The tpi Gene as a Tool for Epidemiological Studies of Giardiasis

Giardia duodenalis is a protozoon that causes infection in humans and animals. It can be transmitted by contaminated water, from person to person or by contact with animals; it being the cause one of the most common intestinal infections in our country, so it is a public health concern. The epidemiological study thereof requires the molecular characterization of parasites, using genes with great variability, such as the one that codes triosephosphate isomerase (tpi), and analizing the homology between isolates.The purpose of this work is to establish the identity criterion for epidemiological comparison of Giardia isolates.2-3 stool samples were collected in alternate days from 26 patients with giardiasis. After DNA extraction, a fragment of the tpi gene and a fragment of the beta-giardin (bg) gene—used for comparison purposes—were amplified by means of PCR techniques. The obtained fragments were sequenced and the sequences analyzed with the BioEdit and DnaSP v.5.0 software.The tpi gene sequences showed a high divergence, with values of diversity Π ranging from 0 to 0.21219. The appearance of multiple peaks in the chromatogram points to the presence of various clones in the same sample. The differences between isolates from the same patient where equal or higher than those found for the collection of all samples.The variability of the tpi gene does not allow identity criteria to be established, which are necessary for isolate identification. Mixed intragenotype infections occur very frequently, which suggests the environmental path is the principal path of transmission and/ or there is very high genetic variability.</p

O gene tpi como ferramenta nos estudos epidemiológicos da giardíase

Giardia duodenalis is a protozoon that causes infection in humans and animals. It can be transmitted by contaminated water, from person to person or by contact with animals; it being the cause one of the most common intestinal infections in our country, so it is a public health concern. The epidemiological study thereof requires the molecular characterization of parasites, using genes with great variability, such as the one that codes triosephosphate isomerase (tpi), and analizing the homology between isolates.The purpose of this work is to establish the identity criterion for epidemiological comparison of Giardia isolates.2-3 stool samples were collected in alternate days from 26 patients with giardiasis. After DNA extraction, a fragment of the tpi gene and a fragment of the beta-giardin (bg) gene—used for comparison purposes—were amplified by means of PCR techniques. The obtained fragments were sequenced and the sequences analyzed with the BioEdit and DnaSP v.5.0 software.The tpi gene sequences showed a high divergence, with values of diversity Π ranging from 0 to 0.21219. The appearance of multiple peaks in the chromatogram points to the presence of various clones in the same sample. The differences between isolates from the same patient where equal or higher than those found for the collection of all samples.The variability of the tpi gene does not allow identity criteria to be established, which are necessary for isolate identification. Mixed intragenotype infections occur very frequently, which suggests the environmental path is the principal path of transmission and/ or there is very high genetic variability.Giardia duodenalis es un protozoo que causa infección en humanos y animales, que se puede transmitir por vía hídrica, de persona a persona o por contacto con animales, siendo una de las infecciones intestinales más frecuentes en nuestro país, por lo que supone una preocupación de Salud Pública. Su estudio epidemiológico, requiere la caracterización molecular de los parásitos, utilizando genes con gran variabilidad como el que codifica la triosafosfatoisomerasa (tpi) y analizando la homología entre aislamientos.El objetivo del trabajo es establecer el criterio de identidad que permita la comparación epidemiológica de los aislamientos de Giardia.Se recogieron 2-3 muestras de heces en días alternos, de 26 pacientes con giardiosis. Tras la extracción de ADN, se amplificaron por técnicas de PCR, un fragmento del gen tpi y un fragmento del gen de la beta-giardina (bg), que se utilizó como comparación. Los fragmentos obtenidos fueron secuenciados y las secuencias analizadas con los programas BioEdit y DnaSP v.5.0.Las secuencias del gen tpi mostraron una elevada divergencia, con valores de diversidad Π entre 0 y 0,21219. La aparición de picos múltiples en el cromatograma, indicaron la presencia de varios clones en la misma muestra. Las diferencias entre aislamientos del mismo paciente fueron iguales o mayores que las encontradas para el conjunto de todas las muestras.La variabilidad del gen tpi no permite establecer unos criterios de identidad, necesarios para la identificación de aislamientos. Las infecciones mixtas intragenotipo ocurren de una forma muy frecuente, sugiriendo una implicación de la vía ambiental como principal fuente de transmisión o una variación genética muy elevada.Giardia duodenalis é um protozoário que causa infeção em humanos e animais, que se pode transmitir por via hídrica, de pessoa a pessoa ou por contacto com animais, sendo uma das infeções intestinais mais frequentes no nosso país, representando uma preocupação de Saúde Pública. O seu estudo epidemiológico requer a caracterização molecular dos parasitas utilizando genes com grande variedade como o que codifica a triose-fosfato isomerase (tpi) e analisando a homologia entre isolamentos.O objetivo do trabalho é estabelecer o critério de identidade que permita a comparação epidemiológica dos isolamentos de Giardia.Recolheram-se 2-3 amostras de fezes, em dias alternados, de 26 pacientes com giardíase. Após a extração de ADN, um fragmento do gene tpi e um fragmento do gene de beta-giardina (bg) foram amplificados por técnicas de PCR, que foram utilizadas como comparação. Os fragmentos obtidos foram sequenciados e as sequências analisadas com os programas BioEdit y DnaSP v.5.0.As sequências do gene tpi mostraram uma elevada divergência, com valores de diversidade Π entre 0 e 0,21219. O aparecimento de múltiplos picos no cromatograma indicaram a presença de vários clones na mesma amostra. As diferenças entres isolamentos do mesmo paciente foram iguais ou maiores que as encontradas para o conjunto de todas as amostras.A variabilidade do gene tpi não permite estabelecer alguns critérios de identidade necessários para a identificação de isolamentos. As infeções mistas intragenótipo ocorrem de uma forma muito frequente, sugerindo uma implicação da via ambiental como principal fonte de transmissão e/ou uma variação genética muito elevada

