Daniela Gutiérrez

Immunoproteomic Analysis To Identify Shiga Toxin-Producing Escherichia coli Outer Membrane Proteins Expressed during Human Infection

ABSTRACT Shiga-toxin producing Escherichia coli (STEC) is the etiologic agent of acute diarrhea, dysentery, and hemolytic-uremic syndrome (HUS). There is no approved vaccine for STEC infection in humans, and antibiotic use is contraindicated, as it promotes Shiga toxin production. In order to identify STEC-associated antigens and immunogenic proteins, outer membrane proteins (OMPs) were extracted from STEC O26:H11, O103, O113:H21, and O157:H7 strains, and commensal E. coli strain HS was used as a control. SDS-PAGE, two-dimensional-PAGE analysis, Western blot assays using sera from pediatric HUS patients and controls, and matrix-assisted laser desorption ionization–tandem time of flight analyses were used to identify 12 immunogenic OMPs, some of which were not reactive with control sera. Importantly, seven of these proteins have not been previously reported to be immunogenic in STEC strains. Among these seven proteins, OmpT and Cah displayed IgG and IgA reactivity with sera from HUS patients. Genes encoding these two proteins were present in a majority of STEC strains. Knowledge of the antigens produced during infection of the host and the immune response to those antigens will be important for future vaccine development.

Chaperone-Usher Pili Loci of Colonization Factor-Negative Human Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrhea worldwide. Among the 25 different ETEC adhesins, 22 are known as “colonization factors” (CFs), of which 17 are assembled by the chaperone-usher (CU) mechanism. Currently, there is no preventive therapy against ETEC, and CFs have been proposed as components for vaccine development. However, studies of diarrhea-causing ETEC strains worldwide indicate that between 15 and 50% of these are negative for known CFs, hindering the selection of the most widespread structures and suggesting that unknown adhesins remain to be identified. Here, we report the result of a comprehensive analysis of 35 draft genomes of ETEC strains which do not carry known adhesin genes; our goal was to find new CU pili loci. The phylogenetic profiles and serogroups of these strains were highly diverse, a majority of which produced only the heat-labile toxin. We identified 10 pili loci belonging to CU families β (1 locus), γ2 (7 loci), κ (1 locus), and π (1 locus), all of which contained the required number of open reading frames (ORFs) to encode functional structures. Three loci were variants of previously-known clusters, three had been only-partially described, and four are novel loci. Intra-loci genetic variability identified would allow the synthesis of up to 14 different structures. Clusters of putative γ2-CU pili were most common (23 strains), followed by putative β-CU pili (12 strains), which have not yet been fully characterized. Overall, our findings significantly increase the number of ETEC adhesion genes associated with human infections.

Mechanosensory organ regeneration in zebrafish depends on a population of multipotent progenitor cells kept latent by Schwann cells

BackgroundRegenerating damaged tissue is a complex process, requiring progenitor cells that must be stimulated to undergo proliferation, differentiation and, often, migratory behaviors and morphological changes. Multiple cell types, both resident within the damaged tissue and recruited to the lesion site, have been shown to participate. However, the cellular and molecular mechanisms involved in the activation of progenitor cell proliferation and differentiation after injury, and their regulation by different cells types, are not fully understood. The zebrafish lateral line is a suitable system to study regeneration because most of its components are fully restored after damage. The posterior lateral line (PLL) is a mechanosensory system that develops embryonically and is initially composed of seven to eight neuromasts distributed along the trunk and tail, connected by a continuous stripe of interneuromastic cells (INCs). The INCs remain in a quiescent state owing to the presence of underlying Schwann cells. They become activated during development to form intercalary neuromasts. However, no studies have described if INCs can participate in a regenerative event, for example, after the total loss of a neuromast.ResultsWe used electroablation in transgenic larvae expressing fluorescent proteins in PLL components to completely ablate single neuromasts in larvae and adult fish. This injury results in discontinuity of the INCs, Schwann cells, and the PLL nerve. In vivo imaging showed that the INCs fill the gap left after the injury and can regenerate a new neuromast in the injury zone. Further, a single INC is able to divide and form all cell types in a regenerated neuromast and, during this process, it transiently expresses the sox2 gene, a neural progenitor cell marker. We demonstrate a critical role for Schwann cells as negative regulators of INC proliferation and neuromast regeneration, and that this inhibitory property is completely dependent on active ErbB signaling.ConclusionsThe potential to regenerate a neuromast after damage requires that progenitor cells (INCs) be temporarily released from an inhibitory signal produced by nearby Schwann cells. This simple yet highly effective two-component niche offers the animal robust mechanisms for organ growth and regeneration, which can be sustained throughout life.

