Validación de ELISA indirecto para evaluación de anticuerpos bovinos y de cobayo contra virus parainfluenza 3 bovino (bpi3). Evaluación de seroprevalencia a virus bpi3 en rodeos bovinos de la provincia de Buenos Aires

In Argentine, livestock farming occupies a preponderant place in the national economy. A sizeable percentage of the losses in livestock is due to the incidence of infectious diseases, whether of viral, bacterial or parasitic origin. Among the diseases that cause great economic losses in the livestock industry are those of the bovine respiratory complex (CRB). Parainfluenza 3 (PI3) is one of the pathogens implicated. The PI-3 virus is endemic in cattle and has a worldwide distribution. Seroprevalence in our country is 60-90 %. The objective was the validation of two indirect ELISAs that quantify bovine and guinea pig antibodies against PI3 to complement other assays. The validation of the bovine ELISA will allow the determination of seroprevalence of PI3 in bovine herds of Buenos Aires and will contribute to the development of the guinea pig model that evaluates the immunogenic quality of commercial vaccines. We continued with the development of the experimental PI3 ELISA of bovine animals. To establish the cutoff point for this assay, a frequency curve was performed with positive and negative sera of known history. The cut-off point was determined at a corrected optical density greater than or equal to 0.3, from which the positive sera are considered. To calculate repeatability, the results of a sample within the same plate were evaluated, making series with the same samples in 6 plates at the same time (variation between plates). For the repetitive parameter, a coefficient of variation (CV, standard deviation of the replicates/means of the replicates) was obtained: 22.8 % between replicates, and a CV of 12 % between the plates. Linearity was evaluated by performing 16 replicates of a positive serum on 6 bases of dilutions 4. The Linearity obtained was 76 % (Linear Regression Analysis, R2) Diagnostic sensitivity and specificity were calculated by testing a series of reference samples, taken from animals with known history and health status regarding specific disease/infection. The sera were evaluated by inhibition of hemagglutination to establish the concordance between both techniques. The degree of agreement was established by means of the Weighted Kappa index. The sensitivity represents the sera detected positive through the technique of the total of positively positive sera, and specificity represents the sera detected negative through the technique of the total of negative sera. In conclusion, the cut-off point of PI3 bovine ELISA assay was calculated. The sensitivity was of 92 % and the specificity was of 100 %.En Argentina, la explotación ganadera ocupa un lugar preponderante de la economía. Un gran porcentaje de las pérdidas se encuentra vinculado a las enfermedades infecciosas. Entre las enfermedades más relevantes se encuentran las del complejo respiratorio bovino (CRB), la parainfluenza 3 (PI3) es uno de los patógenos implicados. El virus de PI3 es endémico del ganado bovino, presenta distribución mundial. La seroprevalencia en nuestro país es 60-90 %. El objetivo fue validar dos ELISA indirectos que cuantifiquen anticuerpos bovinos y de cobayos contra PI3 para complementar otros. La validación del ELISA bovino permitirá determinar seroprevalencia de PI3 en rodeos bovinos de Buenos Aires y aportará al desarrollo del modelo cobayo que evalúa calidad inmunogénica de vacunas comerciales. Se continuó el desarrollo del ELISA PI3 experimental de bovinos planteado en este trabajo. Para establecer el punto de corte se realizó una curva de frecuencia con sueros positivos y negativos de historia conocida. El punto de corte se determinó en una densidad óptica corregida mayor o igual a 0.3, a partir de la cual los sueros se consideran positivos. Para calcular la repetibilidad se evaluaron los resultados de una misma muestra dentro de una misma placa y, efectuando series en 6 placas a la vez, (variación entre placas). Para el parámetro repetibilidad se obtuvo un coeficiente de variación (desviación estándar de las réplicas dividida por la media de las réplicas) de 22.8 % entre réplicas y un coeficiente de variación de 12 % entre placas. La linealidad es la capacidad del ensayo, dentro de un determinado intervalo, de dar un resultado que sea proporcional a la cantidad del analito a determinar. Se evaluó mediante la realización de 16 repeticiones de un suero positivo en 6 diluciones base 4. La linealidad obtenida fue de un 76 % (Regresión lineal, R2). La sensibilidad representa los sueros detectados positivos por la técnica del total de sueros fehacientemente positivos. La especificidad representa los sueros detectados negativos por la técnica del total de sueros fehacientemente negativos. Ambos parámetros fueron calculados evaluando una serie de muestras de referencia, tomadas de animales de situación sanitaria conocida. Se evaluaron los sueros por inhibición de la hemoaglutinación para establecer la concordancia entre ambas técnicas, mediante el índice Kappa Ponderado. Se logró calcular el punto de corte del ELISA y se determinó que la sensibilidad es del 92 % con una especificidad del 100 %

