Subgingival microbiome in periodontal health, gingivitis and different stages of periodontitis.

AIM: To characterize the subgingival microbiome in subjects with different periodontal health statuses. MATERIALS AND METHODS: In this cross-sectional observational study, subgingival samples were harvested from Spanish subjects with different periodontal health statuses, based on the 2018 Classification of Periodontal and Peri-Implant Diseases and Conditions. Samples were processed using high-throughput sequencing technologies (Illumina MiSeq). Taxa differentially abundant were identified using Analysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC). α- and ß-diversity metrics were calculated using q2-diversity in QIIME2. The analyses were adjusted for age, gender and smoking status. RESULTS: The identified subgingival microbiome showed statistically significant differences among subjects, categorized into periodontal health, gingivitis and stages I-II and III-IV periodontitis (p < .05). In patients with severe (stages III-IV) periodontitis, the genera Filifactor and Fretibacterium were detected 24 times more frequently than in periodontally healthy subjects. Similarly, the genera Porphyromonas, Prevotella and Tannerella were detected four times more frequently (p < .05). The genera Granulicatella, Streptococcus, Paracoccus, Pseudomonas, Haemophilus, Actinobacteria, Bergeyella and Capnocytophaga were significantly associated with healthier periodontal status (p < .05). CONCLUSIONS: Significant differences were detected in the subgingival microbiome among periodontal health, gingivitis and stages I-II or III-IV periodontitis, suggesting overlapping, yet distinguishable microbial profiles.

19. Adaptación transcultural y validación del instrumento Self Care of Heart Failure Index 6.2 para su uso en Uruguay

Antecedentes: el tratamiento de la IC es complejo por los múltiples fármacos a utilizar y las medidas no farmacológicas requeridas, por ello es importante que el paciente implemente su autocuidado adecuadamente. Autocuidado es el proceso de toma de decisiones del paciente con elección de comportamientos que mantienen la estabilidad fisiológica y la respuesta a los síntomas cuando ocurren y existen instrumentos para su evaluación. El Self Care of Heart Failure Index 6.2 (SCHFI 6.2), desarrollado en Estados Unidos, ayuda a los profesionales de la salud a identificar la adhesión (o no adhesión) al autocuidado, auxiliando en el redimensionamiento de orientaciones y conductas, pero para su utilización es necesario una adaptación transcultural. Objetivo: adaptar y validar el SCHFI 6.2 para pacientes uruguayos con IC. Material y método: estudio metodológico desarrollado en cuatro fases: traducción de la versión original (inglés) al español por dos traductores oficiales uruguayos; síntesis de las dos traducciones; retrotraducción y evaluación por la autora original; evaluación por el comité de expertos. Se invitó a ocho expertos en IC para evaluar cada ítem del cuestionario, en relación con la equivalencia semántica, idiomática, experimental y conceptual, a través de una escala Likert de cuatro puntos. Los datos fueron tabulados por Microsoft Excel 2013 y evaluados a través del índice de validez de contenido (IVC). El estudio se realizó entre los meses de julio y setiembre de 2017, con la aprobación del comité de ética. Resultados: de los 53 puntos evaluados, cinco presentaron IVC por debajo de 0,75, fueron reformulados y reenviados al comité de expertos para nueva evaluación, obteniéndose la versión final del SCHFI 6.2 español / Uruguay. Conclusiones: los instrumentos para evaluar el autocuidado son herramientas importantes y de bajo costo para el seguimiento de pacientes con IC. Se pretende dar seguimiento al estudio a través de la validación clínica y, con ello, poner a disposición la escala para uso de los profesionales de salud uruguayos, lo que podrá auxiliar en el direccionamiento de las orientaciones a estos pacientes.

Gene Editing for the Efficient Correction of a Recurrent COL7A1 Mutation in Recessive Dystrophic Epidermolysis Bullosa Keratinocytes.

Clonal gene therapy protocols based on the precise manipulation of epidermal stem cells require highly efficient gene-editing molecular tools. We have combined adeno-associated virus (AAV)-mediated delivery of donor template DNA with transcription activator-like nucleases (TALE) expressed by adenoviral vectors to address the correction of the c.6527insC mutation in the COL7A1 gene, causing recessive dystrophic epidermolysis bullosa in a high percentage of Spanish patients. After transduction with these viral vectors, high frequencies of homology-directed repair were found in clones of keratinocytes derived from a recessive dystrophic epidermolysis bullosa (RDEB) patient homozygous for the c.6527insC mutation. Gene-edited clones recovered the expression of the COL7A1 transcript and collagen VII protein at physiological levels. In addition, treatment of patient keratinocytes with TALE nucleases in the absence of a donor template DNA resulted in nonhomologous end joining (NHEJ)-mediated indel generation in the vicinity of the c.6527insC mutation site in a large proportion of keratinocyte clones. A subset of these indels restored the reading frame of COL7A1 and resulted in abundant, supraphysiological expression levels of mutant or truncated collagen VII protein. Keratinocyte clones corrected both by homology-directed repair (HDR) or NHEJ were used to regenerate skin displaying collagen VII in the dermo-epidermal junction.

