Cell- and gene-therapy approaches to inner ear repair

18 páginas.Sensorineural hearing loss is the most common sensory disorder in humans. It is primarily due to the degeneration of highly specialised mechanosensory cells in the cochlea, the so-called hair cells. Hearing problems can also be caused or further aggravated by the death of auditory sensory neurons that convey the information from the hair cells to the brain stem. Despite the discovery of stem/progenitor cells in the mammalian cochlea, no regeneration of either damaged hair cells or auditory neurons has been observed in mammals, in contrast to what is seen in avians and non-mammalian vertebrates. The reasons for this divergence have not yet been elucidated, although loss of stem cells and/or loss of their phenotypic plasticity in adult mammals have been put forward as possible explanations. Given the high incidence of this disorder and its economic and social implications, a considerable number of research lines have been set up aimed towards the regeneration of cochlear sensory cell types. This review summarizes the various routes that have been explored, ranging from the genetic modification of endogenous cells remaining in the inner ear in order to promote their transdifferentiation, to the implantation of exogenous stem or progenitor cells and their subsequent differentiation within the host tissue. Prophylactic treatments to fight against progressive sensory cell degeneration in the inner ear are also discussed.This work was supported by a grant from the MiCINN (PLE2009-0098), Ciberned, Tercel and Red de Terapia Celular de Castilla y León.Peer reviewe

In vivo and in vitro treatments against Sparicotyle chrysophrii (Monogenea: Microcotylidae) parasitizing the gills of gilthead sea bream (Sparus aurata L.)

The effect of in vitro and in vivo treatments against Sparicotyle chrysophrii, a microcotylid parasite of gilthead sea bream (Sparus aurata L.), was studied. In vitro chemical treatments were targeted to eggs, oncomiracidia and adults, and were tested both as disinfectants and therapeutics for infected animals. The compounds were: distilled water, formalin, limoseptic ®, hydrogen peroxide, chlorine, and praziquantel (PZQ). Larvae were sensitive to all the treatments, but adults were more resistant, as chlorine (60 ppm - 1 h), hydrogen peroxide (100 ppm - 30 min) and PZQ (50 ppm - 30 min) produced only 10% mortality. All adults were killed only with distilled water, limoseptic (0.1% - 5 min), formalin (300 ppm - 30 min), or hydrogen peroxide (200 ppm - 30 min). Eggs were the most resistant stage, as only 30 min in limoseptic (0.1% in distilled water) or in formalin (300 ppm) prevented hatching. PZQ was used in vivo either as a curative or preventive treatment. The highest dose tested (400 mg kg- 1 BW; effective dose 116.3 mg kg- 1 BW due to palatability problems leading to 45% reduction in host food intake) did not significantly decrease prevalence of infection when given for 6 consecutive days. A lower dose (200 mg kg- 1 BW) (effective dose 158.1 mg kg- 1 BW) was rejected to a lower degree and decreased the prevalence of infection from 90% to 40%. When a lower dose (40 mg kg- 1 BW) was administered for longer periods (20 days), food intake was reduced slightly, but the infection did not decrease significantly. The oral intubation with PZQ (200 mg kg- 1 BW) once a week for 4 weeks did not prevent the infection of fish by cohabitation. However, a significant reduction in the abundance of the parasite was registered. In view of the results, recommendations for fish treatment and disinfection of aquaculture facilities are discussed.Funding for this study was provided by the Spanish Ministerio de Ciencia y Tecnología (project no. AGL-2002-0475-C02-C01). The authors are grateful to M. Alonso and P. Cabrera for excellent assistance in experimental infections, and to Ramon Soriano (Andrés Pintaluba S.A.) for providing the medicated food

Immune and haematological parameters of Blackbelly ewes infected with gastrointestinal nematodes

