Utilidad de la ecografía diafragmática para predecir el éxito en la extubación / Usefulness of diaphragmatic ultrasound in predicting extubation success

Objetivo Evaluar la exactitud diagnóstica de la ecografía diafragmática para predecir el éxito en la extubación. Diseño Estudio de exactitud diagnóstica. Ámbito Unidad de Cuidado Intensivo Médico de un hospital académico de la ciudad de Bogotá (Colombia). Pacientes o participantes Muestra consecutiva de pacientes mayores de 18 años con ventilación mecánica invasiva durante más de 48h. Intervenciones Evaluación ecográfica diafragmática al finalizar la prueba de ventilación espontánea. Variables de interés principales Se evaluó la excursión diafragmática (ED, cm), el tiempo de inspiración (TPIAdia, s), la velocidad de contracción del diafragma (ED/TPIAdia, cm/s), el tiempo total (Ttot, s) y la fracción de engrosamiento (TFdi, %). Resultados Se incluyeron 84 pacientes, el 79,8% (n=67) con extubación exitosa y el 20,2% (n=17) con extubación fallida. La variable con mejor capacidad discriminatoria para predecir éxito en la extubación fue la velocidad de contracción, con un AUC-ROC de 0,70 (p=0,008). Conclusiones La velocidad de contracción diafragmática mostró una capacidad discriminatoria aceptable. La ultrasonografía podría formar parte de un abordaje multifactorial en el proceso de extubación. (AU)

Objective To evaluate the diagnostic accuracy of diaphragmatic ultrasound in predicting extubation success. Design A diagnostic accuracy study was carried out. Scope Intensive Care Unit of an Academic hospital in the city of Bogotá (Colombia). Patients or participants A consecutive sample of patients >18 years of age subjected to invasive mechanical ventilation for >48h. Interventions Diaphragmatic ultrasound evaluation at the end of spontaneous ventilation testing. Main variables of interest Diaphragmatic excursion (DE, cm), inspiration time (TPIAdia, s), diaphragm contraction speed (DE/TPIAdia, cm/s) and total time (Ttot, s) were evaluated, together with thickening fraction (TFdi, %). Results A total of 84 patients were included, 79.8% (n=67) with successful extubation and 20.2% (n=17) with failed extubation. The variable with the best discriminatory capacity in predicting extubation success was diaphragm contraction speed, with AUC-ROC 0.70 (p=0.008). Conclusions Diaphragm contraction speed exhibited acceptable discriminatory capacity. Ultrasound could be part of a multifactorial approach in the extubation process. (AU)

[Usefulness of diaphragmatic ultrasound in predicting extubation success]. / Utilidad de la ecografía diafragmática para predecir el éxito en la extubación.

OBJECTIVE: To evaluate the diagnostic accuracy of diaphragmatic ultrasound in predicting extubation success. DESIGN: A diagnostic accuracy study was carried out. SCOPE: Intensive Care Unit of an Academic hospital in the city of Bogotá (Colombia). PATIENTS OR PARTICIPANTS: A consecutive sample of patients >18 years of age subjected to invasive mechanical ventilation for >48h. INTERVENTIONS: Diaphragmatic ultrasound evaluation at the end of spontaneous ventilation testing. MAIN VARIABLES OF INTEREST: Diaphragmatic excursion (DE, cm), inspiration time (TPIAdia, s), diaphragm contraction speed (DE/TPIAdia, cm/s) and total time (Ttot, s) were evaluated, together with thickening fraction (TFdi, %). RESULTS: A total of 84 patients were included, 79.8% (n=67) with successful extubation and 20.2% (n=17) with failed extubation. The variable with the best discriminatory capacity in predicting extubation success was diaphragm contraction speed, with AUC-ROC 0.70 (p=0.008). CONCLUSIONS: Diaphragm contraction speed exhibited acceptable discriminatory capacity. Ultrasound could be part of a multifactorial approach in the extubation process.

Genotyping of SNPs associated with meat tenderness: comparison of two PCR-based methods.

