miRNAs and Genes Involved in the Interplay between Ocular Hypertension and Primary Open-Angle Glaucoma. Oxidative Stress, Inflammation, and Apoptosis Networks.

Glaucoma has no cure and is a sight-threatening neurodegenerative disease affecting more than 100 million people worldwide, with primary open angle glaucoma (POAG) being the most globally prevalent glaucoma clinical type. Regulation of gene expression and gene networks, and its multifactorial pathways involved in glaucoma disease are landmarks for ophthalmic research. MicroRNAs (miRNAs/miRs) are small endogenous non-coding, single-stranded RNA molecules (18-22 nucleotides) that regulate gene expression. An analytical, observational, case-control study was performed in 42 patients of both sexes, aged 50 to 80 years, which were classified according to: (1) suffering from ocular hypertension (OHT) but no glaucomatous neurodegeneration (ND) such as the OHT group, or (2) have been diagnosed of POAG such as the POAG group. Participants were interviewed for obtaining sociodemographic and personal/familial records, clinically examined, and their tear samples were collected and frozen at 80 °C until processing for molecular-genetic assays. Tear RNA extraction, libraries construction, and next generation sequencing were performed. Here, we demonstrated, for the first time, the differential expression profiling of eight miRNAs when comparing tears from the OHT versus the POAG groups: the miR-26b-5p, miR-152-3p, miR-30e-5p, miR-125b-2-5p, miR-224-5p, miR-151a-3p, miR-1307-3p, and the miR-27a-3p. Gene information was set up from the DIANA-TarBase v7, DIANA-microT-CDS, and TargetScan v7.1 databases. To build a network of metabolic pathways, only genes appearing in at least four of the following databases: DisGeNet, GeneDistiller, MalaCards, OMIM PCAN, UniProt, and GO were considered. We propose miRNAs and their target genes/signaling pathways as candidates for a better understanding of the molecular-genetic bases of glaucoma and, in this way, to gain knowledge to achieve optimal diagnosis strategies for properly identifying HTO at higher risk of glaucoma ND. Further research is needed to validate these miRNAs to discern the potential role as biomarkers involved in oxidative stress, immune response, and apoptosis for the diagnosis and/or prognosis of OHT and the prevention of glaucoma ND.

Signature of Circulating Biomarkers in Recurrent Non-Infectious Anterior Uveitis. Immunomodulatory Effects of DHA-Triglyceride. A Pilot Study.

The purpose of this study was to identify circulating biomarkers of recurrent non-infectious anterior uveitis (NIAU), and to address the anti-inflammatory effects of triglyceride containing docosahexaenoic acid (DHA-TG). A prospective multicenter study was conducted in 72 participants distributed into: patients diagnosed with recurrent NIAU in the quiescence stage (uveitis group (UG); n = 36) and healthy controls (control group (CG); n = 36). Each group was randomly assigned to the oral supplementation of one pill/day (+) containing DHA-TG (n = 18) or no-pill condition (-) (n = 17) for three consecutive months. Data from demographics, risk factors, comorbidities, eye complications and therapy were recorded. Blood was collected and processed to determine pro-inflammatory biomarkers by bead-base multiplex assay. Statistical processing with multivariate statistical analysis was performed. The mean age was 50, 12 (10, 31) years. The distribution by gender was 45% males and 55% females. The mean number of uveitis episodes was 5 (2). Higher plasma expression of interleukin (IL)-6 was detected in the UG versus the CG (p = 5 × 10-5). Likewise, significantly higher plasma levels were seen for IL-1ß, IL-2, INFγ (p = 10-4), and TNFα (p = 2 × 10-4) in the UG versus the CG. Significantly lower values of the above molecules were found in the +DHA-TG than in the -DHA-TG subgroups, after 3 months of follow-up, TNFα (p = 10-7) and IL-6 (p = 3 × 10-6) being those that most significantly changed. Signatures of circulating inflammatory mediators were obtained in the quiescent stage of recurrent NIAU patients. This 3-month follow-up strongly reinforces that a regular oral administration of DHA-TG reduces the inflammatory load and may potentially supply a prophylaxis-adjunctive mediator for patients at risk of uveitis vision loss.

