Involvement of the Transcriptional Coactivator ThMBF1 in the Biocontrol Activity of Trichoderma harzianum

[EN] Trichoderma harzianum is a filamentous fungus well adapted to different ecological niches. Owing to its ability to antagonize a wide range of plant pathogens, it is used as a biological control agent in agriculture. Selected strains of T. harzianum are also able to increase the tolerance of plants to biotic and abiotic stresses. However, little is known about the regulatory elements of the T. harzianum transcriptional machinery and their role in the biocontrol by this species. We had previously reported the involvement of the transcription factor THCTF1 in the T. harzianum production of the secondary metabolite 6-pentyl-pyrone, an important volatile compound related to interspecies cross-talk. Here, we performed a subtractive hybridization to explore the genes regulated by THCTF1, allowing us to identify a multiprotein bridging factor 1 (mbf1) homolog. The gene from T. harzianum T34 was isolated and characterized, and the generated Thmbf1 overexpressing transformants were used to investigate the role of this gene in the biocontrol abilities of the fungus against two plant pathogens. The transformants showed a reduced antifungal activity against Fusarium oxysporum f. sp. lycopersici race 2 (FO) and Botrytis cinerea (BC) in confrontation assays on discontinuous medium, indicating that the Thmbf1 gene could affect T. harzianum production of volatile organic compounds (VOC) with antifungal activity. Moreover, cellophane and dialysis membrane assays indicated that Thmbf1 overexpression affected the production of low molecular weight secreted compounds with antifungal activity against FO. Intriguingly, no correlation in the expression profiles, either in rich or minimal medium, was observed between Thmbf1 and the master regulator gene cross-pathway control (cpc1). Greenhouse assays allowed us to evaluate the biocontrol potential of T. harzianum strains against BC and FO on susceptible tomato plants. The wild type strain T34 significantly reduced the necrotic leaf lesions caused by BC while plants treated with the Thmbf1-overexpressing transformants exhibited an increased susceptibility to this pathogen. The percentages of Fusarium wilt disease incidence and values of aboveground dry weight showed that T34 did not have biocontrol activity against FO, at least in the 'Moneymaker' tomato variety, and that Thmbf1 overexpression increased the incidence of this disease. Our results show that the Thmbf1 overexpression in T34 negatively affects its biocontrol mechanismsSIThis research project was funded by the Spanish Ministry of Economy and Competitiveness (Project no. AGL2015-70671-C2) and the Junta de Castilla y León (Project no. SA009U16

Determination of Exosome Mitochondrial DNA as a Biomarker of Renal Cancer Aggressiveness

This study is partially supported by ID#53169721 project funded by Pfizer and the exosomes characterization was financed thanks to the grant PY20_00241 (FEDER/Junta de Andalucia-Regional Ministry of Economic Transformation, Industry, Knowledge and Universities).Here, the role of non-invasive biomarkers in liquid biopsy was evaluated, mainly in exosomes and mitochondrial DNA (mtDNA) as promising, novel, and stable biomarkers for renal cell carcinoma (RCC). A total of 140 fractions (named from B to F) obtained by ultracentrifugations of whole blood samples from 28 individuals (13 patients and 15 controls) were included. Nanoparticle Tracking Analysis (NTA) was conducted to characterized exosomal fraction. Subsequently, an analysis of digital PCR (dPCR) using the QuantStudio (TM) 3D Digital PCR platform was performed and the quantification of mtDNA copy number by QuantStudioTM 12K Flex Real-Time PCR System (qPCR) was developed. Moreover, Next Generation Sequencing (NGS) analyses were included using MiSeq system (Illumina, San Diego, CA, USA). An F fraction, which contains all exosome data and all mitochondrial markers, was identified in dPCR and qPCR with statistically significant power (adjusted p values <= 0.03) when comparing cases and controls. Moreover, present analysis in mtDNA showed a relevant significance in RCC aggressiveness. To sum up, this is the first time a relation between exosomal mtDNA markers and clinical management of RCC is analyzed. We suggest a promising strategy for future liquid biopsy RCC analysis, although more analysis should be performed prior to application in routine clinical practice.Pfizer 53169721FEDER/Junta de Andalucia-Regional Ministry of Economic Transformation, Industry, Knowledge and Universities PY20_0024