Background: It is necessary to identify phenotypic traits related to natural resistance against gastrointestinal nematodes (GIN) in order to know the host immunity status in productive ewes. Objetive: To determine haematological and immunological parameters (IgA and IgG) during pregnancy and lactation in Blackbelly ewes naturally infected with GIN Methods: The number of eggs per gram (EPG), packed cell volume (% PCV), plasmatic protein (PP), and peripheral eosinophils were determined during eight months. In addition, sera and saliva samples were collected to establish IgG and IgA kinetics by indirect enzyme-linked immunosorbent assay (ELISA). Results: The results showed 2,592 ± 2,403 EPG and 22.2 ± 4.0% PCV during lactation and 595 ± 901 EPG and 25.1 ± 2.5% PCV during pregnancy. A higher percentage of Trichostrongylus colubriformis larvae were observed in pregnancy (84 to100%) than in lactation (36 to 44%). The IgA activity in serum samples showed a marked reduction (from 80 to 10%) during lambing for both Haemonchus contortus and T. colubriformis antigens. In saliva samples, IgA activity with regard to the standard decreased from 56% at 60 days to 30% at 45 days before lambing and remained low for 45 days during lactation (23 to 32% activity). The eosinophils numbers were 2.0 x 109 cells L-1 in pregnancy and remained low at 0.7 x 109 cells L-1 in lactation. Conclusion: The studied variables reflect the breakdown of immunity against GIN in Blackbelly ewes before and after lambing.Antecedentes: A identificação de traços fenotípicos relacionados à resistência natural contra nematóides gastrintestinais (GIN) é necessária para saber a imunidade do hospedeiro em ovelhas produtivas. Objetivo: Determinar parâmetros hematológicos e imunológicos (IgA e IgG) em períodos de gestação e lactação em ovelhas Blackbelly naturalmente infectados com GIN. Métodos: O número de ovos por grama (EPG), volume empacotado de células (% PCV), proteína plasmática (PP) e eosinófilos periféricos foram determinados durante oito meses. Além disso, as amostras de soro e saliva foram recolhidas para determinar a cinética de IgG e IgA por ELISA indireto. Resultados: Os resultados mostraram 2.592 ± 2.403 EPG e 22,2 ± 4,0% PCV durante a lactação e 595 ± 901 EPG e 25,1 ± 2,5% PCV durante a gravidez. A percentagem mais elevada de larvas de Trichostrongylus colubriformis foi observada na gravidez (84 a 100%) do que na lactação (36 a 44%). A atividade de IgA em amostras de soro mostrou uma redução acentuada (80 a 10%) durante o parto nos antígenos de Haemonchus contortus e T. colubriformis. Em amostras de saliva, a atividade de IgA diminuiu de 56 a 30% do dia 60 ao 45 antes do parto e permaneceu baixa por 45 dias durante a lactação (atividade de 23 a 32%). O número de eosinófilos foi de 2,0 x 109 células L-1 na gravidez e manteve-se baixo, com 0,7 x 109 células L-1 na lactação. Conclusaõ: As variáveis estudadas refletem a quebra da imunidade contra GIN em ovelhas Blackbelly antes e depois do parto.Antecedentes: La identificación del fenotipo relacionado con la resistencia contra nematodos gastrointestinales (GIN) es necesaria para conocer la inmunidad del huésped en ovejas en producción. Objetivo: Determinar los parámetros hematológicos e inmunológicos (IgA e IgG) en gestación y lactancia en ovejas Blackbelly infectadas naturalmente con GIN. Métodos: Se determinó el número de huevos por gramo de heces (EPG), se identificaron las larvas y se registró el porcentaje del volumen celular aglomerado (% PCV), proteína plasmática (PP) y eosinófilos periféricos durante ocho meses. Además, se colectó suero y saliva para determinar la cinética de IgG e IgA por medio de un ensayo inmuno-enzimático (ELISA) indirecto. Resultados: Los resultados mostraron 2.592 ± 2.403 EPG y 22,2 ± 4,0% PCV durante la lactancia y 595 ± 901 EPG y 25,1 ± 2,5% PCV durante la gestación. Se observó un mayor porcentaje de larvas de Trichostrongylus colubriformis en gestación (84 a 100%) que en lactancia (36 a 44%). La actividad de la IgA en las muestras de suero mostró una marcada reducción después del parto para ambos antígenos de Haemonchus contortus y T. colubriformis (80 a 10%). En saliva, la actividad de la IgA disminuyó de 56 a 30% del día 60 al 45 antes del parto y se mantuvo baja en los primeros 45 días de la lactancia (actividad de 23 a 32%). El número de eosinófilos fue de 2,0 x 109 células L-1 durante la gestación, y se redujo a 0,7 x 109 células L-1 en la lactancia. Conclusión: Las variables estudiadas reflejan la ruptura de la inmunidad contra GIN en ovejas Blackbelly antes y después del parto

Immune and haematological parameters of Blackbelly ewes infected with gastrointestinal nematodes

