A novel isolation method for spontaneously released extracellular vesicles from brain tissue and its implications for stress-driven brain pathology.

BACKGROUND: Extracellular vesicles (EVs), including small EVs (sEVs) such as exosomes, exhibit great potential for the diagnosis and treatment of brain disorders, representing a valuable tool for precision medicine. The latter demands high-quality human biospecimens, especially in complex disorders in which pathological and specimen heterogeneity, as well as diverse individual clinical profile, often complicate the development of precision therapeutic schemes and patient-tailored treatments. Thus, the collection and characterization of physiologically relevant sEVs are of the utmost importance. However, standard brain EV isolation approaches rely on tissue dissociation, which can contaminate EV fractions with intracellular vesicles. METHODS: Based on multiscale analytical platforms such as cryo-EM, label-free proteomics, advanced flow cytometry, and ExoView analyses, we compared and characterized the EV fraction isolated with this novel method with a classical digestion-based EV isolation procedure. Moreover, EV biogenesis was pharmacologically manipulated with either GW4869 or picrotoxin to assess the validity of the spontaneous-release method, while the injection of labelled-EVs into the mouse brain further supported the integrity of the isolated vesicles. RESULTS: We hereby present an efficient purification method that captures a sEV-enriched population spontaneously released by mouse and human brain tissue. In addition, we tested the significance of the release method under conditions where biogenesis/secretion of sEVs was pharmacologically manipulated, as well as under animals' exposure to chronic stress, a clinically relevant precipitant of brain pathologies, such as depression and Alzheimer's disease. Our findings show that the released method monitors the drug-evoked inhibition or enhancement of sEVs secretion while chronic stress induces the secretion of brain exosomes accompanied by memory loss and mood deficits suggesting a potential role of sEVs in the brain response to stress and related stress-driven brain pathology. CONCLUSIONS: Overall, the spontaneous release method of sEV yield may contribute to the characterization and biomarker profile of physiologically relevant brain-derived sEVs in brain function and pathology. Video Abstract.

Use of homeopathic products in pigs feed improves the percentage, quality and marbling of meat.

Studies and innovations on alternative feed additives, especially on homeopathic remedies have been highlighted in order to replace or reduce the use of antibiotics in pig production. This paper aimed to assess the addition of homeopathic products in pig diet and their effects on the growth performance, serum metabolites, nutrient and energy digestibility, carcass traits and meat quality. A total of 60 immunocastrated male pigs, weighing on average 30.91 ± 0.95 kg, were distributed in two treatments, 10 replicates and three animals/experimental unit. There was no effect (P≥0.05) of treatment on the growth performance and serum metabolites. The percentage of acid-insoluble ash recovered in the diet was greater (P≤0.01) in diets containing homeopathic products. The apparent digestible energy of diets containing homeopathic products was reduced (P≤0.01) in the growing phase and reduced (P≤0.01) the apparent digestibility coefficients of dry matter, crude protein, soluble neutral and acid detergent fibers, and gross energy in the growing and finishing phases. Pig that received diets with homeopathic products had higher (P≤0.05) amount of meat, percentage of meat and marbling. The use of homeopathic products in diets improves the percentage and quality of meat, as well as the marbling of the pig carcass, maintaining the performance.

Effectiveness of Belimumab After Rituximab in Systemic Lupus Erythematosus : A Randomized Controlled Trial.