Tyr78Phe Transthyretin Mutation with Predominant Motor Neuropathy as the Initial Presentation

Transthyretin (TTR) amyloidosis, the most frequent form of hereditary amyloidosis, is caused by dominant mutations in the TTR gene. More than 100 mutations have been identified. Clinical manifestations of TTR amyloidosis are usually induced by extracellular amyloid deposition in several organs. The major neurological manifestation is motor-sensory neuropathy associated with dysautonomic impairment. Here, we describe a 63-year-old man who came to our institution due to a suspected motor neuron disease. During a 4-year follow-up period, he underwent extensive clinical examination, electromyographic studies, sural nerve biopsy and TTR gene analysis by direct sequencing. Despite the predominant motor involvement, the detailed clinical examination also showed some mild sensory and dysautonomic signs. In addition, his clinical and family history included multiorgan disorders, such as carpal tunnel syndrome, as well as conditions with cardiac, renal, eye, and hepatic involvement. The sural nerve biopsy disclosed amyloid deposition, and the sequence analysis of the TTR gene detected a heterozygous Tyr78Phe substitution. The TTR gene variant found in our patient had only been described once so far, in a French man of Italian origin presenting with late-onset peripheral neuropathy and bilateral carpal tunnel syndrome. The predominant motor involvement presented by our patient is an uncommon occurrence and demonstrates the clinical heterogeneity of TTR amyloidosis

Cosegregation of novel mitochondrial 16S rRNA gene mutations with the age-associated T414G variant in human cybrids

Ever increasing evidence has been provided on the accumulation of mutations in the mitochondrial DNA (mtDNA) during the aging process. However, the lack of direct functional consequences of the mutant mtDNA load on the mitochondria-dependent cell metabolism has raised many questions on the physiological importance of the age-related mtDNA variations. In the present work, we have analyzed the bioenergetic properties associated with the age-related T414G mutation of the mtDNA control region in transmitochondrial cybrids. The results show that the T414G mutation does not cause per se any detectable bioenergetic change. Moreover, three mtDNA mutations clustered in the 16S ribosomal RNA gene cosegregated together with the T414G in the same cybrid cell line. Two of them, namely T1843C and A1940G, are novel and associate with a negative bioenergetic phenotype. The results are discussed in the more general context of the complex heterogeneity and the dramatic instability of the mitochondrial genome during cell culture of transmitochondrial cybrids

Intrinsic and extrinsic factors influencing the clinical course of B-cell chronic lymphocytic leukemia: prognostic markers with pathogenetic relevance

B-cell chronic lymphocytic leukemia (CLL), the most frequent leukemia in the Western world, is characterized by extremely variable clinical courses with survivals ranging from 1 to more than 15 years. The pathogenetic factors playing a key role in defining the biological features of CLL cells, hence eventually influencing the clinical aggressiveness of the disease, are here divided into "intrinsic factors", mainly genomic alterations of CLL cells, and "extrinsic factors", responsible for direct microenvironmental interactions of CLL cells; the latter group includes interactions of CLL cells occurring via the surface B cell receptor (BCR) and dependent to specific molecular features of the BCR itself and/or to the presence of the BCR-associated molecule ZAP-70, or via other non-BCR-dependent interactions, e.g. specific receptor/ligand interactions, such as CD38/CD31 or CD49d/VCAM-1. A putative final model, discussing the pathogenesis and the clinicobiological features of CLL in relationship of these factors, is also provided

A new case of limb girdle muscular dystrophy 2g in a greek patient, founder effect and review of the literature

