Validation of a highly sensitive HaloTag-based assay to evaluate the potency of a novel class of allosteric ß-Galactosidase correctors.

Site-directed Enzyme Enhancement Therapy (SEE-Tx®) technology is a disease-agnostic drug discovery tool that can be applied to any protein target of interest with a known three-dimensional structure. We used this proprietary technology to identify and characterize the therapeutic potential of structurally targeted allosteric regulators (STARs) of the lysosomal hydrolase ß-galactosidase (ß-Gal), which is deficient due to gene mutations in galactosidase beta 1 (GLB1)-related lysosomal storage disorders (LSDs). The biochemical HaloTag cleavage assay was used to monitor the delivery of wildtype (WT) ß-Gal and four disease-related ß-Gal variants (p.Ile51Thr, p.Arg59His, p.Arg201Cys and p.Trp273Leu) in the presence and absence of two identified STAR compounds. In addition, the ability of STARs to reduce toxic substrate was assessed in a canine fibroblast cell model. In contrast to the competitive pharmacological chaperone N-nonyl-deoxygalactonojirimycin (NN-DGJ), the two identified STAR compounds stabilized and substantially enhanced the lysosomal transport of wildtype enzyme and disease-causing ß-Gal variants. In addition, the two STAR compounds reduced the intracellular accumulation of exogenous GM1 ganglioside, an effect not observed with the competitive chaperone NN-DGJ. This proof-of-concept study demonstrates that the SEE-Tx® platform is a rapid and cost-effective drug discovery tool for identifying STARs for the treatment of LSDs. In addition, the HaloTag assay developed in our lab has proved valuable in investigating the effect of STARs in promoting enzyme transport and lysosomal delivery. Automatization and upscaling of this assay would be beneficial for screening STARs as part of the drug discovery process.

Epidemiología de la esclerosis múltiple en España / Epidemiology of multiple sclerosis in Spain

Introducción. La esclerosis múltiple es una enfermedad crónica autoinmune, inflamatoria y degenerativa del sistema nervioso central, y es el trastorno neurológico discapacitante no traumático más común en adultos jóvenes. Los estudios de prevalencia más recientes indican que la frecuencia de la enfermedad ha aumentado en el mundo en las últimas décadas, que dicho incremento de la prevalencia ocurre fundamentalmente a expensas de un mayor número de casos de mujeres con formas remitentes, y que el gradiente latitudinal de la incidencia de la enfermedad se viene atenuando. Objetivo. Revisar los estudios sobre epidemiología de esclerosis múltiple en España para verificar si las tendencias mundiales se confirman en nuestro país. Desarrollo. Búsqueda bibliográfica en las bases de datos PubMed y Teseo usando como palabras clave "epidemiology", "prevalence" e "incidence", cruzándolas con los términos "multiple sclerosis" y "Spain"; se realiza una selección inicial por título y resumen, en castellano e inglés, entre los años 1968 y 2018. Conclusiones. Un buen número de estudios epidemiológicos recientes en España confirman que es una región de prevalencia media-alta de la enfermedad a lo largo de su geografía. Las cifras de prevalencia aumentan progresivamente a lo largo de las últimas décadas hasta alcanzar en la actualidad 80-180 casos por 100.000 habitantes, y ello ha ocurrido a expensas de una mayor frecuencia de la enfermedad en las mujeres

Introduction. Multiple sclerosis is a chronic autoimmune, inflammatory and neurodegenerative disease of the central nervous system and the most common non-traumatic disabling neurological disease in young adults. In the latest decades, multiple sclerosis is increasing worldwide, especially in women. The latitudinal distribution has been progressively attenuated. Aim. To review the epidemiological studies of multiple sclerosis in Spain to verify if this worldwide trend also occurs in Spain. Development. We searched PubMed and Teseo databases using the search terms 'epidemiology', 'prevalence', 'incidence', 'multiple sclerosis' and 'Spain'. We selected articles published in Spanish and English between 1968 and 2018. Conclusions. Recent epidemiological studies confirm that Spain is a medium-high risk area for MS. The prevalence of MS has increased significantly throughout Spain in the latest years, especially in women, and recent studies show prevalence as high as 80-180 cases per 100,000

Multiple sclerosis prevalence and incidence in San Vicente del Raspeig, Spain.