Heme oxygenase-1 and 2 common genetic variants and risk for restless legs syndrome

Varios neurotransmisores, neuropatológicos, neuroimagen, y los datos experimentales, sugieren que la deficiencia de hierro juega un papel importante en la fisiopatología del síndrome de piernas inquietas (RLS). HMOX (Hemeoxygenases) es un importante mecanismo de defensa contra el estrés oxidativo, principalmente a través de la degradación del hemo a biliverdin, libre de hierro, y monóxido de carbono. Hemos analizado si los genes HMOX1 y HMOX2 están relacionados con el riesgo de desarrollar el síndrome de piernas inquietas. Se analizó la distribución de genotipos y las frecuencias alélicas de los HMOX1 rs2071746, HMOX1 rs2071747, HMOX2 rs2270363 y rs1051308 HMOX2 SNPs, así como la presencia de variaciones de número de copia (CNVs) de estos genes en 205 sujetos RLS y 445 controles sanos. Las frecuencias de rs2071746 genotipo TT y R2071746T variante alélica fueron significativamente inferiores en los pacientes de SPI que en controles, aunque los otros 3 SNPs estudiados RLS no difirió entre pacientes y controles. Ninguno de los polimorfismos estudiados influyeron en el inicio de la enfermedad, la gravedad de la RLS, historia familiar de SPI, la ferritina sérica, o respuesta a agonistas dopaminérgicos, clonazepam o GABAergic drogas. El presente estudio sugiere una débil asociación entre el polimorfismo rs2071746 HMOX1 y el riesgo de desarrollar el SPI en la población española.Several neurochemical, neuropathological, neuroimaging, and experimental data, suggest that iron deficiency plays an important role in the pathophysiology of restless legs syndrome (RLS). Hemeoxygenases (HMOX) are an important defensive mechanism against oxidative stress, mainly through the degradation of heme to biliverdin, free iron, and carbon monoxide. We analyzed whether HMOX1 and HMOX2 genes are related with the risk to develop RLS. We analyzed the distribution of genotypes and allelic frequencies of the HMOX1 rs2071746, HMOX1 rs2071747, HMOX2 rs2270363, and HMOX2 rs1051308 SNPs, as well as the presence of Copy number variations (CNVs) of these genes in 205 subjects RLS and 445 healthy controls. The frequencies of rs2071746TT genotype and rs2071746T allelic variant were significantly lower in RLS patients than that in controls, although the other 3 studied SNPs did not differ between RLS patients and controls. None of the studied polymorphisms influenced the disease onset, severity of RLS, family history of RLS, serum ferritin levels, or response to dopaminergic agonist, clonazepam or GABAergic drugs. The present study suggests a weak association between HMOX1 rs2071746 polymorphism and the risk to develop RLS in the Spanish population.• Instituto de Salud Carlos III, Fondo de Investigación Sanitaria: Ayudas PI12/00241, PI12/00324 y RETICS RD12/0013/0002 • Junta de Extremadura: Ayuda GR10068 GR10068 y PRIS10016 (Fundesalud,Mérida, Spain) • Ministerio de Ciencia e Innovación: Ayudas SAF2006-10126 (2006–2009) y SAF2010-22329-C02-01 (2011–2013) • Parcialmente financiado Fondos FEDER – Fondo Europeo de Desarrollo RegionalpeerReviewe

Association between vitamin D receptor rs731236 (Taq1) polymorphism and risk for restless legs syndrome in the Spanish caucasian population

