Live attenuated TB vaccines representing the three modern Mycobacterium tuberculosis lineages reveal that the Euro–American genetic background confers optimal vaccine potential

Background: Human tuberculosis (TB) is caused by a plethora of Mycobacterium tuberculosis complex (MTBC) strains belonging to seven phylogenetic branches. Lineages 2, 3 and 4 are considered “modern” branches of the MTBC responsible for the majority of worldwide TB. Since the current BCG vaccine confers variable protection against pulmonary TB, new candidates are investigated. MTBVAC is the unique live attenuated vaccine based on M. tuberculosis in human clinical trials. Methods: MTBVAC was originally constructed by unmarked phoP and fadD26 deletions in a clinical isolate belonging to L4. Here we construct new vaccines based on isogenic gene deletions in clinical isolates of the L2 and L3 modern lineages. These three vaccine candidates were characterized at molecular level and also in animal experiments of protection and safety. Findings: Safety studies in immunocompromised mice showed that MTBVAC-L2 was less attenuated than BCG Pasteur, while the original MTBVAC was found even more attenuated than BCG and MTBVAC-L3 showed an intermediate phenotype. The three MTBVAC candidates showed similar or superior protection compared to BCG in immunocompetent mice vaccinated with each MTBVAC candidate and challenged with three representative strains of the modern lineages. Interpretation: MTBVAC vaccines, based on double phoP and fadD26 deletions, protect against TB independently of the phylogenetic linage used as template strain for their construction. Nevertheless, lineage L4 confers the best safety profile

Radiographic and histopathological study of gastrointestinal dysmotility in lipopolysaccharide-induced sepsis in the rat

AbstractSepsis is a highly incident condition in which a cascade of proinflammatory cytokines is involved. One of its most frequent consequences is ileus, which can increase mortality. Animal models such as that induced by systemic administration of lipopolysaccharide (LPS) are useful to deeply evaluate this condition. The effects of sepsis on the gastrointestinal (GI) tract have been explored but, to our knowledge, in vivo studies showing the motor and histopathological consequences of endotoxemia in an integrated way are lacking. Our aim was to study in rats the effects of sepsis on GI motility, using radiographic methods, and to assess histological damage in several organs.MethodsMale rats were intraperitoneally injected with saline or E. coli LPS at 0.1, 1, or 5 mg kg−1. Barium sulfate was intragastrically administered, and X‐rays were performed 0–24 h afterwards. Several organs were collected for organography, histopathology, and immunohistochemistry studies.Key ResultsAll LPS doses caused gastroparesia, whereas changes in intestinal motility were dose‐and time‐dependent, with an initial phase of hypermotility followed by paralytic ileus. Lung, liver, stomach, ileum, and colon (but not spleen or kidneys) were damaged, and density of neutrophils and activated M2 macrophages and expression of cyclooxygenase 2 were increased in the colon 24 h after LPS 5 mg kg−1.Conclusions and InferencesUsing radiographic, noninvasive methods for the first time, we show that systemic LPS causes dose‐, time‐, and organ‐dependent GI motor effects. Sepsis‐induced GI dysmotility is a complex condition whose management needs to take its time‐dependent changes into account

Allergenicity to worldwide invasive grass Cortaderia selloana as environmental risk to public health

Allergies to grass pollen affects about 20% of the population worldwide. In the last few decades, the South American grass Cortaderia selloana (CS, Pampas grass) has expanded worldwide in a variety of countries including the USA, Australia and Western Europe. In many of these locations, CS has strikingly spread and has now been classified an invasive species. Many pernicious consequences of CS have been reported for local biodiversity, landscape and structures. However, the effect on human health has not been studied. To investigate this issue, we have chosen a European region on the northern cost of Spain where CS spread is overwhelming, Cantabria. We obtained CS pollen extract and analysed the allergenic reaction of 98 patients that were allergic to pollen of local grasses. We determined the skin reaction and the presence of specific IgE antibodies (sIgE) to CS or to a typical autochthonous grass, Phleum pratense. We also compared the seasonal symptoms with reported grass pollen counts in the area. The results strongly suggest that CS can cause respiratory allergies at a similar extent to the local grasses. Given that CS pollinises later than the local grasses, this would extend the period of grass allergies in the region for about three months every year, as stated by most of the patients. This is the first study reported on the effects of the striking expansion of CS on human health. Considering the strong impact that respiratory allergies have on the population, our results suggest that CS can currently constitute a relevant environmental health issue.We thank Daniel Liebana Uranga (Cantabria, Spain) for permission to include authorship and data obtained by Marta Uranga, sadly deceased before preparation of the manuscript. We thank Thalia Burn (England, UK) for manuscript text revision and Life Stop Cortaderia (Cantabria, Spain) for image in Fig. 2B-right. This work received financial support from ALK-Abello S.A (Madrid, Spain). This article is dedicated to the memory of Marta Uranga