BACKGROUND: Changes in the demographic epidemiology of multiple sclerosis (MS) may challenge the view of a latitudinal gradient in the distribution of MS. The objective of this study was to assess the incidence and prevalence of MS in addition to information on MS phenotypes and the use of disease modifying therapies (DMTs) in San Vicente del Raspeig in south eastern Spain. METHODS: This was a prospective epidemiological study of MS in San Vicente del Raspeig (population of 57,175 inhabitants based on the 2017 census) from 2005 to 2018. Multiple sources were used to identify MS cases. We considered as prevalent and incident cases all patients who satisfied either the criteria of Poser for clinically or laboratory-supported definite MS, or McDonald criteria. MS phenotypes were defined according to the 2013 revisions. RESULTS: For the prevalence data, 64 patients were identified. The non-adjusted prevalence was 111.9 (95% CI: 87.7-142.9) cases per 100,000 inhabitants; the prevalence was 159.3 cases per 100,000 inhabitants for women and 63.6 cases per 100,000 inhabitants for men. The female-to-male ratio was 2.6:1. The age-adjusted prevalence for the European standard population was 107 cases per 100,000 inhabitants. During the study period, the incidence was 5 cases per 100,000 inhabitants per year. Most patients were being treated with DMTs (81.3%). MS was active in at least 12.5% of patients. CONCLUSIONS: The results are consistent with the increased risk of MS in Spain observed over the last three decades, with growing prevalence rates that place the country in the high-risk prevalence zone.

A Prominent Role of the Human Cytomegalovirus UL8 Glycoprotein in Restraining Proinflammatory Cytokine Production by Myeloid Cells at Late Times during Infection.

Human cytomegalovirus (HCMV) persistence in infected individuals relies on a plethora of mechanisms to efficiently reduce host immune responses. To that end, HCMV uses a variety of gene products, some of which have not been identified yet. Here we characterized the UL8 gene, which consists of two exons, sharing the first with the HCMV RL11 family member UL7 UL8 is a transmembrane protein with an N-terminal immunoglobulin (Ig)-like domain in common with UL7 but with an extended stalk and a distinctive cytoplasmic tail. The UL8 open reading frame gives rise to a heavily glycosylated protein predominantly expressed on the cell surface, from where it can be partially endocytosed and subsequently degraded. Infections with UL8-tagged viruses indicated that UL8 was synthesized with late-phase kinetics. By virtue of its highly conserved Ig-like domain, this viral protein interacted with a surface molecule present on activated neutrophils. Notably, when ectopically expressed in THP-1 myeloid cells, UL8 was able to significantly reduce the production of a variety of proinflammatory cytokines. Mutations in UL8 indicated that this functional effect was mediated by the cell surface expression of its Ig-like domain. To investigate the impact of the viral protein in the infection context, we engineered HCMVs lacking the UL8 gene and demonstrated that UL8 decreases the release of a large number of proinflammatory factors at late times after infection of THP-1 cells. Our data indicate that UL8 may exert an immunosuppressive role key for HCMV survival in the host.IMPORTANCE HCMV is a major pathogen that causes life-threatening diseases and disabilities in infected newborns and immunocompromised individuals. Containing one of the largest genomes among all reported human viruses, HCMV encodes an impressive repertoire of gene products. However, the functions of a large proportion of them still remain unknown, a fact that complicates the design of new therapeutic approaches to prevent or treat HCMV-associated diseases. In this report, we have conducted an extensive study of UL8, one of the previously uncharacterized HCMV open reading frames. We found that the UL8 protein is expressed at late times postinfection and utilized by HCMV to reduce the production of proinflammatory factors by infected myeloid cells. Thus, the work presented here points to a key role of UL8 as a novel HCMV immune modulator capable of restraining host antiviral defenses.

