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Inherited hearing impairment is a highly heterogeneous group of disorders with an overall incidence of about 1 in 2000 newborns. (1) Among them, prelingual, severe hearing loss with no other [...]

BCG, the only vaccine licensed against tuberculosis, demonstrates variable efficacy in humans. Recent preclinical studies highlight the potential for mucosal BCG vaccination to improve protection. Lung tissue-resident memory T cells reside within the parenchyma, potentially playing an important role in protective immunity to tuberculosis. We hypothesised that mucosal BCG vaccination may enhance generation of lung tissue-resident T cells, affording improved protection against Mycobacterium tuberculosis. In a mouse model, mucosal intranasal (IN) BCG vaccination conferred superior protection in the lungs compared to the systemic intradermal (ID) route. Intravascular staining allowed discrimination of lung tissue-resident CD4+T cells from those in the lung vasculature, revealing that mucosal vaccination resulted in an increased frequency of antigen-specific tissue-resident CD4+T cells compared to systemic vaccination. Tissue-resident CD4+T cells induced by mucosal BCG displayed enhanced proliferative capacity compared to lung vascular and splenic CD4+T cells. Only mucosal BCG induced antigen-specific tissue-resident T cells expressing a PD-1+KLRG1−cell-surface phenotype. These cells constitute a BCG-induced population which may be responsible for the enhanced protection observed with IN vaccination. We demonstrate that mucosal BCG vaccination significantly improves protection over systemic BCG and this correlates with a novel population of BCG-induced lung tissue-resident CD4+T cells.

Cell membrane proteins are sensitive to their fatty acid environment; however, the effects of those fatty acids are not completely understood. In this work, we have optimized a model for the study of membrane protein regulation, which allows the investigation of lipid metabolism in cultured tumor cells. The membrane fatty acid composition of Reuber H35 hepatoma cells was modified by enriching phospholipids in individual fatty acids of different length and degree of saturation. Lauric, myristic, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids were supplemented to the culture medium of cells growing in presence or absence of lipoproteins. The four fatty acids were incorporated into membrane phospholipids in a dose-dependent manner. Lauric and myristic acids were actively metabolized, increasing percentages of longer and unsaturated chain fatty acids, but they did not modify the unsaturated index. As expected, EPA and DHA significantly decreased the unsaturated index. Incorporation of EPA and DHA was accompanied of a significant decrease of total monounsaturated fatty acids and an increase of total saturated fatty acids. Our results demonstrate that Reuber H35 hepatoma cells constitute a suitable model to manipulate the lipid composition of the cell membranes and perform functionality studies on the membrane proteins in response to their lipid environment.

ABSTRACTPrevious experiments for the identification of novel diagnostic or vaccine candidates for bovine tuberculosis have followed a targeted approach, wherein specific groups of proteins suspected to contain likely candidates are prioritized for immunological assessment (for example, with in silicoapproaches). However, a disadvantage of this approach is that the sets of proteins analyzed are restricted by the initial selection criteria. In this paper, we describe a series of experiments to evaluate a nonbiased approach to antigen mining by utilizing a Gateway clone set for Mycobacterium tuberculosis, which constitutes a library of clones expressing 3,294 M. tuberculosisproteins. Although whole-blood culture experiments using Mycobacterium bovis-infected animals and M. bovisBCG-vaccinated controls did not reveal proteins capable of differential diagnosis, several novel immunogenic proteins were identified and prioritized for efficacy studies in a murine vaccination/challenge model. These results demonstrate that Rv3329-immunized mice had lower bacterial cell counts in their spleens following challenge with M. bovis. In conclusion, we demonstrate that this nonbiased approach to antigen mining is a useful tool for identifying and prioritizing novel proteins for further assessment as vaccine antigens.