Una lesión en el cintigrama renal DMSA 6 meses post fase aguda de una pielonefritis representa siempre una cicatriz: un debate abierto

BACKGROUND Abnormal Dimercaptosuccinic acid (DMSA) renal scintigraphy performed six months after an acute pyelonephritis (AP) is generally interpreted as scarring. AIM To perform a follow up of childhood patients showing scintigraphic renal lesions during the acute phase of pyelonephritis (within 7 days from the beginning of fever). MATERIAL AND METHODS A scintigraphic control was carried out at 5-7 months and, in case of persistent lesions, an additional late scintigraphy at 10-13 months. All patients were followed clinically for one year and those with a relapse of urinary tract infection were excluded from the study. RESULTS Eighty five patients with a median age of 8 months were included. Among these, the first scintigraphic control was normal in 59 (69%) and abnormal in 26 patients (31%). In five of these 26 patients (5/26:19%-5/85: 6%), a considerable regression of the lesions was obvious on the early control, and normalized completely on the late control. When expressing the results in kidney units, 107 showed lesions during the acute phase of infection; 69% was normal at the early control. Thirty three showed lesions persisting at the early control (31%) and 7 out of these 33 (21%) became normal on the late control (7/107: 7%). In total, 25% of the children included in the study (24% of the kidney units) remained with renal sequelae one year after the initial episode of AP. CONCLUSIONS The persistence of scintigraphic lesions six months after an episode of AP, does not necessarily correspond to permanent scars, since normalization can sometimes be observed on late controls.Background: Abnormal Dimercaptosuccinic acid (DMSA) renal scintigraphy performed six months after an acute pyelonephritis (AP) is generally interpreted as scarring. Aim: To perform a follow up of childhood patients showing scintigraphic renal lesions during the acute phase of pyelonephritis (within 7 days from the beginning of fever). Material and Methods: A scintigraphic control was carried out at 5-7 months and, in case of persistent lesions, an additional late scintigraphy at 10-13 months. All patients were followed clinically for one year and those with a relapse of urinary tract infection were excluded from the study. Results: Eighty five patients with a median age of 8 months were included. Among these, the first scintigraphic control was normal in 59 (69%) and abnormal in 26 patients (31%). In five of these 26 patients (5/26:19%-5/85: 6%), a considerable regression of the lesions was obvious on the early control, and normalized completely on the late control. When expressing the results in kidney units, 107 showed lesions during the acute phase of infection; 69% was normal at the early control. Thirty three showed lesions persisting at the early control (31%) and 7 out of these 33 (21%) became normal on the late control (7/107: 7%). In total, 25% of the children included in the study (24% of the kidney units) remained with renal sequelae one year after the initial episode of AP. Conclusions: The persistence of scintigraphic lesions six months after an episode of AP, does not necessarily correspond to permanent scars, since normalization can sometimes be observed on late controls.

Coli Surface Antigen 26 Acts as an Adherence Determinant of Enterotoxigenic Escherichia coli and Is Cross-Recognized by Anti-CS20 Antibodies

The coli surface antigen 26 (CS26) of enterotoxigenic Escherichia coli (ETEC) had been described as a putative adhesive pilus based on the partial sequence of the crsH gene, detected in isolates from children with diarrhea in Egypt. However, its production and activity as adherence determinant has not been experimentally addressed. The crsH was identified as a homolog of genes encoding structural subunits of ETEC colonization factors (CFs) CS12, CS18, and CS20. These CFs, along with the recently discovered CS30, belong to the γ2 family of pili assembled by the chaperone-usher pathway (CU pili). Further, the complete CS26 locus, crsHBCDEFG, was described in an O141 ETEC strain (ETEC 100664) obtained from a diarrhea case in The Gambia, during the Global Enterics Multicenter Study. Here, we report that CS26 is a pilus of ∼10 nm in diameter, with the capacity to increase the cell adherence of the non-pathogenic strain E. coli DH10B. As for other related pili, production of CS26 seems to be regulated by phase variation. Deletion of crsHBCDEFG in ETEC 100664 significantly decreased its adherence capacity, which was recovered by in trans complementation. Furthermore, CrsH was cross-recognized by polyclonal antibodies directed against the major structural subunit of CS20, CsnA, as determined by Western blotting and immunogold labeling. ETEC CS26+ strains were found to harbor the heat-labile enterotoxin only, within three different sequence types of phylogroups A and B1, the latter suggesting acquisition through independent events of horizontal transfer. Overall, our results demonstrate that CS26 is an adhesive pilus of human ETEC. In addition, cross-reactivity with anti-CsnA antibodies indicate presence of common epitopes in γ2-CFs.