BACKGROUND: B-cell depletion with rituximab is commonly used for patients with systemic lupus erythematosus (SLE) that is refractory to conventional therapy, but it yields variable responses. We hypothesized that high B-cell activating factor (BAFF) levels after rituximab can cause disease flares, thereby limiting its effectiveness. OBJECTIVE: To obtain preliminary evidence for efficacy of the anti-BAFF therapeutic belimumab after rituximab in SLE. DESIGN: Phase 2, randomized, double-blind (patients, assessors, researchers, care providers), placebo-controlled, parallel-group, superiority trial. (ISRCTN: 47873003). SETTING: England. PARTICIPANTS: Fifty-two patients who had SLE that was refractory to conventional treatment and whose physicians had recommended rituximab therapy were recruited between 2 February 2017 and 28 March 2019. INTERVENTION: Participants were treated with rituximab and 4 to 8 weeks later were randomly assigned (1:1) to receive intravenous belimumab or placebo for 52 weeks. MEASUREMENTS: The prespecified primary end point was serum IgG anti-double-stranded DNA (anti-dsDNA) antibody levels at 52 weeks. Secondary outcomes included incidence of disease flares and adverse events. RESULTS: At 52 weeks, IgG anti-dsDNA antibody levels were lower in patients treated with belimumab compared with placebo (geometric mean, 47 [95% CI, 25 to 88] vs. 103 [CI, 49 to 213] IU/mL; 70% greater reduction from baseline [CI, 46% to 84%]; P < 0.001). Belimumab reduced risk for severe flare (BILAG-2004 grade A) compared with placebo (hazard ratio, 0.27 [CI, 0.07 to 0.98]; log-rank P = 0.033), with 10 severe flares in the placebo group and 3 in the belimumab group. Belimumab did not increase incidence of serious adverse events. Belimumab significantly suppressed B-cell repopulation compared with placebo (geometric mean, 0.012 [CI, 0.006 to 0.014] vs. 0.037 [CI, 0.021 to 0.081] × 109/L) at 52 weeks in a subset of patients (n = 25) with available data. LIMITATIONS: Small sample size; biomarker primary end point. CONCLUSION: Belimumab after rituximab significantly reduced serum IgG anti-dsDNA antibody levels and reduced risk for severe flare in patients with SLE that was refractory to conventional therapy. The results suggest that this combination could be developed as a therapeutic strategy. PRIMARY FUNDING SOURCE: Versus Arthritis.

Nanobody-Based Theranostic Agents for HER2-Positive Breast Cancer: Radiolabeling Strategies.

The overexpression of human epidermal growth factor 2 (HER2) in breast cancer (BC) has been associated with a more aggressive tumor subtype, poorer prognosis and shorter overall survival. In this context, the development of HER2-targeted radiotracers is crucial to provide a non-invasive assessment of HER2 expression to select patients for HER2-targeted therapies, monitor response and identify those who become resistant. Antibodies represent ideal candidates for this purpose, as they provide high contrast images for diagnosis and low toxicity in the therapeutic setting. Of those, nanobodies (Nb) are of particular interest considering their favorable kinetics, crossing of relevant biological membranes and intratumoral distribution. The purpose of this review is to highlight the unique characteristics and advantages of Nb-based radiotracers in BC imaging and therapy. Additionally, radiolabeling methods for Nb including direct labeling, indirect labeling via prosthetic group and indirect labeling via complexation will be discussed, reporting advantages and drawbacks. Furthermore, the preclinical to clinical translation of radiolabeled Nbs as promising theranostic agents will be reported.

In Vivo Tracking of Extracellular Vesicles by Nuclear Imaging: Advances in Radiolabeling Strategies.

Extracellular vesicles (EVs) are naturally secreted vesicles that have attracted a large amount of interest in nanomedicine in recent years due to their innate biocompatibility, high stability, low immunogenicity, and important role in cell-to-cell communication during pathological processes. Their versatile nature holds great potential to improve the treatment of several diseases through their use as imaging biomarkers, therapeutic agents, and drug-delivery vehicles. However, the clinical translation of EV-based approaches requires a better understanding of their in vivo behavior. Several imaging technologies have been used for the non-invasive in vivo tracking of EVs, with a particular emphasis on nuclear imaging due to its high sensitivity, unlimited penetration depth and accurate quantification. In this article, we will review the biological function and inherent characteristics of EVs and provide an overview of molecular imaging modalities used for their in vivo monitoring, with a special focus on nuclear imaging. The advantages of radionuclide-based imaging modalities make them a promising tool to validate the use of EVs in the clinical setting, as they have the potential to characterize in vivo the pharmacokinetics and biological behavior of the vesicles. Furthermore, we will discuss the current methods available for radiolabeling EVs, such as covalent binding, encapsulation or intraluminal labeling and membrane radiolabeling, reporting the advantages and drawbacks of each radiolabeling approach.