Mesenchymal stromal cells enhance the engraftment of hematopoietic stem cells in an autologous mouse transplantation model.

INTRODUCTION: Studies have proposed that mesenchymal stem cells (MSCs) improve the hematopoietic engraftment in allogeneic or xenogeneic transplants and this is probably due to the MSCs' immunosuppressive properties. Our study aimed to discern, for the first time, whether MSC infusion could facilitate the engraftment of hematopoietic stem cells (HSCs) in autologous transplantations models, where no immune rejection of donor HSCs is expected. METHODS: Recipient mice (CD45.2) mice, conditioned with moderate doses of radiation (5-7 Gy), were transplanted with low numbers of HSCs (CD45.1/CD45.2) either as a sole population or co-infused with increasing numbers of adipose-derived-MSCs (Ad-MSCs). The influence of Ad-MSC infusion on the short-term and long-term engraftment of donor HSCs was investigated. Additionally, homing assays and studies related with the administration route and with the Ad-MSC/HSC interaction were conducted. RESULTS: Our data show that the co-infusion of Ad-MSCs with low numbers of purified HSCs significantly improves the short-term and long-term hematopoietic reconstitution of recipients conditioned with moderate irradiation doses. This effect was Ad-MSC dose-dependent and associated with an increased homing of transplanted HSCs in recipients' bone marrow. In vivo and in vitro experiments also indicate that the Ad-MSC effects observed in this autologous transplant model are not due to paracrine effects but rather are related to Ad-MSC and HSC interactions, allowing us to propose that Ad-MSCs may act as HSC carriers, facilitating the migration and homing of the HSCs to recipient bone marrow niches. CONCLUSION: Our results demonstrate that Ad-MSCs facilitate the engraftment of purified HSCs in an autologous mouse transplantation model, opening new perspectives in the application of Ad-MSCs in autologous transplants, including HSC gene therapy.

The protease inhibitor atazanavir triggers autophagy and mitophagy in human preadipocytes.

BACKGROUND: The association between HAART and lipodystrophy is well established, but lipodystrophy pathogenesis is still poorly understood. Drugs, and in particular protease inhibitors, accumulate in adipose tissue affecting adipocyte physiology and gene expression by several mechanisms. Recent studies have identified autophagy as another process affected by these classes of drugs, but no studies have been performed in adipose cells. METHODS: SW872 preadipocytic human cell line was used to evaluate changes induced by amprenavir (APV), ritonavir (RTV), or atazanavir (ATV), all used at 10-200 µmol/l. A subline was stably transfected with murine stem cell virus (pMSCV)-enhanced green fluorescent protein (EGFP)-LC3 plasmid (to obtain a fluorescent LC3 protein) and treated with ATV at different doses. The distribution of LC3 and the colocalization of mitochondria, lysosome, and autophagosome were assessed by confocal microscopy. Transmission electron microscopy of ATV-treated cells was also performed. The cellular content of lysosomes was assessed using Lysotracker Green; apoptosis was evaluated by annexin V/propidium iodide staining, and mitochondrial superoxide anion (mtO2) was analyzed by mitoSOX red. Lysosomes, apoptosis, and mtO2 were studied by flow cytometry and multispectral imaging flow cytometry. RESULTS: In SW872 cells, RTV caused massive apoptosis, more than autophagy, whereas APV was almost ineffective. ATV induced both apoptosis (high doses) and autophagy (low doses). ATV-treated cells displayed LC3-specific punctae, suggesting the formation of autophagosomes that enclosed mitochondria, as revealed by electron microscopy. At low doses, ATV promoted mitochondrial superoxide generation, whereas at high doses, it induced mitochondrial membrane depolarization. CONCLUSION: Autophagy/mitophagy can be considered a mechanism triggered by ATV in SW872 preadipocytes.

Relevance of testicular sperm DNA oxidation for the outcome of ovum donation cycles.