Perfusión y función ventricular en SPECT cardiaco y coronariografía del mismo día según localización en enfermedad de un vaso

BACKGROUND Single-photon emission computed tomography (SPECT) can be used as a non-invasive tool for the assessment of coronary perfusion. AIM To assess ventricular perfusion and function by SPECT in patients with single vessel coronary artery disease. MATERIAL AND METHODS Among patients with indications for a coronary artery angiography, those with significant lesions in one vessel, were selected for the study. Within 24 hours, cardiac SPECT examinations on basal conditions and after high doses of dipyridamole, were performed. SPECT data from 38 patients with a low probability of coronary artery disease was used for comparisons. RESULTS Ten patients aged 61 ± 8 years (seven men) were studied. Visual analysis of SPECT revealed signs suggestive of ischemia in eight patients. The remaining two patients did not have perfusion disturbances. SPECT detected eight of ten abnormal vessels reported in the coronary artery angiography. There were two false negative results Summed stress, summed rest and summed difference scores were 9.78 ± 6.51, 3.22 ± 5.07 and 6.33 ± 4.97, respectively. The ejection fractions under stress and at rest were 53 ± 11.7% and 61 ± 15.7% respectively (p < 0.01). The figures for the control group were 69.1 ± 13.5% and 75.2 ± 12.04% respectively (significantly different from patients). Two patients had a summed motion score above 14.9. Likewise, two patients had a summed thickening score above 10.9. CONCLUSIONS SPECT detected 80% of coronary lesions found during coronary artery angiography. Visual analysis of perfusion is highly reliable for diagnosis. Quantitative parameters must be considered only as reference parameters.

Bone scintigraphy in children with cat scratch disease.

Aim The objective of this study was to evaluate the degree and incidence of bone involvement in patients with cat scratch disease. Methods Patients admitted between 2004 and 2011 at the pediatric department for cat scratch disease and a positive serology for Bartonella henselae were identified. Only those having undergone a bone scintigraphy (BS) were included in this retrospective study. Results Sixteen girls and 8 boys with a mean age of 7 years were studied. Bone scintigraphy was positive in 6 (25%), but only 2 had bone pain. Axial involvement was present in all 6 patients, and appendicular lesions in 3 of them. Three patients had a BS control, with improvement or normalization after treatment with antibiotics. Conclusions Bone involvement occurs infrequently in patients with cat scratch disease and is not always associated with specific signs. Cat scratch disease must be suspected in patients with fever of unknown origin presenting multifocal lesions on BS.

Influence of extracardiac activity and perfusion abnormalities on myocardial perfusion gated SPECT parameters: Interobserver analysis

OBJECTIVE Extracardiac activity (ECA) may affect interpretation of gated SPECT myocardial perfusion studies (MPSs). To solve this problem, available softwares include myocardial edge delimitation. PURPOSE To evaluate the influence of ECA in automatic myocardial edge detection under normal conditions and with abnormal perfusion and also evaluate the reproducibility of semi-automatic processing. METHODS A total of 100 MPSs, 50 with ECA, were analyzed. Each subgroup included 25 cases with perfusion abnormalities. The cases were processed automatically and by 4 independent operators with different levels of experience. Commercial QGS and QPS softwares were used with tools to mask and relocate the left ventricle area. Functional parameters (final diastolic and systolic volumes and ejection fraction) and perfusion parameters such as the reversibility perfusion score and rest perfusion defect extension were analyzed. The data were compared with Pearsons correlation and Students test. RESULTS Interobserver correlation significantly worsened with the presence of ECA and was moderately affected by perfusion abnormalities. More experienced observers presented better correlation. Reproducibility was greater for the functional perfusion parameters, independently of the observers experience. CONCLUSIONS ECA significantly affects automatic edging delimitation, affecting the MPS values. Interobserver reproducibility with manual processing was more altered regarding functional parameters than in the perfusion scores. Perfusion abnormalities did not interfere with software reproducibility, and when present, better correlation was found. If ECA is not present, manual intervention should be avoided.