Varios trabajos recientes sugieren un posible papel de la deficiencia de vitamina D en la etiología o el síndrome de las piernas inquietas (RLS). Hemos analizado la posible relación de 2 polimorfismos de un solo nucleótido (SNP) en el receptor de la vitamina D3 (GEN VDR) con el riesgo de SPI. Hemos estudiado la variante alélica genotipo y frecuencias de VDR rs2228570 y rs731236 VDR SNPs en 205 RLS pacientes y 445 controles sanos mediante un ensayo TaqMan. Las frecuencias de los rs731236AAgenotype y la variante alélica rs731236un SPI fue significativamente inferior en los pacientes que en los controles (P<0,005 y 0,01, respectivamente). El síndrome de las piernas inquietas pacientes portadoras de la variante alélica rs731236G había una edad temprana en el inicio, y los portadores del genotipo GG731236rs tuvieron mayor severidad de RLS, aunque estos datos desaparecieron después de los análisis multivariados. Ninguno de los SNPs estudiados estaba relacionada con la positividad de la historia familiar de SPI. Estos resultados sugieren una modesta, pero significativa asociación entre rs731236 VDR SNP y el riesgo de síndrome de piernas inquietas.Several recent works suggest a possible role of vitamin D deficiency in the etiology or restless legs syndrome (RLS). We analyzed the possible relationship of 2 common single nucleotide polymorphisms (SNPs) in the vitamin D3 receptor (VDR) gene with the risk for RLS. We studied the genotype and allelic variant frequencies of VDR rs2228570 and VDR rs731236 SNPs in 205 RLS patients and 445 healthy controls using a TaqMan essay. The frequencies of the rs731236AAgenotype and the allelic variant rs731236A were significantly lower in RLS patients than in controls (P<0.005 and<0.01, respectively). Restless legs syndrome patients carrying the allelic variant rs731236G had an earlier age at onset, and those carrying the rs731236GG genotype had higher severity of RLS, although these data disappeared after multivariate analyses. None of the SNPs studied was related with the positivity of family history of RLS. These results suggest a modest, but significant association between VDR rs731236 SNP and the risk for RLS.• Instituto de Salud Carlos III, Madrid, Fondo de Investigación Sanitaria: Ayudas PI12/00241, PI12/00324, y RETICS RD12/0013/0002 • Junta de Extremadura: GR15026 y PRIS10016 • Ministerio de Ciencia e Innovación: Ayudas SAF2006-10126 (2006–2009) y SAF2010-22329-C02-01 (2011-2013) • Parciamente financiado con Fondos FEDERpeerReviewe

Association between vitamin D receptor rs731236 (Taq1) polymorphism and risk for restless legs syndrome in the Spanish caucasian population

Varios trabajos recientes sugieren un posible papel de la deficiencia de vitamina D en la etiología o el síndrome de las piernas inquietas (RLS). Hemos analizado la posible relación de 2 polimorfismos de un solo nucleótido (SNP) en el receptor de la vitamina D3 (GEN VDR) con el riesgo de SPI. Hemos estudiado la variante alélica genotipo y frecuencias de VDR rs2228570 y rs731236 VDR SNPs en 205 RLS pacientes y 445 controles sanos mediante un ensayo TaqMan. Las frecuencias de los rs731236AAgenotype y la variante alélica rs731236un SPI fue significativamente inferior en los pacientes que en los controles (P<0,005 y 0,01, respectivamente). El síndrome de las piernas inquietas pacientes portadoras de la variante alélica rs731236G había una edad temprana en el inicio, y los portadores del genotipo GG731236rs tuvieron mayor severidad de RLS, aunque estos datos desaparecieron después de los análisis multivariados. Ninguno de los SNPs estudiados estaba relacionada con la positividad de la historia familiar de SPI. Estos resultados sugieren una modesta, pero significativa asociación entre rs731236 VDR SNP y el riesgo de síndrome de piernas inquietas.Several recent works suggest a possible role of vitamin D deficiency in the etiology or restless legs syndrome (RLS). We analyzed the possible relationship of 2 common single nucleotide polymorphisms (SNPs) in the vitamin D3 receptor (VDR) gene with the risk for RLS. We studied the genotype and allelic variant frequencies of VDR rs2228570 and VDR rs731236 SNPs in 205 RLS patients and 445 healthy controls using a TaqMan essay. The frequencies of the rs731236AAgenotype and the allelic variant rs731236A were significantly lower in RLS patients than in controls (P<0.005 and<0.01, respectively). Restless legs syndrome patients carrying the allelic variant rs731236G had an earlier age at onset, and those carrying the rs731236GG genotype had higher severity of RLS, although these data disappeared after multivariate analyses. None of the SNPs studied was related with the positivity of family history of RLS. These results suggest a modest, but significant association between VDR rs731236 SNP and the risk for RLS.• Instituto de Salud Carlos III, Madrid, Fondo de Investigación Sanitaria: Ayudas PI12/00241, PI12/00324, y RETICS RD12/0013/0002 • Junta de Extremadura: GR15026 y PRIS10016 • Ministerio de Ciencia e Innovación: Ayudas SAF2006-10126 (2006–2009) y SAF2010-22329-C02-01 (2011-2013) • Parciamente financiado con Fondos FEDERpeerReviewe