Limb girdle muscular dystrophy (LGMD) type 2G is a rare form of muscle disease, described only in a few patients worldwide, caused by mutations in TCAP gene, encoding the protein telethonin. It is characterised by proximal limb muscle weakness associated with distal involvement of lower limbs, starting in the first or second decade of life. We describe the case of a 37-year-old woman of Greek origin, affected by disto-proximal lower limb weakness. No cardiac or respiratory involvement was detected. Muscle biopsy showed myopathic changes with type I fibre hypotrophy, cytoplasmic vacuoles, lipid overload, multiple central nuclei and fibre splittings; ultrastructural examination showed metabolic abnormalities. Next generation sequencing analysis detected a homozygous frameshift mutation in the TCAP gene (c.90_91del), previously described in one Turkish family. Immunostaining and Western blot analysis showed complete absence of telethonin. Interestingly, Single Nucleotide Polymorphism analysis of the 10 Mb genomic region containing the TCAP gene showed a shared homozygous haplotype of both the Greek and the Turkish patients, thus suggesting a possible founder effect of TCAP gene c.90_91del mutation in this part of the Mediterranean area. (C) 2018 Elsevier B.V. All rights reserved.Peer reviewe

Post-Transplant Nivolumab Plus Unselected Autologous Lymphocytes in Refractory Hodgkin Lymphoma: A Feasible and Promising Salvage Therapy Associated With Expansion and Maturation of NK Cells

Immune checkpoint inhibitors (CI) have demonstrated clinical activity in Hodgkin Lymphoma (HL) patients relapsing after autologous stem cell transplantation (ASCT), although only 20% complete response (CR) rate was observed. The efficacy of CI is strictly related to the host immune competence, which is impaired in heavily pre-treated HL patients. Here, we aimed to enhance the activity of early post-ASCT CI (nivolumab) administration with the infusion of autologous lymphocytes (ALI). Twelve patients with relapse/refractory (R/R) HL (median age 28.5 years; range 18-65), underwent lymphocyte apheresis after first line chemotherapy and then proceeded to salvage therapy. Subsequently, 9 patients with progressive disease at ASCT received early post-transplant CI supported with four ALI, whereas 3 responding patients received ALI alone, as a control cohort. No severe adverse events were recorded. HL-treated patients achieved negative PET scan CR and 8 are alive and disease-free after a median follow-up of 28 months. Four patients underwent subsequent allogeneic SCT. Phenotypic analysis of circulating cells showed a faster expansion of highly differentiated NK cells in ALI plus nivolumab-treated patients as compared to control patients. Our data show anti-tumor activity with good tolerability of ALI + CI for R/R HL and suggest that this setting may accelerate NK cell development/maturation and favor the expansion of the "adaptive" NK cell compartment in patients with HCMV seropositivity, in the absence of HCMV reactivation

Tinnitus: Distinguishing between Subjectively Perceived Loudness and Tinnitus-Related Distress

OBJECTIVES: Overall success of current tinnitus therapies is low, which may be due to the heterogeneity of tinnitus patients. Therefore, subclassification of tinnitus patients is expected to improve therapeutic allocation, which, in turn, is hoped to improve therapeutic success for the individual patient. The present study aims to define factors that differentially influence subjectively perceived tinnitus loudness and tinnitus-related distress. METHODS: In a questionnaire-based cross-sectional survey, the data of 4705 individuals with tinnitus were analyzed. The self-report questionnaire contained items about subjective tinnitus loudness, type of onset, awareness and localization of the tinnitus, hearing impairment, chronic comorbidities, sleep quality, and psychometrically validated questionnaires addressing tinnitus-related distress, depressivity, anxiety, and somatic symptom severity. In a binary step-wise logistic regression model, we tested the predictive power of these variables on subjective tinnitus loudness and tinnitus-related distress. RESULTS: The present data contribute to the distinction between subjective tinnitus loudness and tinnitus-related distress. Whereas subjective loudness was associated with permanent awareness and binaural localization of the tinnitus, tinnitus-related distress was associated with depressivity, anxiety, and somatic symptom severity. CONCLUSIONS: Subjective tinnitus loudness and the potential presence of severe depressivity, anxiety, and somatic symptom severity should be assessed separately from tinnitus-related distress. If loud tinnitus is the major complaint together with mild or moderate tinnitus-related distress, therapies should focus on auditory perception. If levels of depressivity, anxiety or somatic symptom severity are severe, therapies and further diagnosis should focus on these symptoms at first