The interplay between glioblastoma and microglia cells leads to endothelial cell monolayer dysfunction via the interleukin-6-induced JAK2/STAT3 pathway.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor, with an average life expectancy of 12-15 months. GBM is highly infiltrated by microglial cells (MG) promoting tumor growth and invasiveness. Moreover, microglia activation and subsequent neuroinflammation seem to be involved in blood-brain barrier (BBB) dysfunction commonly observed in several central nervous system diseases, including brain tumors. Nevertheless, how the crosstalk between microglia and tumor cells interferes with BBB function is far from being clarified. Herein, we evaluated the effects of reciprocal interactions between MG and GBM cells in the barrier properties of brain endothelial cells (ECs), using an in vitro approach. The exposure of ECs to the inflammatory microenvironment mediated by MG-GBM crosstalk induced a decrease in the transendothelial electric resistance and an increase in permeability across the ECs (macromolecular flux of 4 kDa-fluorescein isothiocyanate and 70 kDa-Rhodamine B isothiocyanate-Dextran). These effects were accompanied by a downregulation of the intercellular junction proteins, ß-catenin and zonula occludens. Moreover, the dynamic interaction between microglia and tumor cells triggered the release of interleukin-6 (IL-6) by microglia and subsequent activation of the downstream Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway. Interestingly, the depletion of IL-6 or the blockade of the JAK/STAT3 signaling with AG490 were able to prevent the EC hyperpermeability. Overall, we demonstrated that IL-6 released during MG-GBM crosstalk leads to barrier dysfunction through the activation of the JAK/STAT3 pathway in ECs and downregulation of intercellular junction proteins. These results provide new insights into the mechanisms underlying the disruption of BBB permeability in GBM.

Biodiversity of Penicillium species from marine environments in Portugal and description of Penicillium lusitanum sp. nov., a novel species isolated from sea water.

During an extensive survey of marine fungi in coastal marine environments from Portugal, a collection of Penicillium isolates were obtained from sea water, macroalgae and driftwood. Sixteen distinct Penicillium species were identified with Penicillium terrigenum and Penicillium brevicompactum being the most frequent. A Penicillium species isolated from sea water could not be affiliated to any known species. Phylogenetic analyses based on the ITS region of the rDNA and the beta-tubulin (benA) gene placed it into Penicillium section Ramosa, distinct from all currently known species and with Penicillium tunisiense as its closest relative. Although having similar morphological characteristics, these species differ in micromorphological and molecular characters. Thus, Penicillium lusitanum sp. nov. is proposed as a novel species.

Partial replacement of soybean meal with different protein sources in piglet feed during the nursery phase.

OBJECTIVE: Evaluate the partial replacement of soybean meal with different protein sources in piglet feed during the nursery phase in terms of digestibility of feed, nitrogen balance, growth performance and blood parameters. METHODS: Experiment I involved 24 crossbred entire male pigs with an initial body weight of 18.28 ± 0.7 kg, and used a randomized complete block design consisting of three treatments (Fish Meal - FM, Soybean Protein Concentrate - SPC, and Soybean Meal - SM) and eight replicates, with one pig per experimental unit. Experiment II involved 1843 crossbred male and female pigs with an initial body weight of 6.79 ± 0.90 kg, and was based on a completely randomized design with a 2 × 3 factorial arrangement (two sexes and three protein sources) and 13 replicates. RESULTS: The results of Experiment I indicate a significant effect (p &lt;0.05) of the treatment on digestible protein (FM: 17.84%; SPC: 16.72% and SM: 18.13%) and on total nitrogen excretion (TNE, g/kg BW0.75/day) in which pigs fed with SM-based feed had TNE values that were 5.36% and 3.72% higher than SPC and FM, respectively. In the Experiment II, there was difference (p &lt;0.01) between sexes in the starter phase and total period in daily feed intake (DFI) values, which were higher in females, and between the protein sources in DFI, final weight and daily weight gain, which were higher in piglets fed with SPC. For urea in both phases and glucose in the pre-starter II phase, there was a difference (p &lt;0.05) between protein sources and between sexes, in starter phase in urea levels (females: 57.11 mg/dL and males: 50.60 mg/dL). CONCLUSION: The use of feed only at basis of SM influences larger TNE (g/kg BW0.75/day), promotes a reduction in the growth performance of piglets and increases plasma urea levels in pre-starter II.