Single nucleotide polymorphisms (SNPs) carried in calpain (CAPN1), calpastatin (CAST), and leptin (LEP) genes are associated with meat tenderness. Due to the economic importance of this meat quality attribute, the development of fast, reliable, and affordable methods to identify bovine carriers of favorable alleles is of great importance for genetic improvement. Currently, PCR-RFLP is accepted as the standard gold method for genotyping SNPs associated with meat tenderness. But these SNPs can be detected by other techniques as high-resolution melting (HRM) analysis - a post-PCR method - that offers several advantages and has great application potential in the meat industry. In this study, we standardized, validated, and compared the performance of PCR-HRM to that of PCR-RFLP in genotyping bovine SNPs associated with meat tenderness: CAPN4751, CAPN316, CAST2959, CAST282, LEPE2FB, and LEPE2JW. We analyzed genotypes of a total of 380 bovines, 110 Bos taurus and 270 Bos indicus. Results obtained with PCR-HRM were consistent with those found by PCR-RLFP. Furthermore, HRM was found to be highly sensitive, and our results confirmed the repeatability (intra-assay precision) and reproducibility (inter-assay precision) of this assay. An internal control for endonuclease activity was created using site-directed mutagenesis to generate an additional enzymatic restriction point useful to discriminate SNP alleles. Our results show that PCR-HRM is an efficient method that produces reliable and rapid results. However, should be had in account that the method of DNA extraction, the quality and quantity of DNA, analyst-related variations, and primer design may generate challenges for allele discrimination.

Rehabilitación de músculos orofaríngeos con ejercicios y electroterapia para el síndrome de apnea-hipoapnea obstructiva del sueño / Rehabilitation of oropharyngeal muscles with muscle reeducation and electrostimulation in obstructive sleep apnea syndrome

Introducción. El síndrome de apnea obstructiva del sueño (SAOS) tiene repercusiones importantes en la morbimortalidad de los pacientes. Los tratamientos alternos a la presión positiva continua en la vía aérea (CPAP) han resultado poco eficaces, poco seguros o de alta complejidad. Objetivo. Describir los efectos de un programa ambulatorio de reeducación de la musculatura orofacial y extralaríngea y aplicación de electroestimulación (TENS) aplicado a pacientes con SAOS en un hospital universitario de nivel III. Diseño. Estudio de serie de casos incidentes, prospectivo, con pacientes seleccionados consecutivamente. Material y métodos. Diecisiete pacientes con SAOS leve, moderado o severo que asistieron a 24 sesiones (3 semanales) de 30 minutos, de ejercicios orofaríngeos y electroestimulación. Los pacientes fueron evaluados al inicio y al final de la intervención mediante antropometría, polisomnografía, escala de somnolencia de Epworth, SAQLI y tolerancia al CPAP. La tolerancia al CPAP fue evaluada por una escala visual calificada de 1 a 10 (10 correspondía a tolerancia máxima). Las variables cualitativas se describieron en frecuencias y las variables cuantitativas en promedios o medianas dependiendo del tipo de distribución. Resultados. El índice de apnea-hipoapnea mejoró de 22 a 13 por hora, el SAQLI mejoró de 4,65 a 5,33 y Epworth pasó de 7 a 5 después de la intervención. La tolerancia al CPAP mejoró de 3 a 9. Las sesiones fueron bien toleradas, no se presentaron eventos adversos importantes. Cuatro pacientes presentaron dolor muscular facial. Conclusiones. Los ejercicios de reeducación con electroterapia son fáciles, reproducibles, seguros y potencialmente beneficiosos para el SAOS, con mínimos efectos secundarios (AU)

Introduction. Obstructive sleep apnea syndrome (OSA) is associated with increased morbidity and mortality. Alternative interventions to continuous positive airway pressure (CPAP) lack efficacy and safety or are highly complex. Objective. To describe the effects of an outpatient program of orofacial and extralaryngeal muscle re-education combined with electro-stimulation (TENS) of the same muscle groups in patients with OSA attending a tertiary level university hospital. Design. Prospective, incident case series, with consecutively selected patients. Methods. Participants consisted of 17 patients with mild, moderate or severe OSA who attended 24 thirty-minute sessions (three per week) of oropharyngeal exercises and electro-stimulation. The patients were evaluated at the beginning and end of the intervention by anthropometry, polysomnography, the Epworth sleepiness scale, and the Calgary Sleep Apnea Quality of Life (SAQLI) index. CPAP tolerance was evaluated by a visual scale rated from 1 to 10 (10 indicating maximum tolerance). Qualitative variables were described by frequencies, and quantitative variables by means and medians, depending on the type of distribution. Results. After the intervention, the apnea - hypopnea index improved from 22 to 13 per hour, the SAQLI index improved from 4.65 to 5.33, and the Epworth scale from 7 to 5. CPAP tolerance increased from 3 to 9. The sessions were well tolerated with no adverse events of importance. Four patients reported facial muscular pain. Conclusions. Electrotherapy plus active exercises of the oropharyngeal muscles are easy, feasible, safe, and potentially beneficial for OSA, with minimal side effects (AU)

MHC class I genes in a New World primate, the cotton-top tamarin (Saguinus oedipus), have evolved by an active process of loci turnover.