Prácticas y abandonos

Este artículo presenta uno de los aspectos trabajados en el Primer Informe de Avance del Proyecto de Investigación «No lo quiero tener más, lo vengo a devolver. Características de los circuitos burocráticos-administrativos en el proceso de las  adopciones tramitadas a través del Consejo Provincial del Menor en el período 1973 a 1983 bajo el régimen de la Ley 19.134». Su propósito es realizar una vinculación analítico-reflexiva entre las prácticas descriptas y registradas en loslegajos durante el período estudiado y la construcción de la ctegoría «abandono», siendo esta uno de los motivos principales para declarar el estado de adoptabilidad del menor. El acoplamiento de lo moral a lo material permite observar la trama por la que los circuitos burocráticos administrativos se advienen normalizantes y renovar las posibles razones que dan origen al título este Proyecto: «No lo quiero tener más, lo vengo a devolver[…]»

Phospholipase D1 downregulation by α-synuclein: Implications for neurodegeneration in Parkinson's disease

We have previously shown that phospholipase D (PLD) pathways have a role in neuronal degeneration; in particular, we found that PLD activation is associated with synaptic injury induced by oxidative stress. In the present study, we investigated the effect of α-synuclein (α-syn) overexpression on PLD signaling. Wild Type (WT) α-syn was found to trigger the inhibition of PLD1 expression as well as a decrease in ERK1/2 phosphorylation and expression levels. Moreover, ERK1/2 subcellular localization was shown to be modulated by WT α-syn in a PLD1-dependent manner. Indeed, PLD1 inhibition was found to alter the neurofilament network and F-actin distribution regardless of the presence of WT α-syn. In line with this, neuroblastoma cells expressing WT α-syn exhibited a degenerative-like phenotype characterized by a marked reduction in neurofilament light subunit (NFL) expression and the rearrangement of the F-actin organization, compared with either the untransfected or the empty vector-transfected cells. The gain of function of PLD1 through the overexpression of its active form had the effect of restoring NFL expression in WT α-syn neurons. Taken together, our findings reveal an unforeseen role for α-syn in PLD regulation: PLD1 downregulation may constitute an early mechanism in the initial stages of WT α-syn-triggered neurodegeneration.Fil: Conde, Melisa Ailén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Alza, Natalia Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Química; ArgentinaFil: Iglesias González, Pablo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Scodelaro Bilbao, Paola Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Sánchez Campos, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Uranga, Romina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Salvador, Gabriela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentin

New insights into the transposition mechanisms of IS<i>6110</i> and its dynamic distribution between <i>Mycobacterium tuberculosis</i> Complex lineages

<div><p>The insertion Sequence IS<i>6110</i>, only present in the pathogens of the <i>Mycobacterium tuberculosis</i> Complex (MTBC), has been the gold-standard epidemiological marker for TB for more than 25 years, but biological implications of IS<i>6110</i> transposition during MTBC adaptation to humans remain elusive. By studying 2,236 clinical isolates typed by IS<i>6110</i>-RFLP and covering the MTBC, we remarked a lineage-specific content of IS<i>6110</i> being higher in modern globally distributed strains. Once observed the IS<i>6110</i> distribution in the MTBC, we selected representative isolates and found a correlation between the normalized expression of IS<i>6110</i> and its abundance in MTBC chromosomes. We also studied the molecular regulation of IS<i>6110</i> transposition and we found a synergistic action of two post-transcriptional mechanisms: a -1 ribosomal frameshift and a RNA pseudoknot which interferes translation. The construction of a transcriptionally active transposase resulted in 20-fold increase of the transposition frequency. Finally, we examined transposition in <i>M</i>. <i>bovis</i> and <i>M</i>. <i>tuberculosis</i> during laboratory starvation and in a mouse infection model of TB. Our results shown a higher transposition in <i>M</i>. <i>tuberculosis</i>, that preferably happens during TB infection in mice and after one year of laboratory culture, suggesting that IS<i>6110</i> transposition is dynamically adapted to the host and to adverse growth conditions.</p></div

IS<i>6110</i> gene expression and determination of transposition dynamics in the MTBC.