OBJECTIVE: To determine the relevance of sperm DNA oxidation caused by free radicals in samples obtained via testicular biopsies by means of flow cytometry by correlating the measurements of 8-hydroxy-2'-deoxyguanosine (8-OHdG) with embryo features and pregnancy achievement. DESIGN: Prospective cross-sectional study. SETTING: Private University-affiliated setting. PATIENT(S): Fifty-seven azoospermic patients undergoing testicular sperm extraction (TESE) were analyzed in their corresponding assisted reporductive technology cycles using ovum donation to standardize female's characteristics. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Quantification of the adduct 8-OHdG in testicular tissue samples, and its effect on markers of embryo quality and reproductive success, and its relevance as marker of TESE sperm quality. RESULT(S): We found the status of sperm DNA oxidation to have very little clinical relevance for several parameters of embryo quality, fertilization rates, early (days 2-3) and late (days 5-6) development, and achievement of pregnancy. CONCLUSION(S): The TESE obtained cells from azoospermic males do not possess a DNA oxidation status of significant importance in the success of assisted reproduction treatments, as determined by 8-OHdG measurement of each category of cell ploidy.

Influence of aging on murine neutrophil and macrophage function against Candida albicans.

Previous work by our group showed that aged C57BL/6 mice develop an altered innate and adaptive immune response to Candida albicans and are more susceptible to systemic primary candidiasis. In this work, we used young (2-3 months old) and aged (18-20 months old) C57BL/6 mice to study in vitro the influence of aging on (1) the fungicidal activity of neutrophils and macrophages, (2) the production of cytokines by resident peritoneal macrophages in response to C. albicans, and (3) cell surface Toll-like receptor (TLR) 2 expression on resident peritoneal macrophages. Our results indicate that murine phagocytes have a fungicidal activity well preserved with aging. In vitro production of proinflammatory cytokines (IL-6, IL-1beta, and tumor necrosis factor-alpha and chemokines (MIP-2) by purified (CD11b(+)) peritoneal macrophages in response to yeasts and hyphae of C. albicans was significantly lower in aged mice as compared with young mice. However, the production of IL-10 by macrophages, in response to C. albicans, was similar in both young and aged animals. Moreover, baseline TLR2 surface expression level was lower on aged macrophages than on control macrophages. Taken together, these data indicate that the increased susceptibility to C. albicans disseminated infections in aged mice is correlated with defects in TLR2 expression and in cytokine production, but not with an impaired fungicidal activity.

In vitro response to Candida albicans in cultures of whole human blood from young and aged donors.

Invasive infections with opportunistic fungi, such as Candida albicans, have become an increasing problem in aged adults in recent years. This work investigates the influence of human ageing on C. albicans recognition by toll-like receptors (TLRs), essential components of the innate immune system, using a cohort of 96 young (15-42 years) and aged (>70 years) human volunteers. No significant differences between aged and young donors were observed on (1) cell surface TLR2, TLR6 and TLR4 expression on lymphocytes, monocytes and granulocytes, (2) production of cytokines [IL-8, IL-1beta, IL-6, IL-10, tumour necrosis factor (TNF)-alpha and IL-12p70] and prostaglandin E(2) (PGE(2)) by whole human blood in response to C. albicans and (3) fungicidal activity of whole blood. A statistically significant higher titre of natural anti-C. albicans antibodies was found in plasma of volunteers between 80 and 95 years old when compared with other age groups, probably as a consequence of the increased levels of serum Ig that has been described in elderly subjects. Therefore, the results indicate that the increased susceptibility to C. albicans infections in the elderly is not a consequence of defects in TLRs expression or signalling, nor of an impaired fungicidal activity of blood.

Efficient selection of silenced primary cells by flow cytometry.

BACKGROUND: RNA interference has emerged as a new and potent tool to knockdown the expression of target genes and to investigate their functions. For short time experiments with mammalian cell lines, RNA interference is typically induced by transfecting small interfering RNAs (siRNAs). Primary cells constitute important experimental systems in many studies because of their similarity to their in vivo counterparts; however, transfection of these cells has been found to be difficult. As a consequence, RNA interference of primary cells may result in mixed phenotypes because of the simultaneous presence in the same preparation of transfected and nontransfected cells. This may be particularly inconvenient when certain experiments (for example, biochemical analysis) should be performed. METHODS: We use fluorescently labeled siRNAs to induce RNA interference in fibroblasts, and flow-cytometry associated cell sorting to separate subpopulations of transfected cells according to fluorescence intensity. RESULTS: Flow cytometry allows one to discriminate between strongly- and weakly- or nonsilenced fibroblasts, since the fluorescence intensity of transfected cells is related to the number of internalized siRNA copies and to the mRNA knockdown efficiency. CONCLUSIONS: The use of fluorescently labeled siRNAs may allow one to isolate by flow-cytometry associated cell sorting the most efficiently silenced primary cells for subsequent analysis.