Immunotherapy reduces allergen-mediated CD66b expression and myeloperoxidase levels on human neutrophils from allergic patients.

UNLABELLED: CD66b is a member of the carcinoembryonic antigen family, which mediates the adhesion between neutrophils and to endothelial cells. Allergen-specific immunotherapy is widely used to treat allergic diseases, and the molecular mechanisms underlying this therapy are poorly understood. The present work was undertaken to analyze A) the in vitro effect of allergens and immunotherapy on cell-surface CD66b expression of neutrophils from patients with allergic asthma and rhinitis and B) the in vivo effect of immunotherapy on cell-surface CD66b expression of neutrophils from nasal lavage fluid during the spring season. Myeloperoxidase expression and activity was also analyzed in nasal lavage fluid as a general marker of neutrophil activation. RESULTS: CD66b cell-surface expression is upregulated in vitro in response to allergens, and significantly reduced by immunotherapy (p<0.001). Myeloperoxidase activity in nasal lavage fluid was also significantly reduced by immunotherapy, as were the neutrophil cell-surface expression of CD66b and myeloperoxidase (p<0.001). Interestingly, CD66b expression was higher in neutrophils from nasal lavage fluid than those from peripheral blood, and immunotherapy reduced the number of CD66+MPO+ cells in nasal lavage fluid. Thus, immunotherapy positive effects might, at least in part, be mediated by the negative regulation of the CD66b and myeloperoxidase activity in human neutrophils.

Allergen immunotherapy decreases LPS-induced NF-κB activation in neutrophils from allergic patients.

BACKGROUND: Allergen-specific immunotherapy (IT) is widely used to treat allergic diseases. The molecular mechanisms have not been clarified yet completely. The present work was undertaken to analyze the effect of IT in the activation of NF-κB. METHODS: Neutrophils from 15 pollen-allergic IT-treated patients, 10 untreated pollen-allergic patients, and 10 healthy donors were in vitro stimulated with LPS. NF-κB activation (p65/p52) was measured in their nuclear extracts by enzyme-linked immunosorbent assay (ELISA). IκBα phosphorylation, NF-κB-repressing factor (NRF) activation, and thromboxane A2 (TXA2 ) and Interleukin-8 (IL-8) release were measured by ELISA. RESULTS: There was a positive correlation between the score of symptoms and NF-κB activation in human neutrophils. IT significantly decreased NF-κB activation levels in neutrophils compared with neutrophils from untreated patients. IκBα phosphorylation and NRF activation levels were, respectively, significantly lower and higher in neutrophils from IT-treated patients than from untreated patients. IL-8 and TXA2 release were significantly lower in neutrophils from IT-treated patients than from untreated patients. CONCLUSIONS: IT positive effects are at least in part mediated by the negative regulation of NF-κB activation in human neutrophils. These observations represent a novel view of neutrophils as possible cell target to treat IgE-dependent diseases through NF-κB downmodulation.

Nivel de conocimiento de la enfermedad en una cohorte de pacientes con insuficiencia cardíaca / Level of disease awareness in a cohort of patients with heart failure

La causa más frecuente de reingresos por insuficiencia cardíaca es el mal cumplimiento terapéutico del paciente. Ello puede atribuirse a falta de conocimiento, a la complejidad del tratamiento, a deterioro cognitivo, a depresión o a una comunicación poco efectiva o inadecuada para el paciente por parte del equipo asistencial. Estudios previos muestran altos porcentajes de inadecuado conocimiento de la enfermedad en estos pacientes. En Uruguay no hay estudios que verifiquen esta situación. En el presente trabajo se analizó el conocimiento acerca de su enfermedad entre los pacientes controlados en una unidad especializada de insuficiencia cardíaca utilizando el método de encuesta mediante cuestionario autogestionado diseñado por los investigadores. Un elevado número de pacientes no conocen el diagnóstico de su enfermedad ni sus características, los factores de riesgo para enfermar y/o los factores de riesgo de descompensación. Esto debe obligar a otros análisis en cuanto a barreras de comunicación médico-paciente y/o a instrumentar planes educativos que puedan ser evaluados.