Taxonomic Repositioning of Xanthomonas campestris pv. viticola (Nayudu 1972) Dye 1978 as Xanthomonas citri pv. viticola (Nayudu 1972) Dye 1978 comb. nov. and Emendation of the Description of Xanthomonas citri pv. anacardii to Include Pigmented Isolates Pathogenic to Cashew Plant.

Grapevine bacterial canker, which is caused by Xanthomonas campestris pv. viticola, is one of the most important grapevine diseases in the northeastern region of Brazil. This disease causes severe damage and represents a high potential risk to the development of Brazilian viticulture. In turn, pigmented isolates pathogenic to cashew plant, making cashew fruit unfit for sale, also have been detected in Northeastern Brazil. Given that the taxonomic position of these bacteria is unclear, the multilocus sequence analysis (MLSA) technique, average nucleotide identity (ANI) values and tetranucleotide frequency correlation coefficients (TETRA) were used to analyze their phylogenetic relationship in relation to other Xanthomonas species. X. campestris pv. viticola was closely related to X. citri pv. mangiferaeindicae (repetitive-polymerase chain reaction [rep-PCR], MLSA, and ANI) and X. citri subsp. citri (MLSA and ANI). Pigmented isolates pathogenic to cashew plant were closely related to X. citri pv. anacardii (rep-PCR, MLSA, ANI, and TETRA). The results obtained in this study support the emendation of the description of X. citri pv. anacardii to include pigmented isolates of Xanthomonas pathogenic to cashew plant. In addition, the reclassification of X. campestris pv. viticola as X. citri pv. viticola comb. nov. is suggested.

Congenital Shunts of the Portal Venous System: Case-series of Uncommon Shunts.

BACKGROUND AND AIMS: Congenital shunts of the portal venous system are rare entities that can present in children with clinical heterogeneity. To evaluate the clinical course of children with uncommon shunts presenting to our institution and examine the available literature on this topic. Medical records of children with rare forms of congenital shunts were retrospectively reviewed for demographics, symptoms, management, and outcome between 2003 and 2016. RESULTS: Three female patients with congenital shunts, including a congenital mesenterico-portal Rex shunt (n = 1) and congenital portosystemic shunts (CPSS) (n = 2), were referred for surgical evaluation between ages 4 and 9. Median follow-up was 8 years (range, 6-13 years). One asymptomatic patient did not require treatment and remained disease-free during long-term follow-up. The other 2 patients with CPSS and unusual symptoms, including liver focal nodular hyperplasia (FNH) in infancy (n = 1) and bleeding from esophageal varices (n = 1), showed subsequent progression to liver nodules that were managed by endovascular shunt occlusion. One patient showed symptom resolution and the other showed stable lesions at last follow-up. Literature yielded descriptions of two cases of congenital mesenterico-portal Rex shunt, one case of coincident CPSS and FNH in infancy, but zero reports of bleeding from esophageal varices. CONCLUSIONS: This case series examines each distinct patient's presentation, discusses the diagnosis, management and outcome and compares findings while discussing literature on this topic. A high index of suspicion and familiarity with unusual forms and treatment options is required to allow timely diagnosis and appropriate treatment.

Serotonergic signalling suppresses ataxin 3 aggregation and neurotoxicity in animal models of Machado-Joseph disease.

Polyglutamine diseases are a class of dominantly inherited neurodegenerative disorders for which there is no effective treatment. Here we provide evidence that activation of serotonergic signalling is beneficial in animal models of Machado-Joseph disease. We identified citalopram, a selective serotonin reuptake inhibitor, in a small molecule screen of FDA-approved drugs that rescued neuronal dysfunction and reduced aggregation using a Caenorhabditis elegans model of mutant ataxin 3-induced neurotoxicity. MOD-5, the C. elegans orthologue of the serotonin transporter and cellular target of citalopram, and the serotonin receptors SER-1 and SER-4 were strong genetic modifiers of ataxin 3 neurotoxicity and necessary for therapeutic efficacy. Moreover, chronic treatment of CMVMJD135 mice with citalopram significantly reduced ataxin 3 neuronal inclusions and astrogliosis, rescued diminished body weight and strikingly ameliorated motor symptoms. These results suggest that small molecule modulation of serotonergic signalling represents a promising therapeutic target for Machado-Joseph disease.

