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Abstract Background A prolonged and complicated second stage of labour is associated with serious perinatal complications. The Odon device is an innovation intended to perform instrumental vaginal delivery presently under development. We present an evaluation of the feasibility and safety of delivery with early prototypes of this device from an early terminated clinical study. Methods Hospital-based, multi-phased, open-label, pilot clinical study with no control group in tertiary hospitals in Argentina and South Africa. Multiparous and nulliparous women, with uncomplicated singleton pregnancies, were enrolled during the third trimester of pregnancy. Delivery with Odon device was attempted under non-emergency conditions during the second stage of labour. The feasibility outcome was delivery with the Odon device defined as successful expulsion of the fetal head after one-time application of the device. Results Of the 49 women enrolled, the Odon device was inserted successfully in 46 (93%), and successful Odon device delivery as defined above was achieved in 35 (71%) women. Vaginal, first and second degree perineal tears occurred in 29 (59%) women. Four women had cervical tears. No third or fourth degree perineal tears were observed. All neonates were born alive and vigorous. No adverse maternal or infant outcomes were observed at 6-weeks follow-up for all dyads, and at 1 year for the first 30 dyads. Conclusions Delivery using the Odon device is feasible. Observed genital tears could be due to the device or the process of delivery and assessment bias. Evaluating the effectiveness and safety of the further developed prototype of the BD Odon Device™ will require a randomized-controlled trial. Trial registration ANZCTR ACTRN12613000141741 Registered 06 February 2013. Retrospectively registered.

Iron is an essential mineral that supports numerous biological functions. Studies have reported associations between iron dysregulation and certain cardiovascular and neurodegenerative diseases, but the direction of influence is not clear. Our goal was to use computational approaches to better understand the role of genetically predicted iron levels on disease risk. We meta-analyzed genome-wide association study summary statistics for serum iron levels from two cohorts and two previous meta-analyses. We then obtained summary statistics from 11 neurodegenerative, cerebrovascular, cardiovascular or lipid traits to assess global and regional genetic correlation between iron levels and these traits. We used two-sample Mendelian randomization (MR) to estimate causal effects. Sex-stratified analyses were also carried out to identify effects potentially differing by sex. Overall, we identified three significant global correlations between iron levels and (i) coronary heart disease, (ii) triglycerides, and (iii) high-density lipoprotein (HDL) cholesterol levels. A total of 194 genomic regions had significant (after correction for multiple testing) local correlations between iron levels and the 11 tested traits. MR analysis revealed two potential causal relationships, between genetically predicted iron levels and (i) total cholesterol or (ii) non-HDL cholesterol. Sex-stratified analyses suggested a potential protective effect of iron levels on Parkinson's disease risk in females, but not in males. Our results will contribute to a better understanding of the genetic basis underlying iron in cardiovascular and neurological health in aging, and to the eventual identification of new preventive interventions or therapeutic avenues for diseases which affect women and men worldwide., (© 2024. The Author(s).)

Background: Usher syndrome (USH) encompasses a group of disorders characterized by congenital sensorineural hearing loss (SNHL) and retinitis pigmentosa (RP). We described the clinical findings, natural history, and molecular analyses of USH patients identified during a large-scale screening to identify quantitative traits related to ocular disorders in the SardiNIA project cohort., Methods: We identified 3 USH-affected families out of a cohort of 6,148 healthy subjects. 9 subjects presented a pathological phenotype, with SNHL and RP. All patients and their family members underwent a complete ophthalmic examination including best-corrected visual acuity, slit-lamp biomicroscopy, fundoscopy, fundus autofluorescence, spectral-domain optical coherence tomography, and electrophysiological testing. Audiological evaluation was performed with a clinical audiometer. Genotyping was performed using several arrays integrated with whole genome sequence data providing approximately 22 million markers equally distributed for each subject analyzed. Molecular diagnostics focused on analysis of the following candidate genes: MYO7A, USH1C, CDH23, PCDH15, USH1G, CIB2, USH2A, GPR98, DFNB31, CLRN1, and PDZD7., Results: A single missense causal variant in USH2A gene was identified in homozygous status in all patients and in heterozygous status in unaffected parents. The presence of multiple homozygous patients with the same phenotypic severity of the syndromic form suggests that the Sardinian USH phenotype is the result of a founder effect on a specific pathogenic variant related haplotype. The frequency of heterozygotes in general Sardinian population is 1.89. Additionally, to provide new insights into the structure of usherin and the pathological mechanisms caused by small pathogenic in-frame variants, like p.Pro3272Leu, molecular dynamics simulations of native and mutant protein-protein and protein-ligand complexes were performed that predicted a destabilization of the protein with a decrease in the free energy change., Conclusions: Our results suggest that our approach is effective for the genetic diagnosis of USH. Based on the heterozygous frequency, targeted screening of this variant in the general population and in families at risk or with familial USH can be suggested. This can lead to more accurate molecular diagnosis, better genetic counseling, and improved molecular epidemiology data that are critical for future intervention plans., Trial Registration: We did not perform any health-related interventions for the participants., (© 2024. The Author(s).)

