Ulcerogenic Helicobacter pylori Strains Isolated from Children: A Contribution to Get Insight into the Virulence of the Bacteria

Infection with Helicobacter pylori is the major cause for the development of peptic ulcer disease (PUD). In children, with no other etiology for the disease, this rare event occurs shortly after infection. In these young patients, habits of smoking, diet, consumption of alcohol and non-steroid anti-inflammatory drugs and stress, in addition to the genetic susceptibility of the patient, represent a minor influence. Accordingly, the virulence of the implicated H. pylori strain should play a crucial role in the development of PUD. Corroborating this, our in vitro infection assays comparing a pool of five H. pylori strains isolated from children with PUD to a pool of five other pediatric clinical isolates associated with non-ulcer dyspepsia (NUD) showed the greater ability of PUD strains to induce a marked decrease in the viability of gastric cells and to cause severe damage in the cells cytoskeleton as well as an impairment in the production/secretion of mucins. To uncover virulence features, we compared the proteome of these two groups of H. pylori strains. Two-dimensional gel electrophoresis followed by mass-spectrometry allowed us to detect 27 differentially expressed proteins between them. In addition to the presence of genes encoding well established virulence factors, namely cagA, vacAs1, oipA “on” status, homB and jhp562 genes, the pediatric ulcerogenic strains shared a proteome profile characterized by changes in the abundance of: motility-associated proteins, accounting for higher motility; antioxidant proteins, which may confer increased resistance to inflammation; and enzymes involved in key steps in the metabolism of glucose, amino acids and urea, which may be advantageous to face fluctuations of nutrients. In conclusion, the enhanced virulence of the pediatric ulcerogenic H. pylori strains may result from a synergy between their natural ability to better adapt to the hostile human stomach and the expression of the established virulence factors

Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.status: publishe

Proteome analysis of clustered helicobacter pylori strains

Genomic-methylation typing method, based on strains’ Restriction/Modification systems, confirmed the genetic variability of Helicobacter pylori. According to this, strains isolated from patients of the same family, or from the same geographic region, cluster together. The analysis of proteome’s variability of these clusters has been a missing topic. We applied the Minimum-Common-Restriction-Modification (MCRM) algorithm to genomic-methylation data of 30 H. pylori strains, isolated from Portuguese patients, presenting different gastric diseases. 100% of generated dendrograms presented three incipient clusters (C1, C2 and C3), which is characteristic of strains sharing the geographic origin. The same pattern was observed when the MCRM algorithm was applied to a subset of strains (2 of C1, 2 of C2, 4 of C3 and two outsiders). These were heterogeneous regarding their cagA and vacA genotypes and in terms of patient’s age, gender and gastric disease. Comparative analysis of two-dimensional-gel-electrophoresis (2-DE) maps, obtained for total-protein extracts of each strain, revealed that among 70 matched protein spots (in a universe of 300), 16 were differently abundant (p < .05) among clusters. These proteins’ abundance was then compared having the 2DE-maps regrouped according to the strain’s cagA-genotype or its association with gastric disease. We concluded that abundance variations of at least 12 proteins were dictated by differences in virulence, rather than cluster proximity. Therefore, although the genome-methylation typing method discriminates differences in restriction/modification enzymes, strains of each generated cluster do not share a marked particular proteome, arguing that strains with common geographic origin vary greatly in virulence

Proteome variability among Helicobacter pylori isolates clustered according to genomic methylation

Aims: To understand whether the variability found in the proteome of Helicobacter pylori relates to the genomic methylation, virulence and associated gastric disease. Methods and Results: We applied the Minimum-Common-Restriction- Modification (MCRM) algorithm to genomic methylation data of 30 Portuguese H. pylori strains, obtained by genome sensitivity to Type II restriction enzymes’ digestion. All the generated dendrograms presented three clusters with no association with gastric disease. Comparative analysis of twodimensional gel electrophoresis (2DE) maps obtained for total protein extracts of 10 of these strains, representative of the three main clusters, revealed that among 70 matched protein spots (in a universe of 300), 16 were differently abundant (P < 0 05) among clusters. Of these, 13 proteins appear to be related to the cagA genotype or gastric disease. The abundance of three protein species, DnaK, GlnA and HylB, appeared to be dictated by the methylation status of their gene promoter. Conclusions: Variations in the proteome profile of strains with common geographic origin appear to be related to differences in cagA genotype or gastric disease, rather than to clusters organized according to strain genomic methylation. Significance and Impact of the Study: The simultaneous study of the genomic methylation and proteome is important to correlate epigenetic modifications with gene expression and pathogen virulence.FC