<p>(<b>a</b>) Total IS<i>6110</i> expression in representative strains from the MTBC. Data are relative to BCG Pasteur. Columns and error bars are the average and standard deviation from three independent cultures. (<b>b</b>) IS<i>6110</i> RFLP from MTBC strains analysed in panel (a). (<b>c</b>) IS<i>6110</i> expression values normalised to the copy number content of this element. Columns represent normalised expression of IS<i>6110</i> according to the left Y-axis. Red squares show the IS<i>6110</i> copy number in each strain indicated in the right Y-axis. Normalised expression of BCG Pasteur is used as reference. (<b>d</b>) Expression per <i>IS6110</i> copy relative to the copy number content in MTBC strains. Data fit with an exponential curve (r² = 0.80) indicated by a grey shadowed line.</p

IS<i>6110</i> in the MTBC.

<p>(<b>a</b>) Schematic phylogenetic relationships of MTBC members arisen from a most recent common ancestor (MRCA) after an evolutionary bottleneck. For <i>M</i>. <i>tuberculosis</i> different lineages and families are indicated. The position of IS<i>6110</i> sequences in fully assembled genomes in indicated by black dots. The arrow indicates the position of IS<i>6110</i> in the Direct Repeat region of the CRISPR-Cas locus, which is common to most MTBC strains. For the remaining 17 <i>M</i>. <i>tuberculosis</i> strains different from H37Rv, the number of IS<i>6110</i> sequences is indicated by a box plot (median = 17). (<b>b</b>) Box plots showing the IS<i>6110</i> copies in MTBC families. For each family, the lineage according to panel (a) is provided in parenthesis in the X-axis. For clarity, L4 have been subdivided into 5 different families according to spoligotyping.</p

Transposition in the laboratory and in a mouse infection model using reference <i>M</i>. <i>bovis</i> and <i>M</i>. <i>tuberculosis</i> strains.

<p>(<b>a</b>) Experimental model to measure transposition rates in <i>M</i>. <i>tuberculosis</i> H37Rv (15 IS<i>6110</i> copies) and <i>M</i>. <i>bovis</i> AF2122/97 (1 IS<i>6110</i> copy). Both strains are transformed with pIR-Km and used to inoculate liquid media or to intranasally infect C57BL/6 mice. After the indicated time points aliquots are plated in kanamycin and sucrose containing plates to ensure pIR-km loss and to recover colonies resulting from transposition. (<b>b</b>) Transposition frequencies in laboratory medium in <i>M</i>. <i>bovis</i> and <i>M</i>. <i>tuberculosis</i> are indicated by red and blue columns respectively. Error bars indicate the standard deviation of the mean value from three independent cultures. Transposition preferentially occurs in <i>M</i>. <i>tuberculosis</i> after the stationary phase reaching it maximum in the starvation period. (<b>c</b>) Expression of <i>M</i>. <i>tuberculosis</i> IS<i>6110</i> during exponential, stationary and starvation periods <i>in vitro</i>. Expression of ORF1 and ORF2 are indicated by dark and light blue columns respectively. Results are from three independent cultures. (<b>d</b>) Transposition frequencies during mouse infection with <i>M</i>. <i>bovis</i> or <i>M</i>. <i>tuberculosis</i> are indicated by red and blue columns respectively. Data from lung and spleen are shown and error bars indicate the standard deviation of the mean value from three independent mice. <i>M</i>. <i>bovis</i> does not exhibit increased transposition rates <i>in vivo</i> relative to liquid culture. Conversely <i>M</i>. <i>tuberculosis</i> show 10-fold higher transposition rates compared to exponential growth <i>in vitro</i>. In all cases, transposition frequencies were calculated relative to the total number of CFU in either cultures or mouse organs.</p