Biologic Therapy in Refractory Non-Multiple Sclerosis Optic Neuritis Isolated or Associated to Immune Mediated Inflammatory Diseases. A Multicenter Study

We aimed to assess the e cacy of biologic therapy in refractory non-Multiple Sclerosis (MS) Optic Neuritis (ON), a condition more infrequent, chronic and severe than MS ON. This was an open-label multicenter study of patients with non-MS ON refractory to systemic corticosteroids and at least one conventional immunosuppressive drug. The main outcomes were Best Corrected Visual Acuity (BCVA) and both Macular Thickness (MT) and Retinal Nerve Fiber Layer (RNFL) using Optical Coherence Tomography (OCT). These outcome variables were assessed at baseline, 1 week, and 1, 3, 6 and 12 months after biologic therapy initiation. Remission was defined as the absence of ON symptoms and signs that lasted longer than 24 h, with or without an associated new lesion on magnetic resonance imaging with gadolinium contrast agents for at least 3 months. We studied 19 patients (11 women/8 men; mean age, 34.8 13.9 years). The underlying diseases were Bechet?s disease (n = 5), neuromyelitis optica (n = 3), systemic lupus erythematosus (n = 2), sarcoidosis (n = 1), relapsing polychondritis (n = 1) and anti-neutrophil cytoplasmic antibody -associated vasculitis (n = 1). It was idiopathic in 6 patients. The first biologic agent used in each patient was: adalimumab (n = 6), rituximab (n = 6), infliximab (n = 5) and tocilizumab (n = 2). A second immunosuppressive drug was simultaneously used in 11 patients: methotrexate (n = 11), azathioprine (n = 2), mycophenolate mofetil (n = 1) and hydroxychloroquine (n = 1). Improvement of the main outcomes was observed after 1 year of therapy when compared with baseline data: mean SD BCVA (0.8 0.3 LogMAR vs. 0.6 0.3 LogMAR; p = 0.03), mean SD RNFL (190.5 175.4 m vs. 183.4 139.5 m; p = 0.02), mean SD MT (270.7 23.2 m vs. 369.6 137.4 m; p = 0.03). Besides, the median (IQR) prednisone-dose was also reduced from 40 (10?61.5) mg/day at baseline to. 2.5 (0?5) mg/day after one year of follow-up; p = 0.001. After a mean SD follow-up of 35 months, 15 patients (78.9%) achieved ocular remission, and 2 (10.5%) experienced severe adverse events. Biologic therapy is e ective in patients with refractory non-MS ON

Tumor targeted 4-1BB agonist antibody-albumin fusions with high affinity to FcRn induce anti-tumor immunity without toxicity