Acute and chronic sensitivity, avoidance behavior and sensitive life stages of bullfrog tadpoles exposed to the biopesticide abamectin.

As compared to other aquatic organism groups, relatively few studies have been conducted so far evaluating the toxicity of pesticides to amphibians. This may at least partly be due to the fact that regulations for registering pesticides usually do not require testing amphibians. The sensitivity of amphibians is generally considered to be covered by that based on toxicity tests with other aquatic organisms (e.g. fish) although the impact of a pesticide on amphibians may be very different. In the present study, acute and chronic laboratory tests were conducted to evaluate the acute and chronic toxicity of abamectin (as Vertimec(®) 18EC) to bullfrog (Lithobates catesbeianus) tadpoles. Acute tests were conducted at two tadpole stages (Gosner stage 21G and 25G) and avoidance tests were also conducted with stage Gosner stage 21G tadpoles. Calculated acute toxicity values were greater than those reported for standard fish test species, hence supporting the use of fish toxicity data as surrogates for amphibians in acute risk assessments. Given the limited number and extent of available amphibian toxicity studies, however, research needs to increase our understanding of pesticide toxicity to amphibians are discussed.

Study of 16 Portuguese activated sludge systems based on filamentous bacteria populations and their relationships with environmental parameters.

A survey in 16 activated sludge wastewater treatment plants (WWTP) was conducted to contribute to the knowledge of the environmental parameters that determine the composition of the filamentous community. A total of 128 samples of mixed liquor from municipal WWTP were collected during 2 years, and 22 filamentous morphotypes were identified. The most frequent and abundant filamentous bacteria were, in both cases and by this order, type 0041/0675, type 0092, Microthrix parvicella and 1851, nocardioforms and Haliscomenobacter hydrossis. Concerning dominance, type 1851 was the most frequently dominant morphotype, followed by M. parvicella and types 0092 and 0041/0675. These were also, and by this order, the dominant morphotypes during bulking occurrences. Significant correlations were obtained between the abundance of filamentous bacteria and environmental parameters, but multivariate statistical analysis only confirmed the correlation between type 0092 and Sludge Volume Index (SVI), emphasizing the association of this filament with bulking. The discussion of the results in light of published works was complicated by the random use of terms such as frequency, abundance, and dominance with different and often unclear meanings. This reinforces the need of clarifying these terms when discussing the causes of filamentous overgrowth in WWTP.

Moko Disease-Causing Strains of Ralstonia solanacearum from Brazil Extend Known Diversity in Paraphyletic Phylotype II.

The epidemic situation of Moko disease-causing strains in Latin America and Brazil is unclear. Thirty-seven Ralstonia solanacearum strains from Brazil that cause the Moko disease on banana and heliconia plants were sampled and phylogenetically typed using the endoglucanase (egl) and DNA repair (mutS) genes according to the phylotype and sequevar classification. All of the strains belonged to phylotype II and a portion of the strains was typed as the Moko disease-related sequevars IIA-6 and IIA-24. Nevertheless, two unsuspected sequevars also harbored the Moko disease-causing strains IIA-41 and IIB-25, and a new sequevar was described and named IIA-53. All of the strains were pathogenic to banana and some of the strains of sequevars IIA-6, IIA-24, and IIA-41 were also pathogenic to tomato. The Moko disease-causing strains from sequevar IIB-25 were pathogenic to potato but not to tomato. These results highlight the high diversity of strains of Moko in Brazil, reinforce the efficiency of the egl gene to reveal relationships among these strains, and contribute to a better understanding of the diversity of paraphyletic Moko disease-causing strains of the R. solanacearum species complex, where the following seven distinct genetic clusters have been described: IIA-6, IIA-24, IIA-41, IIA-53, IIB-3, IIB-4, and IIB-25.

Brain metastasis: An insight into novel molecular targets for theranostic approaches.