To further unravel the mechanisms responsible for attenuation of the tuberculosis vaccine Mycobacterium bovis BCG, comparative genomics was used to identify single nucleotide polymorphisms (SNPs) that differed between sequenced strains of Mycobacterium bovis and M. bovis BCG. SNPs were assayed in M. bovis isolates from France and the United Kingdom and from different BCG vaccines in order to identify those that arose during the attenuation process which gave rise to BCG. Informative data sets were obtained for 658 SNPs from 21 virulent M. bovis strains and 13 BCG strains; these SNPs showed phylogenetic clustering that was consistent with the geographical origin of the strains and previous schemes for BCG genealogies. The data revealed a closer relationship between BCG Tice and BCG Pasteur than was previously appreciated, while we were able to position BCG Beijing within a grouping of BCG Denmark-derived strains. Only 186 SNPs were identified between virulent M. bovis strains and all BCG strains, with 115 nonsynonymous SNPs affecting important functions such as global regulators, transcriptional factors, and central metabolism, which might impact on virulence. We therefore refine previous genealogies of BCG vaccines and define a minimal set of SNPs between virulent M. bovis strains and the attenuated BCG strain that will underpin future functional analyses.

ABSTRACTTo further unravel the mechanisms responsible for attenuation of the tuberculosis vaccine Mycobacterium bovisBCG, comparative genomics was used to identify single nucleotide polymorphisms (SNPs) that differed between sequenced strains of Mycobacterium bovisand M. bovisBCG. SNPs were assayed in M. bovisisolates from France and the United Kingdom and from different BCG vaccines in order to identify those that arose during the attenuation process which gave rise to BCG. Informative data sets were obtained for 658 SNPs from 21 virulent M. bovisstrains and 13 BCG strains; these SNPs showed phylogenetic clustering that was consistent with the geographical origin of the strains and previous schemes for BCG genealogies. The data revealed a closer relationship between BCG Tice and BCG Pasteur than was previously appreciated, while we were able to position BCG Beijing within a grouping of BCG Denmark-derived strains. Only 186 SNPs were identified between virulent M. bovisstrains and all BCG strains, with 115 nonsynonymous SNPs affecting important functions such as global regulators, transcriptional factors, and central metabolism, which might impact on virulence. We therefore refine previous genealogies of BCG vaccines and define a minimal set of SNPs between virulent M. bovisstrains and the attenuated BCG strain that will underpin future functional analyses.

The mechanisms by which saturated and polyunsaturated fatty acids may exert their effects on levels of blood cholesterol and human atherosclerosis have not been fully established. In this work, we studied the translational effects of myristic (14:0) and eicosapentaenoic (20:5) acids on 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Reuber H35 hepatoma cells. This enzyme is an intrinsic membrane, 96-kDa protein whose proteolysis releases an enzymatically active, 52- to 56-kDa, soluble fragment. We optimized an immunoblot procedure for quantifying small amounts of both the native and the soluble forms of HMG-CoA reductase from Reuber H35 hepatoma cells. We demonstrated that the upregulation of HMG-CoA reductase by myristic acid is due to an increase of the HMG-CoA reductase protein; therefore, protein synthesis would be required for the increase of HMG-CoA reductase activity caused by this fatty acid. In contrast, the downregulation of HMG-CoA reductase caused by eicosapentaenoic acid is not due to decreased protein synthesis, since similar levels of protein were found in the presence and absence of this fatty acid. Results obtained with cycloheximide as a protein-synthesis inhibitor confirm these findings.