Genome size determination in chagas disease transmitting bugs (hemiptera-triatominae) by flow cytometry.

Because information about genome size in triatomines is scarce and contradictory, we performed DNA quantification by flow cytometry in 13 species belonging to five genera (Dipetalogaster, Eratyrus, Panstrongylus, Rhodnius, and Triatoma) to infer overall tendencies and phylogenetic associations. The results show that the haploid DNA content of the subfamily Triatominae varies nearly 4-fold, from<0.7 pg in Rhodnius species (0.6x10(9) bp) to 2.7 pg in Triatoma delpontei (2.6x10(9) bp). Considering that triatomines present similar chromosome numbers, we suggest that genome size differences are the result of variation in the quantity of repetitive DNA sequences localized in hetero and euchromatin. Changes in heterochromatin are particularly important when considering populations or closely related species; in more distant taxa, euchromatic changes also play a role. Our analyses indicate that flow cytometry is a useful tool for population, taxonomic, and evolutionary studies in this subfamily.

Endothelial nitric oxide synthase regulates T cell receptor signaling at the immunological synapse.

The role of nitric oxide (NO) in T cells remains controversial, and the origin and localization of endogenous NO and whether it regulates lymphocyte activation are unclear. We show here that, within minutes of binding to antigen, T cells produce NO via endothelial nitric oxide synthase (eNOS). This process required increased intracellular Ca2+ and phosphoinositide3-kinase activity. By using an eNOS-green fluorescent fusion protein and fluorescent probes to detect NO, we show that eNOS translocates with the Golgi apparatus to the immune synapse of T helper cells engaged with antigen-presenting cells (APC), where it was fully activated. Overexpression of eNOS prevented the central coalescence of CD3 at the T cell-APC contact site, which was accompanied by increased phosphorylation of CD3zeta chain, ZAP-70, and extracellular signal-regulated kinases and increased IFN-gamma synthesis, but reduced production of IL-2. Therefore, eNOS-derived NO selectively potentiates T cell receptor signaling to antigen at the immunological synapse.

Cytokine production by peripheral lymphocytes in melanoma.

BACKGROUND: The differentiation of T cells towards a T helper 1 (Th1) or Th2 phenotype based on their profile of cytokine production, is of great relevance in the regulation of immune responses. We have determined by flow cytometry, the expression of selected Th1 and Th2 cytokines by activated T cells in whole blood samples (WB) from normal donors and from patients with different clinical stages of melanoma in different clinical stages. METHODS: WB samples from 6 normal donors and 19 patients with melanoma were activated over 4 hours with PMA + ionomycin in presence or absence of a protein secretion inhibitor. Following surface staining (CD3-Cy5+CD8-FITC), fixation and permeabilization, cells were stained with PE-labelled antibodies against Th1 cytokines (IL-2, IFN-gamma, TNF-alpha) and Th2 cytokines (IL-4, IL-10). RESULTS: The most relevant results were related to IFN-gamma and IL-10 production. The percentage of IFN-gamma producer cells was significantly lower in melanoma patients, independent of the stage, than in controls. IL-10 production was significantly increased in melanoma patients with respect to normal donors. CONCLUSIONS: Our data support the notion that the pattern of cytokines produced by lymphocytes from melanoma patients may help to explain the impairment in their T cell immune response. More extensive studies regarding the pattern of cytokines, not only in peripheral blood, but also in tumour tissue and sentinel lymph nodes, are needed to confirm these data.