The most common cause of readmission for heart failure is poor patient compliance. This can be attributed to lack of knowledge, the complexity of treatment, cognitive impairment, depression or to an ineffective or inadequate communication to the patient by the healthcare team. Previous studies show a high percentage of inadequate knowledge about the disease in these patients. In Uruguay there are no studies that verify this. In this paper we analyzed the knowledge about their disease among the patients managed in a specialist heart failure unit using the survey method through self-administered questionnaire designed by the researchers.
A large number of patients do not know the diagnosis of the disease or the characteristics of the same, the risk factors for disease and / or risk factors of decompensation This should force other barriers analysis for doctor-patient communication and/or implement educational plans can be evaluated.

Keratinocyte cell lines derived from severe generalized recessive epidermolysis bullosa patients carrying a highly recurrent COL7A1 homozygous mutation: models to assess cell and gene therapies in vitro and in vivo.

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by deficiency of type VII collagen due to COL7A1 mutations such as c.6527insC, recurrently found in the Spanish RDEB population. Assessment of clonal correction-based therapeutic approaches for RDEB requires large expansions of cells, exceeding the replication capacity of human primary keratinocytes. Thus, immortalized RDEB cells with enhanced proliferative abilities would be valuable. Using either the SV40 large T antigen or papillomavirus HPV16-derived E6-E7 proteins, we immortalized and cloned RDEB keratinocytes carrying the c.6527insC mutation. Clones exhibited high proliferative and colony-forming features. Cytogenetic analysis revealed important differences between T antigen-driven and E6-E7-driven immortalization. Immortalized cells responded to differentiation stimuli and were competent for epidermal regeneration and recapitulation of the blistering RDEB phenotype in vivo. These features make these cell lines useful to test novel therapeutic approaches including those aimed at editing mutant COL7A1.

Enhancing membrane disruption by targeting and multivalent presentation of antimicrobial peptides.

In order to enhance the membrane disruption of antimicrobial peptides both targeting and multivalent presentation approaches were explored. The antimicrobial peptides anoplin and temporin L were conjugated via click chemistry to vancomycin and to di- and tetravalent dendrimers. The vancomycin unit led to enhanced membrane disruption of large unilamellar vesicles (LUVs) displaying the vancomycin target lipid II, but only for temporin L and not for anoplin. The multivalent presentation led to enhanced LUV membrane disruption in the case of anoplin but not for temporin L.

Structure-activity relationship studies on a novel family of specific HIV-1 reverse transcriptase inhibitors.

We have previously reported the discovery and preliminary structure-activity relationships of a new class of specific HIV-1 reverse transcriptase (RT) inhibitors whose prototype compound is the 1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3-N-[(carboxy) methyl]-thymine. In an attempt to increase the inhibitory efficacy against HIV-1 RT of this new class of nucleosides, and to further explore the structural features required for anti-HIV-1 activity, different types of modifications have been carried out on the prototype compound. These include substitution of the tert-butyldimethylsilyl groups by other liphophilic groups, replacement of the carboxy group at the N-3 position of the nucleobase by other functional groups, change in the length of the spacer between the thymine and the carboxylic acid residue and substitution of the thymine moiety by other pyrimidine (uracil, 5-ethyluracil) or purine (hypoxanthine) nucleobases. In addition, the most salient structural features of this new class of HIV-1-specific nucleosides have been incorporated into classical HIV RT nucleoside inhibitors such as ddl, AZT, d4T. Our studies demonstrate that both the carboxymethyl moiety at the nucleobase and tert-butyldimethylsilyl groups at the sugar are important structural components since deletion of either of them is detrimental to the antiviral activity.

L-tryptophan reacts with naturally occurring and food-occurring phenolic aldehydes to give phenolic tetrahydro-beta-carboline alkaloids: activity as antioxidants and free radical scavengers.

The reaction between the essential amino acid l-tryptophan and flavoring or naturally occurring phenyl and phenolic aldehydes was studied, and the alkaloidal reaction products were characterized by NMR and HPLC-MS. Benzaldehyde, vanillin, syringaldehyde, salicylaldehyde, and anisaldehyde condensed with l-tryptophan in aqueous-acidic media affording the corresponding phenolic tetrahydro-beta-carboline-3-carboxylic acid as two diastereoisomers, 1S,3S-cis and 1R,3S-trans. With the exception of benzaldehyde, the rest of the aldehydes needed heating conditions (70 degrees C) to significantly form tetrahydro-beta-carbolines over time with the cyclization highly favored at low pH. This suggests a likely formation of these compounds under conditions that may occur in foods, food processing, or cooking. The new phenolic tetrahydro-beta-carboline alkaloids were assayed, for the first time, for their activity as free radical scavengers and antioxidants and showed good antioxidant properties with Trolox equivalent antioxidant capacity (TEAC) values much higher than those of ascorbic acid and the water soluble vitamin E analogue, Trolox, in the 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) assay.