Antigenic diversity among Portuguese clinical isolates of Helicobacter pylori

BACKGROUND: The human gastroduodenal pathogen, Helicobacter pylori, is characterized by an unusual extent of genetic heterogeneity. This dictates differences in the antigenic pattern of strains resulting in heterogeneous human humoral immune responses. Here, we examined the antigenic variability among a group of 10 strains isolated from Portuguese patients differing in age, gender, and H. pylori-associated gastric diseases. MATERIAL AND METHODS:  Immunoassays were performed on two-dimensional electrophoresis gels obtained for the proteome of each strain, using a commercial pool of antibodies produced in rabbit, against the whole cell lysate of an Australian H. pylori strain. Relevant proteins were identified by mass spectrometry. RESULTS: Immunoproteomes of the Portuguese strains showed no correlation between the number of antigenic proteins or the antigenic profile, and the disease to which each strain was associated. The Heat shock protein B was the unique immunoreactive protein common to all of them. Additionally, seven proteins were found to be antigenic in at least 80% of strains: enoyl-(acyl-carrier-protein) reductase (NADH); Catalase; Flagellin A; 2 isoforms of alkyl hydroperoxide reductase; succinyl-CoA transferase subunit B; and an unidentified protein. These proteins were present in the proteome of all tested strains, suggesting that differences in their antigenicity are related to antigenic variance. CONCLUSIONS:  This study showed evidence of the variability of antigenic pattern among H. pylori strains. We believe that this fact contributes to the failure of anti-H. pylori vaccines and the low accuracy of serological tests based on a low number of proteins or antigens of only one strain

Effect of Carbamylated Erythropoietin in a Chronic Model of TNBS-Induced Colitis

Background: Inflammatory bowel disease (IBD) is a public health issue with a growing prevalence, which can be divided into two phenotypes, namely Crohn’s disease (CD) and ulcerative colitis (UC). Currently, used therapy is based only on symptomatic and/or palliative pharmacological approaches. These treatments seek to induce and maintain remission of the disease and ameliorate its secondary effects; however, they do not modify or reverse the underlying pathogenic mechanism. Therefore, it is essential to investigate new potential treatments. Carbamylated erythropoietin (cEPO) results from the modification of the Erythropoietin (EPO) molecule, reducing cardiovascular-related side effects from the natural erythropoiesis stimulation. cEPO has been studied throughout several animal models, which demonstrated an anti-inflammatory effect by decreasing the production of several pro-inflammatory cytokines. Aim: This study aimed to evaluate the efficacy and safety of cEPO in a chronic TNBS-induced colitis model in rodents. Methods: Experimental colitis was induced by weekly intrarectal (IR) administrations of 1% TNBS for 5 weeks in female CD-1 mice. Then, the mice were treated with 500 IU/kg/day or 1000 IU/kg/day of cEPO through intraperitoneal injections for 14 days. Results: cEPO significantly reduced the concentration of alkaline phosphatase (ALP), fecal hemoglobin, tumor necrosis factor (TNF)-α, and interleukin (IL)-10. Also, it demonstrated a beneficial influence on the extra-intestinal manifestations, with the absence of significant side effects of its use. Conclusion: Considering the positive results from cEPO in this experiment, it may arise as a new possible pharmacological approach for the future management of IBD

Thermal Conductivity of Metastable Ionic Liquid [C2mim][CH3SO3]

Ionic liquids have been suggested as new engineering fluids, namely in the area of heat transfer, as alternatives to current biphenyl and diphenyl oxide, alkylated aromatics and dimethyl polysiloxane oils, which degrade above 200 &deg;C and pose some environmental problems. Recently, we have proposed 1-ethyl-3-methylimidazolium methanesulfonate, [C2mim][CH3SO3], as a new heat transfer fluid, because of its thermophysical and toxicological properties. However, there are some interesting points raised in this work, namely the possibility of the existence of liquid metastability below the melting point (303 K) or second order-disorder transitions (&lambda;-type) before reaching the calorimetric freezing point. This paper analyses in more detail this zone of the phase diagram of the pure fluid, by reporting accurate thermal-conductivity measurements between 278 and 355 K with an estimated uncertainty of 2% at a 95% confidence level. A new value of the melting temperature is also reported, Tmelt = 307.8 &plusmn; 1 K. Results obtained support liquid metastability behaviour in the solid-phase region and permit the use of this ionic liquid at a heat transfer fluid at temperatures below its melting point. Thermal conductivity models based on Bridgman theory and estimation formulas were also used in this work, failing to predict the experimental data within its uncertainty

The ulcerogenic profile of Helicobacter pylori paediatric strains associated with peptic ulcer disease.

Helicobacter pylori infection is the major cause of paediatric peptic ulcer disease (PUD). In children with no other aetiology for the disease, this rare event occurs shortly after infection, presuming a still poorly understood higher susceptibility of the patient and highlighting the virulence of the implicated strain. Recently, we showed that the enhanced virulence of a group of paediatric ulcerogenic-strains result from a synergy between their ability to better adapt to the hostility of their niche and the expression of cagA, vacAs1, oipA ‘‘on’’ status, homB and jhp5621. Accordingly, these ulcerogenic strains share a particular proteome profile, providing them with better antioxidant defences, a metabolism favouring the biosynthesis of aromatic amino acids and higher motility1. Corroborating these findings, our preliminary data on electronic microscopic analyses demonstrated the presence of more abundant flagella in PUD-associated paediatric strains, in contrast to the control strain, a paediatric strain associated with non-ulcer dyspepsia (NUD). Compared with paediatric NUD-associated isolates, ulcerogenic H. pylori strains present a greater ability to induce a marked decrease in the gastric cells’ viability and to cause them severe cytoskeleton damage and mucins’ production/secretion impairment1. To uncover the underlying molecular mechanisms, we are now characterizing the modifications induced by these strains in the proteome of human gastric cells, during in vitro infection, by two-dimensional gel electrophoresis followed by mass-spectrometry.Research Grant 2011 – Sociedade Portuguesa de Gastrenterologia