Estudio del patrón de citocinas (Th1/Th2) producido por linfocitos T periféricos y del existente en tejido tumoral de pacientes con melanoma en diferentes estadios* / Th1/Th2 Cytokine production by peripheral T lymphocytes and tumoral tissue from patients with melanoma in different clinical stages

Objetivos. La diferenciación de las células T en los patrones Th1 o Th2 según su patrón de producción de citocinas se ha demostrado muy importante en la regulación de las respuestas inmunes. Hemos estudiado si existe una diferencia entre sujetos controles y pacientes con melanoma en la expresión de estas citocinas, tanto en sangre periférica (mediante citometría de flujo) como en biopsias de los tumores (mediante RTPCR cuantitativa a tiempo real).Métodos. En la primera parte del estudio se incluyeron 25 pacientes: 6 eran controles y 19 habían sido diagnosticados de melanoma. Para la citometría de flujo se obtuvieron muestras de sangre de los 25 pacientes y se realizó una activación durante 4 horas con PMA + ionomicina en presencia o ausencia de un inhibidor de la secreción de proteínas (GolgiStop). Tras realizar un marcaje de superficie (CD3-Cy5+CD8-FITC) y una fijación y permeabilización (CytoFix-CytoPerm) las células fueron teñidas con anticuerpos marcados con ficoeritrina frente a las citocinas caracteríticas del patrón Th1 (IL-2, IFN, TNF-) y frente a las del patrón Th2 (IL-4, IL-10). En la segunda parte del estudio se incluyeron ocho biopsias cutáneas: dos controles, tres melanomas cutáneos localizados y tres metástasis cutáneas de melanoma. Tras extraer el ARN total, se estudiaron mediante una técnica de RT-PCR cuantitativa a tiempo real los tránscritos (mARN) de las siguientes citocinas: IL-2, IFN- , TNF-, IL-4, IL-5, IL-10.Resultados. Los datos más relevantes de la primera parte del estudio se relacionan con la producción de IFN y de IL-10. El porcentaje de células productoras de IFN era significativamente menor en los pacientes con melanoma, independientemente de su estadio, en relación con los controles. La producción de IL-10 se encontraba significativamente elevada en los pacientes con melanoma en comparación con los donantes sanos. Se observó una tendencia a un aumento en la producción de IL-10 en los estadios más avanzados de la enfermedad. La segunda parte del estudio mostró la existencia de un patrón Th2 o Th0 en las biopsias tanto de melanomas localizados como en las metástasis cutáneas. Conclusiones. Nuestros datos sugieren que los linfocitos activados de los pacientes donantes sanos tienden a exhibir un fenotipo Th1, mientras que en los pacientes con melanoma se observa un aumento en la proporción de linfocitos productores de una citocina característica del patrón Th2 como es la IL-10, con reconocidas propiedades inmunosupresoras. El análisis del propio tumor apoya estos resultados, ya que el patrón hallado con mayor frecuencia es el Th2 (AU)

The glyceraldehyde-3-phosphate dehydrogenase polypeptides encoded by the Saccharomyces cerevisiae TDH1, TDH2 and TDH3 genes are also cell wall proteins.

The authors show that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Saccharomyces cerevisiae, previously thought to be restricted to the cell interior, is also present in the cell wall. GAPDH activity, proportional to cell number and time of incubation, was detected in intact wild-type yeast cells. Intact cells of yeast strains containing insertion mutations in each of the three structural TDH genes (tdh1, tdh2 and tdh3) and double mutants (tdh1 tdh2 and tdh1 tdh3) also displayed a cell-wall-associated GAPDH activity, in the range of parental wild-type cells, although with significant differences among strains. A cell wall location of GAPDH was further confirmed in wild-type and tdh mutants by indirect immunofluorescence and flow cytometry analysis with a polyclonal antibody against S. cerevisiae GAPDH. By immunoelectron microscopy, the GAPDH protein was detected at the outer surface of the cell wall of wild-type cells, as well as in the cytoplasm. Western immunoblot analysis of cell wall extracts and cytosol showed that Tdh2 and Tdh3 polypeptides are present in the cell wall, as well as in the cytosol, of exponentially growing cells. Tdh1 is only detected in stationary-phase cells, again in both cytosol and cell wall extracts. The results incorporate the GAPDH of S. cerevisiae, encoded by TDH1-3, into the newly emerging family of multifunctional cell-wall-associated GAPDHs which retain their catalytic activity.