Abstract:  The defective major histocompatibility complex (MHC) DRB6 gene is transcribed into mRNA in human [peripheral blood lymphocytes, transfected and Epstein-Barr virus (EBV)] and chimpanzee EBV cell lines. MHC-DRB6 presents several anomalies, which include stop codons in exon 2, lack of the usual polyadenilation signal of other MHC-DRB genes, and a promoter region and exon 1 taken from a locally inserted retrovirus. The complete cDNA sequences from human DRB6*0201 and three common chimpanzee alleles (Patr-DRB6*0108, Patr-DRB6*0109, Patr-DRB6*0111) have been obtained; two exon 1-exon 2 cDNA sequences from bonobos (Papa-DRB6*0101 and Papa-DRB6*0102) are also shown. In contrast to chimpanzee DRB6 transcripts, the human ones: (1) present an exon 1-exon 2 splicing site that includes the transcription of the first 141 nucleotides of intron 1, rendering a longer exon 1, and (2) show a duplication of exon 6, which would render a longer cytoplasmic tail in a putative DRB6 protein. These two characteristics are found in all the human sequences obtained, regardless of the cellular type tested, and they are not present in any of the chimpanzee alleles reported; consequently, they are human-specific. All the alleles reported here bear stop codons in the three possible reading frames; however, a certain level of expression of DRB6 has been observed by cytofluorometry. This could be due to the presence of a selenocysteine insertion sequence (SECIS) stem-loop structure located at the 3 untranslated region of the DRB6 mRNA, which directs selenocysteine incorporation at UGA codons. DRB6 transcription and translation would be the first gene model of a readingthrough stop codon mechanism in primate MHC. It is also feasible that the DRB6 gene might generate a population of short polypeptides, bound to plasmatic membranes, having non-antigen-presenting functions or which are presented by other MHC molecules as HLA-E presents HLA-G and -B leader sequence-derived peptides.

Two batches ofHelicella (H.) itala (adult specimens) and two ofCepaea nemoralis (adult and young specimens) were experimentally infected with larvae I (L-I) ofMuellerius sp. andNeostrongylus linearis obtained from the lungs and faeces ofRupicapra rupicapra. In assess larval development, the number and percentage of the total number of larvae (L-I+L-II+L-III) per mollusc were studied, together with the number and percentage of L-III per snail and the days on which the different larval stages were reached. The development ofMuellerius sp. andN. linearis was greater in larvae from faeces. For both species of molluscs, the values for the percentages of the total number of larvae and L-III were higher inN. linearis than inMuellerius sp., but there were no notable differences in the days on which the various larval stages were reached. Both nematodes achieved a greater degree of development in young specimens ofC. nemoralis than in adults. Whether the larvae came from facces or the lungs,H. (H.) itala was a better intermediate host thanC. nemoralis forMuellerius sp. andN. linearis.

La encefalopatía traumática crónica (ETC) es una enfermedad neurodegenerativa que afecta a personas que han padecido traumatismos craneales repetitivos. No obstante, también durante el seguimiento de los pacientes con traumatismo craneoencefálico (TCE) único se pueden observar cambios respecto de su situación previa. Presentamos cuatro casos clínicos de pacientes visitados en la consulta externa del Instituto Guttmann entre 2017 y 2019, afectos de secuelas leves de TCE grave y único que han desarrollado posteriormente una enfermedad neurodegenerativa sin un diagnóstico concreto y que pudiesen cumplir criterios clínicos de síndrome de encefalopatía traumática crónica. Los médicos rehabilitadores son los profesionales con mayor posibilidad de identificar estos pacientes, indicando las exploraciones complementarias necesarias y estableciendo nuevos objetivos de rehabilitación.

The optimal conditions for measuring 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity in Reuber H35 hepatoma cells are described in this paper. Cells in the exponential phase of growth were lysed by incubation with Brij 97 detergent for 30 min. We used imidazole buffer supplemented with EDTA and leupeptine, two inhibitors of proteases. Disrupted cells were then centrifuged at 12000g. Although microsomes are usually reported as enzyme preparations for measuring HMG-CoA reductase, our data showed that hepatoma cells may be used without previous isolation of microsomes. The 12000g supernatant showed similar levels of total and specific activities to those found in the microsomal fraction obtained after 105000g centrifugation. The soluble fraction showed less than 10% of reductase activity. Reductase activity from Reuber H35 hepatoma cells increased proportionally to the reaction time from 30 to 90 min and to the amount of protein added in a range of 50–500 μg. Our modified method was very sensitive and reproducible, because very low specific activity (about 15–100 pmol min⁻¹ per mg protein) could be quantified in different assay conditions obtaining similar values.