Fingolimod: efectividad y seguridad en la práctica clínica habitual. Estudio observacional, retrospectivo y multicéntrico en la provincia de Alicante / Fingolimod: effectiveness and safety in routine clinical practice. An observational, retrospective, multi-centre study in the province of Alicante

INTRODUCCIÓN: Los estudios postautorización son importantes para confirmar si los resultados de los ensayos clínicos se reproducen en la práctica clínica habitual. OBJETIVO: Evaluar la efectividad y seguridad del fingolimod en la práctica clínica en la provincia de Alicante. PACIENTES Y MÉTODOS: Estudio multicéntrico retrospectivo de pacientes con esclerosis múltiple remitente tratados con fingolimod. Se recogen las características demográficas, clínicas y farmacológicas. Se describe la efectividad del fármaco -tasa anualizada de brotes (TAB) y porcentaje de pacientes libres de brotes- al año y a los dos años de tratamiento en relación con el año previo y datos de efectos secundarios. RESULTADOS: Se incluyó a 89 pacientes. El tratamiento previo fue inmunomodulador (interferón beta o acetato de glatiramero) en 54 pacientes y natalizumab en 32. Cincuenta pacientes cambiaron por fracaso con el inmunomodulador y 31 por serología positiva del virus JC (VJC+). La TAB global disminuyó el 67,3% el primer año (p < 0,0001) y el 84,1% el segundo (p = 0,0078). Disminuyó en los pacientes con fracaso del inmunomodulador (el 85,6% el primer año, p < 0,0001; el 88,9% el segundo año, p = 0,0039) y aumentó de forma no significativa en los pacientes VJC+ en el primer año. El porcentaje de pacientes libres de brotes en la población global aumentó del 32,6 al 68,1% en el primer año (p < 0,0019) y al 82,6% en el segundo (p = 0,0215). Este aumento no se observó en los pacientes VJC+. Trece pacientes tuvieron efectos secundarios, que obligaron a la retirada del fármaco en dos de ellos. CONCLUSIÓN: En la práctica clínica de la provincia de Alicante, el fingolimod mostró una efectividad y una seguridad ligeramente superiores a las de los ensayos clínicos

INTRODUCTION: Post-authorisation studies are important to confirm whether the outcomes of clinical trials are reproduced in usual clinical practice. AIMS: To evaluate the effectiveness and safety of fingolimod in clinical practice in the province of Alicante. PATIENTS AND METHODS: A retrospective multi-centre study was conducted with remitting multiple sclerosis patients treated with fingolimod. Demographic, clinical and pharmacological data were collected. We report on the effectiveness of the drug -annualised relapse rate (ARR) and percentage of patients free from attacks- at one and at two years after treatment in relation to the previous year, and data concerning side effects are also provided. RESULTS: The sample consisted of 89 PATIENTS: Previous treatment was with immunomodulators (interferon beta or glatiramer acetate) in 54 patients and natalizumab in 32. Fifty patients changed due to failure with the immunomodulator and 31 owing to positive serology for JC virus (JCV+). Overall ARR decreased by 67.3% the first year (p < 0.0001) and by 84.1% the second (p = 0.0078). It diminished in patients with immunomodulator failure (85.6% the first year, p < 0.0001; 88.9% the second year, p = 0.0039) and increased in a non-significant manner in JCV+ patients in the first year. The percentage of patients free from relapses in the overall population increased from 32.6% to 68.1% in the first year (p < 0.0019) and to 82.6% in the second (p = 0.0215). This increase was not observed in JCV+ PATIENTS: Side effects were reported by 13 patients, which led to the drug being withdrawn in two of them. CONCLUSIONS: In clinical practice in the province of Alicante, levels of effectiveness and safety of fingolimod proved to be slightly higher than those found in clinical trials (AU)

Signaling Lymphocytic Activation Molecule Family Receptor Homologs in New World Monkey Cytomegaloviruses.