17 p.-4 fig.-1 tab.-1 grph. abst.Costimulation of tumor-infiltrating T lymphocytes by anti-4-1BB monoclonal antibodies (mAbs) has shown anti-tumor activity in human trials, but can be associated with significant off-tumor toxicities involving FcγR interactions. Here, we introduce albumin-fused mouse and human bispecific antibodies with clinically favorable pharmacokinetics designed to confine 4-1BB costimulation to the tumor microenvironment. These Fc-free 4-1BB agonists consist of an EGFR-specific VHH antibody, a 4-1BB-specific scFv, and a human albumin sequence engineered for high FcRn binding connected in tandem (LiTCo-Albu). We demonstrate in vitro cognate target engagement, EGFR-specific costimulatory activity, and FcRn-driven cellular recycling similar to non-fused FcRn high-binding albumin. The mouse LiTCo-Albu exhibited a prolonged circulatory half-life and in vivo tumor inhibition, with no indication of 4-1BB mAb-associated toxicity. Furthermore, we show a greater therapeutic effect when used in combination with PD-1-blocking mAbs. These findings demonstrate the feasibility of tumor-specific LiTCo-Albu antibodies for safe and effective costimulatory strategies in cancer immunotherapy.Financial support for this work was obtained from the MCIN/AEI/10.13039/501100011033 (SAF2017-89437-P and PDC2021-121711-100 to LA-V, PID2019-104544GB-I00 to CA, and PID2020-113225GB-I00 to FJB), partially supported by the European Regional Development Fund (ERDF); the Carlos III Health Institute (ISCIII) (PI19/00132 to LS; PI20/01030 to BB), partially supported by the ERDF; the ISCIII-RICORS within the Next Generation EU program (plan de Recuperación, Transformación y Resilencia); the Spanish Association Against Cancer (AECC 19084 to LA-V); the CRIS Cancer Foundation (FCRIS-2018-0042 and FCRIS-2021-0090 to LA-V), the BBVA Foundation (Ayudas Fundación BBVA a Equipos de Investigación Científica SARS-CoV-2 years COVID-19 to LA-V); and the Fundació “La Caixa” (HR21-00761 project IL7R_LungCan to LA-V). AD, OAM, and KAH were funded by the Novo Nordisk Foundation, Grant; CEMBID (Center for Multifunctional Biomolecular Drug Design, Grant Number: NNF17OC0028070). OH was supported by an industrial PhD fellowship from the Comunidad de Madrid (IND2020/BMD-17668). AE-L was supported industrial PhD fellowship from the Carlos III Health Institute (IFI18/00045). CD-A was supported by a predoctoral fellowship from the Spanish Ministry of Science Innovation and Universities (PRE2018-083445). LR-P was supported by a predoctoral fellowship from the Immunology Chair, Universidad Francisco de Vitoria/Merck. LD-A was supported by a Rio Hortega fellowship from the Carlos III Health Institute (CM20/00004).Peer reviewe

Post-mortem findings in Spanish patients with COVID-19; a special focus on superinfections

IntroductionWhole-body autopsies may be crucial to understand coronavirus disease 2019 (COVID-19) pathophysiology. We aimed to analyze pathological findings in a large series of full-body autopsies, with a special focus on superinfections. MethodsThis was a prospective multicenter study that included 70 COVID-19 autopsies performed between April 2020 and February 2021. Epidemiological, clinical and pathological information was collected using a standardized case report form. ResultsMedian (IQR) age was 70 (range 63.75-74.25) years and 76% of cases were males. Most patients (90%,) had at least one comorbidity prior to COVID-19 diagnosis, with vascular risk factors being the most frequent. Infectious complications were developed by 65.71% of the patients during their follow-up. Mechanical ventilation was required in most patients (75.71%) and was mainly invasive. In multivariate analyses, length of hospital stay and invasive mechanical ventilation were significantly associated with infections (p = 0.036 and p = 0.013, respectively). Necropsy findings revealed diffuse alveolar damage in the lungs, left ventricular hypertrophy in the heart, liver steatosis and pre-infection arteriosclerosis in the heart and kidneys. ConclusionOur study confirms the main necropsy histopathological findings attributed to COVID-19 in a large patient series, while underlining the importance of both comorbid conditions and superinfections in the pathology

A PD-L1/EGFR bispecific antibody combines immune checkpoint blockade and direct anti-cancer action for an enhanced anti-tumor response