Clinical and molecular characterization of a cohort of patients with novel nucleotide alterations of the Dystrophin gene detected by direct sequencing

<p>Abstract</p> <p>Background</p> <p>Duchenne and Becker Muscular dystrophies (DMD/BMD) are allelic disorders caused by mutations in the dystrophin gene, which encodes a sarcolemmal protein responsible for muscle integrity. Deletions and duplications account for approximately 75% of mutations in DMD and 85% in BMD. The implementation of techniques allowing complete gene sequencing has focused attention on small point mutations and other mechanisms underlying complex rearrangements.</p> <p>Methods</p> <p>We selected 47 patients (41 families; 35 DMD, 6 BMD) without deletions and duplications in <it>DMD </it>gene (excluded by multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction analysis). This cohort was investigated by systematic direct sequence analysis to study sequence variation. We focused our attention on rare mutational events which were further studied through transcript analysis.</p> <p>Results</p> <p>We identified 40 different nucleotide alterations in DMD gene and their clinical correlates; altogether, 16 mutations were novel. DMD probands carried 9 microinsertions/microdeletions, 19 nonsense mutations, and 7 splice-site mutations. BMD patients carried 2 nonsense mutations, 2 splice-site mutations, 1 missense substitution, and 1 single base insertion. The most frequent stop codon was TGA (n = 10 patients), followed by TAG (n = 7) and TAA (n = 4). We also analyzed the molecular mechanisms of five rare mutational events. They are two frame-shifting mutations in the <it>DMD </it>gene 3'end in BMD and three novel splicing defects: IVS42: c.6118-3C>A, which causes a leaky splice-site; c.9560A>G, which determines a cryptic splice-site activation and c.9564-426 T>G, which creates pseudoexon retention within IVS65.</p> <p>Conclusion</p> <p>The analysis of our patients' sample, carrying point mutations or complex rearrangements in <it>DMD </it>gene, contributes to the knowledge on phenotypic correlations in dystrophinopatic patients and can provide a better understanding of pre-mRNA maturation defects and dystrophin functional domains. These data can have a prognostic relevance and can be useful in directing new therapeutic approaches, which rely on a precise definition of the genetic defects as well as their molecular consequences.</p

Exome-wide Rare Variant Analysis Identifies TUBA4A Mutations Associated with Familial ALS

Exome sequencing is an effective strategy for identifying human disease genes. However, this methodology is difficult in late-onset diseases where limited availability of DNA from informative family members prohibits comprehensive segregation analysis. To overcome this limitation, we performed an exome-wide rare variant burden analysis of 363 index cases with familial ALS (FALS). The results revealed an excess of patient variants within TUBA4A, the gene encoding the Tubulin, Alpha 4A protein. Analysis of a further 272 FALS cases and 5,510 internal controls confirmed the overrepresentation as statistically significant and replicable. Functional analyses revealed that TUBA4A mutants destabilize the microtubule network, diminishing its repolymerization capability. These results further emphasize the role of cytoskeletal defects in ALS and demonstrate the power of gene-based rare variant analyses in situations where causal genes cannot be identified through traditional segregation analysis

Analysis of shared common genetic risk between amyotrophic lateral sclerosis and epilepsy

Because hyper-excitability has been shown to be a shared pathophysiological mechanism, we used the latest and largest genome-wide studies in amyotrophic lateral sclerosis (n = 36,052) and epilepsy (n = 38,349) to determine genetic overlap between these conditions. First, we showed no significant genetic correlation, also when binned on minor allele frequency. Second, we confirmed the absence of polygenic overlap using genomic risk score analysis. Finally, we did not identify pleiotropic variants in meta-analyses of the 2 diseases. Our findings indicate that amyotrophic lateral sclerosis and epilepsy do not share common genetic risk, showing that hyper-excitability in both disorders has distinct origins