alpha-chymotrypsin is taken as a model protein to investigate three aspects of the protein extraction by reverse micelles: (1) the comparison between the two forward transfer techniques, i.e., the liquid-liquid and the solid state-liquid transfer; (2)the back-transfer, i.e., the capability of the protein to be recovered from the micellar solution; and (3) the maintainance of the enzyme activity at the end of the extraction cycle. Concerning the forward transfer from the liquid phase, we study first the effect of salt initially present in the aqueous phase on the equilibrium concentration of the extracted species; further, we study the forward protein extraction from the solid state, and the effect of pH, salt, and protein concentration on the transfer efficiency. Concerning the back transfer, we find the somewhat surprising result, that the percentage of protein back-extraction depends on the type and concentration of salt used for the forward transfer. Preliminary data concerning an alternative method for the back-transfer using silica gel to liberate the protein from the micellar environment, are presented. Finally, it is found that the enzyme activity depends again on the type and concentration of salt used for the forward transfer.     

Candida albicans-secreted lipase induces injury and steatosis in immune and parenchymal cells

Virulence depends on opposing reactions between host and pathogen and is intrinsically linked to the host immune status. Virulence factors rely upon microbial attributes that mediate cell damage. While the activity of several Candida albicans hydrolytic enzymes is well characterized, the biological role of lipases is uncertain. In this report, we identified, isolated, and characterized a C. albicans 70 kDa lipase that exhibited maximal activity at physiological pH and temperature. We evaluated the ability of C. albicans lipase to interact with two types of mammalian host cells: macrophages, as crucial immune effector cells involved in fungal control, and hepatocytes, as examples of parenchymal cells compromised during fungal dissemination. Herein, we demonstrate for the first time that an extracellular lipase released by C. albicans directly induced cytotoxicity and promoted the deposition of lipid droplets in the cytoplasm of macrophages and hepatocytes.     

Toxoplasma gondii: partial cross-protection among several strains of the parasite against congenital transmission in a rat model

Rats were immunized with cysts of two Toxoplasma strains or with RH strain tachyzoites prior to pregnancy. The litters of the 13 rats that received homologous challenges with cysts during pregnancy, were all protected, whereas of 173 rats that received heterologous challenges with cysts or oocysts, only 21 protected their litters. 38.3 and 17% of rats immunized with the RH and with complete strains respectively, and 57% of control rats challenged with cysts, transmitted the infection congenitally. The percentages when similar groups were challenged with oocysts, were 33.3, 48.2, and 56.2%, respectively. Immunization with cysts did not completely protect against challenge with oocysts, even if the same strain was used. The divergence of these results from the complete protection against congenital toxoplasmosis observed in immune women and ewes, might be due to the use of excessive challenge doses in the model.     

Detection and typing of Helicobacter pylori cagA/vacA genes by radioactive,one-step polymerase chain reaction in stool samples from children

The detection and molecular typing of Helicobacter pylori virulence genes in human stool specimens by polymerase chain reaction (PCR) require an adequate amount of bacterial DNA and an appropriately adjusted PCR protocol. DNA was isolated from stool samples of 39 H. pylori-infected and nine uninfected Colombian children using the QIAamp Kit following the manufacturer's instructions but with modifications. DNA templates were amplified for the vacA s and m regions and for the cagA gene by PCR using radioactively labeled (32P) primers. The modifications in the standard Qiagen protocol of stool DNA extraction increased the final concentration of eluted total stool DNA 4.7 times (117 +/- 17 versus 22 +/- 3 ng/microl; P < 0.0001). Nevertheless, its amplification by regular PCR programs (30-40 cycles) did not generate visible signals because of the very low ratio of H. pylori DNA to other DNA. PCR for 80 cycles successfully amplified vacA in 36/39 samples (sensitivity, 92.3%) and cagA fragments in 21/39 (53.8%) fecal DNA samples. Both s and m vacA regions were amplified in 33/36 (91.7%) DNA samples. The s1m1 genotype was the most commonly isolated variant, accounting for 17/36 or 47.2% of positive samples. The s2m2 genotype was ascertained to be frequent also (14/36 or 38.9%). Almost all (94.1%) s1m1 genotypes were cagA positive. The majority of s2m2 genotypes (78.6%) were not associated with the cagA gene. Neither cagA nor vacA fragments were amplified from DNA isolates of H. pylori-uninfected children nor from DNA isolated from six gastrointestinal bacterial strains (specificity, 100%). The data suggest that the proposed modified technique of DNA extraction and PCR assay of stool samples may be an effective and reliable noninvasive tool for the detection and typing of H. pylori cagA/vacA virulence genes in infected individuals.     

Genome analysis and clinical implications of the bacterial communities in early biofilm formation on dental implants restored with titanium or zirconia abutments

This cross-sectional study aimed to identify and quantify up to 42 target species colonizing the early biofilm of dental implants restored with titanium or zirconia abutments. A total of 720 samples from 20 healthy individuals were investigated. Biofilm samples were collected from the peri-implant sulci, inner parts of implants, abutment surfaces and prosthetic crowns over a functioning period of 30 days. Checkerboard DNA–DNA hybridization was used for microbial detection and quantitation. Clinical characteristics (probing depth, bleeding on probing, clinical attachment level and marginal bone loss) were also investigated during the monitoring period. Genome counts were low at the implant loading time point for both the abutment materials, and increased over time. Both the titanium and the zirconia groups presented similar microbial counts and diversity over time, and the microbiota was very similar to that colonizing the remaining teeth. Clinical findings were consistent with a healthy condition with no significant difference regarding marginal bone loss between the two materials.     

The role of fluoride on the process of titanium corrosion in oral cavity

Titanium is known to possess excellent biocompatibility as a result of corrosion resistance, lack of allergenicity when compared with many other metals. Fluoride is well known as a specific and effective caries prophylactic agent and its systemic application has been recommended widely over recent decades. Nevertheless, high fluoride concentrations impair the corrosion resistance of titanium. The purpose of this article is to summarize the current data regarding the influence of fluoride on titanium corrosion process in the last 5?years. These data demonstrate noxious effects induced by high fluoride concentration as well as low pH in the oral cavity. Therefore, such conditions should be considered when prophylactic actions are administrated in patients containing titanium implants or other dental devices.     

A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens,Parvalbumins, and Identification of a Major IgE-Binding Epitope

Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients’ sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. ¹H/¹⁵N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.     

Evolution of Substrate Preparation Behaviors for Cultivation of Symbiotic Fungus in Attine Ants (Hymenoptera: Formicidae)

Despite their importance for the evolution of the symbiosis between Attine ants and their fungal cultivar, substrate preparation behaviors have been the focus of few studies. This study aimed to comparatively examining these behaviors in Acromyrmex disciger, Apterostigma pilosum, Mycetarotes parallelus, Myrmicocrypta sp., Trachymyrmex fuscus and Trachymyrmex sp. Nov. to describe the patterns of their evolution. Behavioral observations were carried out with a set of micro cameras and the behavioral frequencies were analyzed by principal components. Our findings revealed that the process can be divided into three parts: physical treatment, chemical treatment, and incorporation. Two behavioral patterns were revealed. The first is exhibited by basal species (Myrmicocrypta sp, A. pilosum and M. parallelus) and is characterized by the absence or low frequency of chemical treatment behaviors, while the second pattern is exhibited by derived species (Trachymyrmex sp. Nov., T. fuscus and A. disciger) and is characterized by great fragmentation of the substrate and deposit of fecal fluid. This suggests that the evolution of the process is marked by an increase in the importance of the chemical treatment, leading to the adaptations observed in leaf-cutting ants.