Brain metastases (BrM) are common malignant lesions in the central nervous system, and pose a significant threat in advanced-stage malignancies due to delayed diagnosis and limited therapeutic options. Their distinct genomic profiles underscore the need for molecular profiling to tailor effective treatments. Recent advances in cancer biology have uncovered molecular drivers underlying tumor initiation, progression, and metastasis. This, coupled with the advances in molecular imaging technology and radiotracer synthesis, has paved the way for the development of innovative radiopharmaceuticals with enhanced specificity and affinity for BrM specific targets. Despite the challenges posed by the blood-brain barrier to effective drug delivery, several radiolabeled compounds have shown promise in detecting and targeting BrM. This manuscript provides an overview of the recent advances in molecular biomarkers used in nuclear imaging and targeted radionuclide therapy in both clinical and preclinical settings. Additionally, it explores potential theranostic applications addressing the unique challenges posed by BrM.

Biomechanical properties of vaginal tissue in women with pelvic organ prolapse.

BACKGROUND/AIMS: To compare biomechanical properties of vaginal tissues between women with and without pelvic organ prolapse (POP) and investigate factors that may influence these properties. METHODS: Forty patients submitted to POP surgery and 15 non-POP cadavers were evaluated. The tissue was excised from anterior and posterior middle third vagina. The biomechanical properties considered were stiffness (E) and maximum stress (S), and they were evaluated by means of uniaxial tension tests. RESULTS: POP patients were associated with higher values of E (13.1 ± 0.8 vs. 9.5 ± 0.7 MPa; p < 0.001) and S (5.3 ± 0.5 vs. 3.2 ± 0.9 MPa; p < 0.001) in the anterior vaginal wall compared to the posterior wall. In contrast, non-POP women presented lower values of E (6.9 ± 1.1 vs. 10.5 ± 1.0 MPa; p = 0.01) and S (2.6 ± 0.4 vs. 3.5 ± 0.4 MPa; p = 0.043) in the anterior wall. The occurrence of POP was the only independent predictor of higher values of E and S in anterior vaginal samples (p = 0.003 and p = 0.008, respectively). Women with severe anterior vaginal prolapse presented higher levels of E and S in the anterior sample compared to those with lower POP stages (p = 0.001 and p = 0.01; respectively). CONCLUSION: Women with POP present significant changes of biomechanical properties in the vagina.

Strength of round and uterosacral ligaments: a biomechanical study.

PURPOSE: To investigate the tensile biomechanical properties of round and uterosacral ligaments. METHODS: Tissue samples were obtained from 15 female cadavers without pelvic organ prolapse. Uniaxial tensile tests were performed to obtain stiffness and maximum stress of round and uterosacral ligaments. Correlations were calculated using the Pearson correlation coefficient. Statistical differences between groups were tested using Student's paired and unpaired t test. RESULTS: There was a great variability in the measurements of stiffness and maximum stress in pelvic ligaments. The round ligaments demonstrated stiffness of 9.1 ± 1.6 MPa (mean ± SEM) (ranging from 2 to 25.6 MPa) and maximum stress of 4.3 ± 0.7 MPa (ranging from 1.2 to 11.5 MPa). The stiffness of the uterosacral ligaments was 14.1 ± 1.4 MPa (ranging from 5.7 to 26.1 MPa) with maximum stress of 6.3 ± 0.8 MPa (ranging from 2.2 to 11.9 MPa). There was a strong positive correlation between stiffness and maximum stress in female pelvic ligaments (ρ = 0.851; p < 0.001). The uterosacral ligaments demonstrated higher stiffness and maximum stress compared to the round ligaments (p = 0.006 and p = 0.034; respectively). Age, body mass index and menopausal status were not associated with the biomechanical proprieties of round and uterosacral ligaments. Nulliparous women had lower uterosacral stiffness (15.5 ± 1.3 vs. 10 ± 1.8 MPa; p = 0.033) and maximum stress (8.2 ± 0.9 vs. 4.2 ± 1.1 MPa; p = 0.028) compared to parous women. CONCLUSION: The uterosacral ligaments are significantly more resistant than round ligaments. Parturition seems to enhance the stiffness and maximum stress of the ligaments.

Survey on selenium clinical supplementation in autoimmune thyroid disease.