Lymphocytes of a New World primate, the cotton-top tamarin (Saguinus oedipus), express classical G-related major histocompatibility complex (MHC) class I molecules with unusually limited polymorphism and variability. Three G-related loci, an F locus, an E locus, and two pseudogenes (So-N1 and So-N3) have been identified by cDNA library screening and extensive PCR analysis of both cDNA and genomic DNA from the cotton-top tamarin. Furthermore, each genus of the subfamily Callitrichinae (tamarins and marmosets) appears to express its own unique set of MHC class I genes, likely due to a rapid turnover of loci. The rapid emergence of unique MHC class I genes in the Callitrichinae genera, resulting from an active process of duplication and inactivation of loci, may account for the limited diversity of the MHC class I genes in the cotton-top tamarin. To determine the nature of the entire complement of MHC class I genes in the cotton-top tamarin, we synthesized a genomic DNA library and screened it with MHC class I-specific probes. We isolated nine new MHC class I pseudogenes from this library. These newly isolated tamarin G-related MHC class I pseudogenes are not closely related to any of their functional counterparts in the tamarin, suggesting that they do not share a recent common ancestral gene with the tamarin's currently expressed MHC class I loci. In addition, these tamarin sequences display a high rate of nonsynonymous substitutions in their putative peptide binding region. This indicates that the genes from which they have derived were likely subject to positive selection and, therefore, were once functional. Our data support the notion that an extremely high rate of loci turnover is largely responsible for the limited diversity of the MHC class I genes in the cotton-top tamarin.

The MHC-E locus is the most well conserved of all known primate class I histocompatibility genes.

The HLA-E locus is characterized by limited polymorphism and low levels of cell surface expression. However, the function of the products of this nonclassical MHC class I gene remains unknown. To evaluate the conservation of the MHC-E locus throughout anthropoid primate evolution, we identified the homologue of the HLA-E locus in six different New World monkey species. Full-length sequencing of MHC-EcDNAs in four unrelated cotton-top tamarins (Saguinus oedipus) revealed no evidence for polymorphism. Using the PCR, denaturing gradient gel electrophoresis, and direct sequencing, we also identified MHC-E alleles in five other New World monkey species, representing all extant platyrrhine families. In contrast to all other classical and nonclassical MHC class I genes in primates, the rate of synonymous nucleotide substitution is much greater than the rate of nonsynonymous nucleotide substitution within exons 2 and 3 encoding the peptide binding region (PBR) in MHC-E genes. The PBR of the MHC-E molecule, therefore, has evolved under purifying selective pressures, and the very unusual evolutionary history of this ancient gene provides further evidence that the products of the HLA-E locus serve a critical immunological function. Given the remarkable conservation of the PBR during primate evolution, this critical immunological function is probably related to the peptide binding ability of the MHC-E protein.

Two different primate species express an identical functional MHC class I allele.

The products of the highly polymorphic and variable major histocompatibility complex (MHC) class I loci play a crucial role in host defenses against infectious disease. While similar alleles have been found in closely related species, sharing of a functional MHC class I allele between two species has never been reported. Here we show that an identical functional MHC class I molecule is present in two different primate species with an approximate divergence time of 0.7 million years. Lymphocytes from the red-crested tamarin (Saguinus geoffroyi) expressed an MHC class I allele (Sage-G*01) that was identical in coding sequence to an MHC class I allele (Saoe-G*08) found in the cotton-top tamarin (Saguinus oedipus). Furthermore, influenza virus-specific cytotoxic T lymphocytes (CTLs) generated in the cotton-top tamarin killed lymphocytes expressing the influenza virus nucleoprotein (NP) from the red-crested tamarin. Since the influenza virus NP epitope is bound by Saoe-G*08 in the cotton-top tamarin, it is likely that this molecule is functional in both species. These data provide the first evidence that functional MHC class I molecules can be maintained entirely intact in two separate species.

Identification of new mamu-DRB alleles using DGGE and direct sequencing.