LPS-induced down-regulation of NO-sensitive guanylyl cyclase in astrocytes occurs by proteasomal degradation in nuclear bodies

From 3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications.-- This abstract is available from: http://www.biomedcentral.com/1471-2210/7/S1/P3[Background] We have previously shown that inflammatory agents (LPS, IL-1β, β-amyloid peptides) that induce reactivity and NOS-2 expression in glial cells down-regulate astroglial soluble guanylyl cyclase (sGC) in vitro and in vivo.[Results] Here we show that the decrease in sGC activity and β1 subunit protein induced by LPS (10 ng/ml, 24 h) in cultured rat cerebellar astrocytes is prevented by inhibitors of proteasome activity (MG132 5 μM; lactacystin 10 μM) whereas other protease inhibitors (calpain inhibitor 25 μM; ICE inhibitor II 100 μM and leupeptin 5 μM) were not effective. Furthermore, immunocytochemistry and confocal laser microscopy revealed that in LPS-treated cells a strong sGC β1 immunorreactivity is evident in aggregates in the cell nuclei where it co-localizes with 20S proteasomes and ubiquitin in clastosomes, nucleoplasmic substructures involved in ubiquitin-proteasome-dependent nuclear proteolysis, but do not colocalize with others proteasome-enriched structures include promyelocytic leukaemia bodies and splicing speckles. In contrast, in untreated astrocytes clastosomes are scarce and sGC β1 immunorectivity shows a diffuse cytoplasmic pattern, while in the nucleus it is very weak. A similar distribution is observed when cells are treated with LPS and the proteasome inhibitor MG132 or the protein synthesis inhibitor cycloheximide.[Conclusion] LPS orchestrates the recruitment of sGC-β1 protein and components of the ubiquitin-proteasome system to specialized nuclear bodies, clastosomes, suggesting a mechanism for inflammation-induced down-regulation of sGC in astrocytes.This work was supported by a SAF2004-01717 grant (Spain).Peer reviewe

Use of Oral Bisphosphonates in Primary Prevention of Fractures in Postmenopausal Women: A Population-Based Cohort Study

<div><p>Oral bisphosphonates are first-line drugs in the treatment of osteoporosis under most guidelines, and have been shown to decrease risk of first fracture only in asymptomatic vertebral fractures and in clinical trial populations that are generally very different from the general population.</p><p>Objective</p><p>To compare incidence of first osteoporotic fracture in two cohorts of postmenopausal women, one treated with bisphosphonates and the other only with calcium and vitamin D.</p><p>Design</p><p>Retrospective population cohort study with paired matching based on data from electronic health records.</p><p>Setting</p><p>Women aged 60 years and older in 2005, from 21 primary care centers in a healthcare region of Spain.</p><p>Participants</p><p>Two groups of women aged 60 years and older (n = 1208), prescribed either calcium and vitamin D (CalVitD) or bisphosphonates (BIPHOS) with or without calcium and vitamin D, were compared for the end point of first recorded osteoporotic-related fracture, with 5-years follow-up.</p><p>Main Outcome Measure</p><p>Incidence of first fracture: Vertebral fracture, osteoporosis with pathological fracture, fracture of the upper humeral epiphysis, fracture of the lower radial epiphysis, or femur fracture.</p><p>Results</p><p>Estimated 10-year risk of fracture was 11.4% (95% confidence interval: 9.6 to 13.2), 11.8% (9.2 to 14.3) in the BIPHOS group and 11.1% (8.6 to 13.6) in the CalVitD group. No significant differences were found between groups in total fractures (Hazard ratio = 0.934 (0.67 to 1.31)) or location (vertebral, femoral, radial or humeral).</p><p>Conclusions</p><p>In postmenopausal women, bisphosphonates have not been shown to better decrease risk of first fracture compared with calcium and vitamin D therapy alone.</p></div

Frequency and fracture risk according to study group and basal comorbidity.

<p>Frequency and fracture risk according to study group and basal comorbidity.</p

Study variables.

<p>Study variables.</p

Incidence curves of fracture during 5 years follow-up by group (BIPHOS / CalVitD).

<p>Incidence curves of fracture during 5 years follow-up by group (BIPHOS / CalVitD).</p

Absolute differences between groups (BIPHOS-CalVitD) pre and post matching.

<p>BMI: Body mass index; DM: Type II diabetes mellitus; PF: Behavioural syndromes associated with physiological disturbances and physical factors; EM: Extrapyramidal and movement disorders; EP: Episodic and paroxysmal disorders; HT: Hypertension; IC: Ischaemic heart diseases; CV: Cerebrovascular disease; COPD: Chronic lower respiratory diseases; IP: Inflammatory polyarthropathies; ART: Arthrosis; AD: Antidepressants; PPIs: Proton-pump inhibitors; GC: Glucocorticoids; BZ: Benzodiazepine; OP: Opiates.</p