Abstract Background: It is necessary to identify phenotypic traits related to natural resistance against gastrointestinal nematodes (GIN) in order to know the host immunity status in productive ewes. Objetive: To determine haematological and immunological parameters (IgA and IgG) during pregnancy and lactation in Blackbelly ewes naturally infected with GIN Methods: The number of eggs per gram (EPG), packed cell volume (% PCV), plasmatic protein (PP), and peripheral eosinophils were determined during eight months. In addition, sera and saliva samples were collected to establish IgG and IgA kinetics by indirect enzyme-linked immunosorbent assay (ELISA). Results: The results showed 2,592 ± 2,403 EPG and 22.2 ± 4.0% PCV during lactation and 595 ± 901 EPG and 25.1 ± 2.5% PCV during pregnancy. A higher percentage of Trichostrongylus colubriformis larvae were observed in pregnancy (84 to100%) than in lactation (36 to 44%). The IgA activity in serum samples showed a marked reduction (from 80 to 10%) during lambing for both Haemonchus contortus and T. colubriformis antigens. In saliva samples, IgA activity with regard to the standard decreased from 56% at 60 days to 30% at 45 days before lambing and remained low for 45 days during lactation (23 to 32% activity). The eosinophils numbers were 2.0 x 109 cells L-1 in pregnancy and remained low at 0.7 x 109 cells L-1 in lactation. Conclusion: The studied variables reflect the breakdown of immunity against GIN in Blackbelly ewes before and after lambing

Generation of inner ear sensory cells from bone marrow-derived human mesenchymal stem cells

[Aim]: Hearing loss is the most common sensory disorder in humans, its main cause being the loss of cochlear hair cells. We studied the potential of human mesenchymal stem cells (hMSCs) to differentiate towards hair cells and auditory neurons. [Materials & methods]: hMSCs were first differentiated to neural progenitors and subsequently to hair cell- or auditory neuron-like cells using in vitro culture methods. [Results]: Differentiation of hMSCs to an intermediate neural progenitor stage was critical for obtaining inner ear sensory lineages. hMSCs generated hair cell-like cells only when neural progenitors derived from nonadherent hMSC cultures grown in serum-free medium were exposed to EGF and retinoic acid. Auditory neuron-like cells were obtained when treated with retinoic acid, and in the presence of defined growth factor combinations containing Sonic Hedgehog. [Conclusion]: The results show the potential of hMSCs to give rise to inner ear sensory cells. © 2012 Future Medicine Ltd.Peer Reviewe

Discovery of a splicing regulator required for cell cycle progression.

In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms

Structure-function characterization of TgRRM1.

<p>(A) The designs of wild type and five TgRRM1 deletion constructs are shown. All polypeptides were expressed under control of the α-tubulin promoter and a DDmyc_3X tag was fused to the N-terminal end of each protein for visualization (DD = FKBP destabilization domain) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen.1003305-HermGotz1" target="_blank">[46]</a>. The residue numbers refer to the proportion of TgRRM1 coding included in each construct. Note omitting shield 1 reduced wt-TgRRM1^DDmyc protein levels ∼3-fold, however, this minimal change was not sufficient to prevent genetic complementation (not shown). Thus, representative results of parasites cultured in standard culture media plus 100 nM shield 1 are shown only. Immunofluorescent images of stable transgenic clones expressing the corresponding constructs on the left were developed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen-1003305-g003" target="_blank">Figure 3</a>. The right panels show Nomarski images of the plaques formed by the transgenic parasites grown at 40°C. Magnification bar (5 µm) is shown. Note that genetic complementation failed only in constructs (ΔCa and ΔNCa) where C-terminal residues 210 to 257 were deleted. (B) <i>P. falciparum</i> PfRRM1 and human RBM42 are functional orthologs of <i>Toxoplasma</i> protein TgRRM1. Schematic description of PfRRM1 and RBM42 expression constructs are indicated along with the TgRRM1 reference. The construction of these expression plasmids followed the designs described for TgRRM1 above and were transfected into mutant 12-109C6 parasites. Numbered residues indicate the start and end of each polypeptide. Red boxes = polypeptide of TgRRM1 origin, green = PfRRM1 regions, and blue = RBM42 regions. Immunofluorescent images were from transgenic clones cultured for 24 h in standard media plus 100 nM shield 1. The co-stains were myc tagged PfRRM1, Tg/PfRRM1 chimera, or RBM42 transgene expression (green = anti-myc), red = anti-IMC1, blue = DAPI. Nomarski images on the right show plaques formed by the clones cultured for 7 days at 40°C (no shield 1). Chimeric Tg/PfRRM1 and human RBM42 constructs were able to rescue high temperature sensitivity of mutant 12-109C6, while PfRRM1 delayed the growth arrest of the mutant (not shown) but this was not sufficient to support long term plaque formation.</p

Splicing defects in TgRRM1 null parasites occur across the parasite transcriptome.