UNLABELLED: Throughout evolution, large DNA viruses have been usurping genes from their hosts to equip themselves with proteins that restrain host immune defenses. Signaling lymphocytic activation molecule (SLAM) family (SLAMF) receptors are involved in the regulation of both innate and adaptive immunity, which occurs upon engagement with their ligands via homotypic or heterotypic interactions. Here we report a total of seven SLAMF genes encoded by the genomes of two cytomegalovirus (CMV) species, squirrel monkey CMV (SMCMV) and owl monkey CMV (OMCMV), that infect New World monkeys. Our results indicate that host genes were captured by retrotranscription at different stages of the CMV-host coevolution. The most recent acquisition led to S1 in SMCMV. S1 is a SLAMF6 homolog with an amino acid sequence identity of 97% to SLAMF6 in its ligand-binding N-terminal Ig domain. We demonstrate that S1 is a cell surface glycoprotein capable of binding to host SLAMF6. Furthermore, the OMCMV genome encodes A33, an LY9 (SLAMF3) homolog, and A43, a CD48 (SLAMF2) homolog, two soluble glycoproteins which recognize their respective cellular counterreceptors and thus are likely to be viral SLAMF decoy receptors. In addition, distinct copies of further divergent CD48 homologs were found to be encoded by both CMV genomes. Remarkably, all these molecules display a number of unique features, including cytoplasmic tails lacking characteristic SLAMF signaling motifs. Taken together, our findings indicate a novel immune evasion mechanism in which incorporation of host SLAMF receptors that retain their ligand-binding properties enables viruses to interfere with SLAMF functions and to supply themselves with convenient structural molds for expanding their immunomodulatory repertoires. IMPORTANCE: The way in which viruses shape their genomes under the continual selective pressure exerted by the host immune system is central for their survival. Here, we report that New World monkey cytomegaloviruses have broadly captured and duplicated immune cell receptors of the signaling lymphocyte activation molecule (SLAM) family during host-virus coevolution. Notably, we demonstrate that several of these viral SLAMs exhibit exceptional preservation of their N-terminal immunoglobulin domains, which results in maintenance of their ligand-binding capacities. At the same time, these molecules present distinctive structural properties which include soluble forms and the absence of typical SLAM signaling motifs in their cytoplasmic domains, likely reflecting the evolutionary adaptation undergone to efficiently interfere with host SLAM family activities. The observation that the genomes of other large DNA viruses might bear SLAM family homologs further underscores the importance of these molecules as a novel class of immune regulators and as convenient scaffolds for viral evolution.

Cytomegalovirus m154 hinders CD48 cell-surface expression and promotes viral escape from host natural killer cell control.

Receptors of the signalling lymphocyte-activation molecules (SLAM) family are involved in the functional regulation of a variety of immune cells upon engagement through homotypic or heterotypic interactions amongst them. Here we show that murine cytomegalovirus (MCMV) dampens the surface expression of several SLAM receptors during the course of the infection of macrophages. By screening a panel of MCMV deletion mutants, we identified m154 as an immunoevasin that effectively reduces the cell-surface expression of the SLAM family member CD48, a high-affinity ligand for natural killer (NK) and cytotoxic T cell receptor CD244. m154 is a mucin-like protein, expressed with early kinetics, which can be found at the cell surface of the infected cell. During infection, m154 leads to proteolytic degradation of CD48. This viral protein interferes with the NK cell cytotoxicity triggered by MCMV-infected macrophages. In addition, we demonstrate that an MCMV mutant virus lacking m154 expression results in an attenuated phenotype in vivo, which can be substantially restored after NK cell depletion in mice. This is the first description of a viral gene capable of downregulating CD48. Our novel findings define m154 as an important player in MCMV innate immune regulation.

Human cytomegalovirus UL7, a homologue of the SLAM-family receptor CD229, impairs cytokine production.

Human cytomegalovirus (HCMV), the ß-herpesvirus prototype, has evolved a wide spectrum of mechanisms to counteract host immunity. Among them, HCMV uses cellular captured genes encoding molecules capable of interfering with the original host function or of fulfilling new immunomodulatory tasks. Here, we report on UL7, a novel HCMV heavily glycosylated transmembrane protein, containing an Ig-like domain that exhibits remarkable amino acid similarity to CD229, a cell-surface molecule of the signalling lymphocyte-activation molecule (SLAM) family involved in leukocyte activation. The UL7 Ig-like domain, which is well-preserved in all HCMV strains, structurally resembles the SLAM-family N-terminal Ig-variable domain responsible for the homophilic and heterophilic interactions that trigger signalling. UL7 is transcribed with early-late kinetics during the lytic infectious cycle. Using a mAb generated against the viral protein, we show that it is constitutively shed, through its mucine-like stalk, from the cell-surface. Production of soluble UL7 is enhanced by PMA and reduced by a broad-spectrum metalloproteinase inhibitor. Although UL7 does not hold the ability to interact with CD229 or other SLAM-family members, it shares with them the capacity to mediate adhesion to leukocytes, specifically to monocyte-derived DCs. Furthermore, we demonstrate that UL7 expression attenuates the production of proinflammatory cytokines TNF, IL-8 and IL-6 in DCs and myeloid cell lines. Thus, the ability of UL7 to interfere with cellular proinflammatory responses may contribute to viral persistence. These results enhance our understanding of those HCMV-encoded molecules involved in sustaining the balance between HCMV and the host immune system.