Rhesus macaques represent important animal models for biomedical research. The ability to identify macaque major histocompatibility complex (Mhc) alleles is crucial for fully understanding these models of autoimmune and infectious disease. Here we describe a rapid and unambiguous way to distinguish DRB alleles in the rhesus macaque using the polymerase chain reaction, denaturing gradient gel electrophoresis (DGGE), and direct sequencing. The highly variable second exon of Mamu-DRB alleles was amplified using generic DRB primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and alleles were determined using fluorescent-based sequencing. Validity of this typing procedure was confirmed by identification of all DRB alleles for three macaques previously characterized by cloning and sequencing techniques. Importantly, our analysis revealed DRB alleles not previously identified in the three reference animals. Using this technique, we identified 40 alleles in fifteen unrelated macaques. On the basis of phylogenetic tree analyses, 14 new DRB alleles were assigned to 10 different Mhc-DRB lineages. Interestingly, two of the new DRB6 lineages had previously been identified in prosimians and pigtailed macaques. Whereas traditional DRB typing methods provide limited information, our new technique provides a simple and relatively rapid way of identifying DRB alleles for tissue typing, determining individual identification and studies of disease association and susceptibility. This new technique should also contribute to ongoing studies of Mhc function and evolution in many different species of nonhuman primates.

Evolutionary instability of the major histocompatibility complex class I loci in New World primates.

Homologues of the human major histocompatibility complex (MHC) HLA-A, -B, -E, -F, and -G loci are present in all the Catarrhini (Old World primates, apes, and humans), and some of their allelic lineages have survived several speciation events. Analysis of 26 MHC class I cDNAs from seven different genera of New World primates revealed that the Callitrichinae (tamarins and marmosets) are an exception to these rules of MHC stability. In gene trees of primate MHC class I genes, sequences from the Callitrichinae cluster in a genus-specific fashion, whereas in the other genera of New World primates, as in the Catarrhini, they cluster in a transgeneric way. The genus-specific clustering of the Callitrichinae cDNAs indicates that there is no orthology between MHC class I loci in genera of this phyletic group. Additionally, the Callitrichinae genera exhibit limited variability of their MHC class I genes, in contrast to the high variability displayed by all other primates. Each Callitrichinae genus, therefore, expresses its own set of MHC class I genes, suggesting that an unusually high rate of turnover of loci occurs in this subfamily. The limited variability of MHC class I genes in the Callitrichinae is likely the result of the recent origin of these loci.

MHC class I-processed pseudogenes in New World primates provide evidence for rapid turnover of MHC class I genes.

The MHC class I genes of the New World primate, the cotton-top tamarin (Saguinus oedipus), are an exception to the high polymorphism and variability displayed by this multigene family. We report the isolation of the first two processed pseudogenes from the MHC region in primates. These two MHC class I-processed pseudogenes (MHC-PS1 and -PS2) were found in several species of New World primates, suggesting a possible explanation for the cotton-top tamarin's limited MHC class I diversity. The pattern of synonymous and nonsynonymous substitutions in PS1 suggests that the gene that gave rise to this processed pseudogene was once subject to selection for variability in the peptide binding region and might, therefore, have been functional. Additionally, PSI is not closely related to the expressed cotton-top tamarin's MHC class I genes, but does show some similarity to So-N1, a tamarin pseudogene from which no transcript has been found. Thus, PS1 may represent a remnant of a once active MHC class I gene that is no longer functional in the cotton-top tamarin. The MHC class I loci in primates, therefore, appear to be evolving by a continual process of duplication and inactivation. This process seems to be exaggerated in New World primates and may in part be responsible for the cotton-top tamarin's limited MHC class I diversity.

The MHC class I genes of the rhesus monkey. Different evolutionary histories of MHC class I and II genes in primates.

Homologues of the human HLA-A and -B MHC class I loci have been found in great apes and Old World primates suggesting that these two loci have existed for at least 30 million years. The C locus, however, shows some sequence similarity to the B locus and has been found only in gorillas, chimpanzees, and humans. To determine the age of the MHC class I C locus and to examine the evolution of the A and B loci we have cloned, sequenced, and in vitro translated 16 MHC class I cDNAs from two unrelated rhesus monkeys (Macaca mulatta) using both cDNA library screening and PCR amplification. Analyses of these sequences suggest that the C locus is not present in the rhesus monkey, indicating that this locus may be of recent origin in gorillas, chimpanzees, and humans. The rhesus monkey's complement of MHC class I genes includes the products of at least one expressed A locus and at least two expressed B loci, indicating that a duplication of the B locus has taken place in the lineage leading to these Old World primates. Comparison of rhesus monkey MHC class I cDNAs to their primate counterparts reveals fundamental differences between MHC class I and class II evolution in primates. Although MHC class II allelic lineages are shared between humans and Old World primates, no such trans-species sharing of allelic lineages is seen at the MHC class I loci.