Objective: Previous trials show that selenium could be a very useful tool in the control and treatment of autoimmune thyroid diseases. In this cross-sectional study, through a survey, we aim to evaluate Portuguese endocrinologists' perception and pattern of prescription of selenium supplements in these diseases and verify its agreement with current guidelines. Methods: The endocrinologists registered in the Portuguese Medical Association were sent an email with a web-based questionnaire, regarding their knowledge and use of selenium supplements in thyroid autoimmune pathology. Results: A total of 105 physicians (33% of the total) submitted the survey. The selenium serum concentration in the general population was unknown to 80% of respondents. Over a third of respondents have never prescribed selenium for autoimmune thyroid disease. However, 89% are not afraid of recommending it, and 61% indicate Graves' orbitopathy as the pathology they would supplement. In Hashimoto's thyroiditis, 36% of respondents use selenium occasionally or frequently, and this percentage rises to 60% in Graves' disease. Conclusions: Although recommendations only encompass mild Graves' orbitopathy, selenium is prescribed across the spectrum of autoimmune thyroid diseases, probably due to recent studies that consistently show improvement of biochemical hallmarks in these patients. Further investigation is required on the impact of selenium supplements on primarily clinical outcomes and to identify disorders and/or patients who will benefit the most. Also, there is still insufficient knowledge of this field in the medical community, and evidence-based practice should continue to be promoted by endocrinology societies.

Identification of biomarkers to stratify response to B-cell-targeted therapies in systemic lupus erythematosus: an exploratory analysis of a randomised controlled trial.

Background: Systemic lupus erythematosus (SLE) is a complex autoimmune disease associated with widespread immune dysregulation and diverse clinical features. Immune abnormalities might be differentially associated with specific organ involvement or response to targeted therapies. We aimed to identify biomarkers of response to belimumab after rituximab to facilitate a personalised approach to therapy. Methods: In this exploratory analysis of a randomised controlled trial (BEAT-LUPUS), we investigated immune profiles of patients with SLE recruited to the 52-week clinical trial, which tested the combination of rituximab plus belimumab versus rituximab plus placebo. We used machine learning and conventional statistics to investigate relevant laboratory and clinical biomarkers associated with major clinical response. BEAT LUPUS is registered at ISRCTN, 47873003, and is now complete. Findings: Between Feb 2, 2017, and March 28, 2019, 52 patients were recruited to BEAT-LUPUS, of whom 44 provided clinical data at week 52 and were included in this analysis. 21 (48%) of 44 participants were in the belimumab group (mean age 39·5 years [SD 12·1]; 17 [81%] were female, four [19%] were male, 13 [62%] were White) and 23 (52%) were in the placebo group (mean age 42·1 years [SD 10·5]; 21 [91%] were female, two [9%] were male, 16 [70%] were White). Ten (48%) of 21 participants who received belimumab after rituximab and eight (35%) of 23 who received placebo after rituximab had a major clinical response at 52 weeks (between-group difference of 13% [95% CI -15 to 38]). We found a predictive association between baseline serum IgA2 anti-double stranded DNA (dsDNA) antibody concentrations and clinical response to belimumab after rituximab, with a between-group difference in major clinical response of 48% (95% CI 10 to 70) in patients with elevated baseline serum IgA2 anti-dsDNA antibody concentrations. Moreover, among those who had a major clinical response, serum IgA2 anti-dsDNA antibody concentrations significantly decreased from baseline only in the belimumab group. Increased circulating IgA2 (but not total) plasmablast numbers, and T follicular helper cell numbers predicted clinical response and were both reduced only in patients who responded to belimumab after rituximab. Serum IgA2 anti-dsDNA antibody concentrations were also associated with active renal disease, whereas serum IgA1 anti-dsDNA antibody and IFN-α concentrations were associated with mucocutaneous disease activity but did not predict response to B-cell targeted therapy. Patients with a high baseline serum interleukin-6 concentration were less likely to have a major clinical response, irrespective of therapy. Interpretation: This exploratory study revealed the presence of distinct molecular networks associated with renal and mucocutaneous involvement, and response to B-cell-targeted therapies, which, if confirmed, could guide precision targeting of advanced therapies for this heterogenous disease. Funding: Versus Arthritis, UCLH Biomedical Research Centre, LUPUS UK, and GSK.