<p>Splicing of mRNAs is globally disrupted in 12-109C6 parasites grown at 40°C (null for TgRRM1, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen-1003305-g004" target="_blank">Figure 4</a>). (A) Intron∶exon (I/E) values for genes expressed in 12-109C6 mutant parasites grown at the permissive temperature (34°C, x-axis) compared to the non-permissive temperature (40°C, y-axis). (B) I/E ratios for genes from the permissive condition (x-axis) compared to those of a genetically complemented strain at the non-permissive temperature (y-axis). (C) As in (A), but selecting only those genes with peak expression in the G1 phase of the cell cycle <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen.1003305-Behnke1" target="_blank">[13]</a>. (D) As in (A), but selecting only those genes with peak expression in the S and M phases of the cell cycle. All axis values are plotted in log scale. Note that the axis scale reflects the inherent range of intron/exon content and length for the 5,833 genes in this dataset.</p

Expression of TgRRM1 is cell cycle–dependent.

<p>(A) A genetically rescued mutant 12-109C6 clone expressing wt-TgRRM1^myc under control of the native TgRRM1 promoter was evaluated for cell cycle expression. Parasites were grown for 24 h at 34°C and then processed for IFA by co-staining with anti-myc (green = TgRRM1^myc protein), anti-IMC1 (red) and DAPI (blue) as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen-1003305-g001" target="_blank">Figure 1</a>. Four image panels (G1 to C phases) show the basic cell cycle profile of wt-TgRRM1^myc expression. Magnification bar (2 µm) is shown. Note that intravacuolar parasites were tightly synchronized allowing the cell cycle position of each vacuole (defined on the left) to be assigned based on known characteristics <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen.1003305-Gubbels1" target="_blank">[6]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen.1003305-Striepen1" target="_blank">[7]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen.1003305-Hu1" target="_blank">[49]</a>. Peak expression of wt-TgRRM1^myc was observed in the G1 panel, while the protein was nearly undetectable in parasites undergoing cytokinesis (M/C and C panels) demonstrating this factor is tightly cell cycle regulated. A fifth image panel (bottom) is included that pinpoints wt-TgRRM1^myc expression with respect to the G1 and S transition; co-staining in the panel is red = anti-myc, green = anti-centrin1, and blue = DAPI. The marker guide panel included here is an inverse image of the merged blue (DAPI) and green (centrin1) images to highlight the centrosome content marked by adjacent red dots. Note, parasites in these two separate vacuoles have single nuclei with no internal daughters, which places their cell cycle position on either side of the G1/S boundary based on single versus double centrosomes (G1 versus S phase, respectively). Strong wt-TgRRM1^myc expression was detected in the vacuole where parasites possessed a single centrosome (vacuole of 4 in G1), whereas wt-TgRRM1^myc was downregulated in S phase parasites associated with recently duplicated centrosomes (vacuole of 2). (B) Cyclical profile of TgRRM1 mRNA spanning nearly two tachyzoite division cycles also shows G1 phase peak expression. The graph is based on expression values obtained from our <i>Toxoplasma</i> cell cycle transcriptome microarray dataset <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen.1003305-Behnke1" target="_blank">[13]</a>. (C) Immunostaining of <i>Plasmodium falciparum</i> merozoites shows distinct cell cycle distribution of PfRRM1 in the nucleus of the ring stage parasites. While the protein is detected in discrete nuclear bodies in ring stages (see Inset), it appears diffused in the nuclei and cytoplasm of trophozoites and schizont stages and barely detectable by IFA. (R- ring; T- trophozoite; S- schizont; Hoechst- nucleic acid stain). (D) A time course immunoblot analysis of <i>P. falciparum</i> ring (R) (8–16 hours post-invasion), trophozoites (T) (24–32 hours post-invasion), and schizont (S) (36–44 hours post-invasion) stages shows constitutive overall expression of PfRRM1 throughout the intraerythrocytic cycle. Anti-Histone H3 antibody was used as a loading control. (E) The graph represents the percentile value of PF13_0318 mRNA measured in the synchronized population of <i>P. falciparum</i> 3D7 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen.1003305-Bartfai1" target="_blank">[50]</a>.</p