Immune checkpoint blockade (ICB) with antibodies has shown durable clinical responses in a wide range of cancer types, but the overall response rate is still limited. Other effective therapeutic modalities to increase the ICB response rates are urgently needed. New bispecific antibody (bsAb) formats combining the ICB effect and a direct action on cancer cells could improve the efficacy of current immunotherapies. Here, we report the development of a PD-L1/EGFR symmetric bsAb by fusing a dual-targeting tandem trimmer body with the human IgG1 hinge and Fc regions. The bsAb was characterized in vitro and the antitumor efficacy was evaluated in humanized mice bearing xenografts of aggressive triple-negative breast cancer and lung cancer. The IgG-like hexavalent bsAb, designated IgTT-1E, was able to simultaneously bind both EGFR and PD-L1 antigens, inhibit EGF-mediated proliferation, effectively block PD-1/PD-L1 interaction, and induce strong antigen-specific antibody-dependent cellular cytotoxicity activity in vitro. Potent therapeutic efficacies of IgTT-1E in two different humanized mouse models were observed, where tumor growth control was associated with a significantly increased proportion of CD8+ T cells. These results support the development of IgTT-1E for the treatment of EGFR+ cancers.L.A-V. was supported by grants from the MCIN/AEI/10.13039/ 501100011033 (PID2020-117323RB-100 and PDC2021-121711-100), the Instituto de Salud Carlos III (DTS20/00089), the CRIS Cancer Foundation (FCRIS-2021-0090), the Spanish Association Against Cancer (PROYE19084ALVA), the Fundación ‘‘La Caixa’’ (HR21-00761 project IL7R_LungCan) and the Fundación de Investigación Biomédica 12 de Octubre Programa Investiga (2022-0082). B.B and L.S. were supported by grants PI20/01030 and PI19/00132 from the Instituto de Salud Carlos III (PI20/01030). FJB and MF-G were supported by grants PID2020- 113225GB-I00 and PRE2018-085788 funded by MCIN/AEI/10.13039/ 501100011033. L.R-P. was supported by a predoctoral fellowship from the Immunology Chair, Universidad Francisco de Vitoria/Merck. C. D-A. was supported by a predoctoral fellowship from the MCIN/AEI/ 10.13039/501100011033 (PRE2018-083445). L.D-A. was supported by a Rio Hortega fellowship from the Instituto de Salud Carlos III (CM20/ 00004). O.H. was supported by an industrial PhD fellowship from the Comunidad de Madrid (IND2020/BMD-17668). AE-L was supported industrial PhD fellowship from the Instituto de Salud Carlos III (IFI18/ 00045)Peer reviewe

Tocilizumab in refractory Caucasian Takayasu's arteritis: a multicenter study of 54 patients and literature review

Objective: To assess the efficacy and safety of tocilizumab (TCZ) in Caucasian patients with refractory Takayasu's arteritis (TAK) in clinical practice. Methods: A multicenter study of Caucasian patients with refractory TAK who received TCZ. The outcome variables were remission, glucocorticoid-sparing effect, improvement in imaging techniques, and adverse events. A comparative study between patients who received TCZ as monotherapy (TCZMONO) and combined with conventional disease modifying anti-rheumatic drugs (cDMARDs) (TCZCOMBO) was performed. Results: The study comprised 54 patients (46 women/8 men) with a median [interquartile range (IQR)] age of 42.0 (32.5-50.5) years. TCZ was started after a median (IQR) of 12.0 (3.0-31.5) months since TAK diagnosis. Remission was achieved in 12/54 (22.2%), 19/49 (38.8%), 23/44 (52.3%), and 27/36 (75%) patients at 1, 3, 6, and 12 months, respectively. The prednisone dose was reduced from 30.0 mg/day (12.5-50.0) to 5.0 (0.0-5.6) mg/day at 12 months. An improvement in imaging findings was reported in 28 (73.7%) patients after a median (IQR) of 9.0 (6.0-14.0) months. Twenty-three (42.6%) patients were on TCZMONO and 31 (57.4%) on TCZCOMBO: MTX (n = 28), cyclosporine A (n = 2), azathioprine (n = 1). Patients on TCZCOMBO were younger [38.0 (27.0-46.0) versus 45.0 (38.0-57.0)] years; difference (diff) [95% confidence interval (CI) = -7.0 (-17.9, -0.56] with a trend to longer TAK duration [21.0 (6.0-38.0) versus 6.0 (1.0-23.0)] months; diff 95% CI = 15 (-8.9, 35.5), and higher c-reactive protein [2.4 (0.7-5.6) versus 1.3 (0.3-3.3)] mg/dl; diff 95% CI = 1.1 (-0.26, 2.99). Despite these differences, similar outcomes were observed in both groups (log rank p = 0.862). Relevant adverse events were reported in six (11.1%) patients, but only three developed severe events that required TCZ withdrawal. Conclusion: TCZ in monotherapy, or combined with cDMARDs, is effective and safe in patients with refractory TAK of Caucasian origin.Funding: This work was partially supported by RETICS Programs, RD08/0075 (RIER), RD12/0009/0013 and RD16/0012 from “Instituto de Salud Carlos III” (ISCIII) (Spain)