Introduction: Disease-modifying therapies (DMTs) have been shown to improve disease outcomes in multiple sclerosis (MS) patients. They may also impair the immune response to vaccines, including the SARS-CoV-2 vaccine. However, available data on both the intrinsic immune effects of DMTs and their influence on cellular response to the SARS-CoV-2 vaccine are still incomplete., Methods: Here, we evaluated the immune cell effects of 3 DMTs on the response to mRNA SARS-CoV-2 vaccination by comparing MS patients treated with one specific therapy (fingolimod, dimethyl fumarate, or natalizumab) with both healthy controls and untreated patients. We profiled 23 B-cell traits, 57 T-cell traits, and 10 cytokines, both at basal level and after stimulation with a pool of SARS-CoV-2 spike peptides, in 79 MS patients, treated with DMTs or untreated, and 32 healthy controls. Measurements were made before vaccination and at three time points after immunization., Results and Discussion: MS patients treated with fingolimod showed the strongest immune cell dysregulation characterized by a reduction in all measured lymphocyte cell classes; the patients also had increased immune cell activation at baseline, accompanied by reduced specific immune cell response to the SARS-CoV-2 vaccine. Also, anti-spike specific B cells progressively increased over the three time points after vaccination, even when antibodies measured from the same samples instead showed a decline. Our findings demonstrate that repeated booster vaccinations in MS patients are crucial to overcoming the immune cell impairment caused by DMTs and achieving an immune response to the SARS-CoV-2 vaccine comparable to that of healthy controls., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Orrù, Serra, Marongiu, Lai, Lodde, Zoledziewska, Steri, Loizedda, Lobina, Piras, Virdis, Delogu, Marini, Mingoia, Floris, Masala, Castelli, Mostallino, Frau, Lorefice, Farina, Fronza, Carmagnini, Carta, Pilotto, Chessa, Devoto, Castiglia, Solla, Zarbo, Idda, Pitzalis, Cocco, Fiorillo and Cucca.)

Gene co-expression network analysis enables identification of biologically meaningful clusters of co-regulated genes (modules) in an unsupervised manner. We present here the largest study conducted thus far of co-expression networks in white blood cells (WBC) based on RNA-seq data from 624 individuals. We identify 41 modules, 13 of them related to specific immune-related functions and cell types (e.g. neutrophils, B and T cells, NK cells, and plasmacytoid dendritic cells); we highlight biologically relevant lncRNAs for each annotated module of co-expressed genes. We further characterize with unprecedented resolution the modules in T cell sub-types, through the availability of 95 immune phenotypes obtained by flow cytometry in the same individuals. This study provides novel insights into the transcriptional architecture of human leukocytes, showing how network analysis can advance our understanding of coding and non-coding gene interactions in immune system cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Forabosco, Pala, Crobu, Diana, Marongiu, Cusano, Angius, Steri, Orrù, Schlessinger, Fiorillo, Devoto and Cucca.)

Background and Aims: Arterial stiffness (AS), quantified by pulse wave velocity (PWV), arises due to impaired arterial elastic tissue and smooth muscle dysfunction. We aimed to examine the longitudinal association of genetic, lipid and inflammation biomarkers with PWV and how these associations may change with aging., Materials and Methods: We utilized genotype and four time-point biomarker data from the SardiNIA cohort [ n  = 6,301; mean baseline age 43.3 (SD 17.3); 58% females]. To investigate the association of PWV with genetic variants, lipid, and inflammation biomarkers, we employed linear mixed modeling, using age as the time scale. Biomarkers exhibiting significant longitudinal associations were categorized into tertiles and individuals within the second tertile or those with heterozygous alleles were excluded, leaving a cohort of 2,000 individuals. This cohort was further divided into four risk groups: low genetic and low biomarker (L-L), low genetic and high biomarker (L-H), high genetic and low biomarker (H-L), and high genetic and high biomarker risk (H-H). Subsequent analyses focused on these risk groups to assess their association to PWV with time., Results: Using the complete dataset, we found a significant longitudinal association of total cholesterol (TC), triglycerides (TG), fibrinogen (FGN), and total white blood cell count (TWBC) with PWV, all with p  < 3.33 × 10 -3 . After grouping, individuals with homogeneous risk alleles of SNP rs3742207 and high baseline TG levels (H-H group) exhibited a 1.39-fold higher PWV (m/s) (95% CI, 1.17-1.64, p =  1.21 × 10 -4 ) compared to the L-L group. Similarly, individuals in the H-H group of rs3742207-TWBC combination showed 1.75 times higher PWV (95% CI, 1.48-0.2.07, p =  1.01 × 10 -10 ) compared to the L-L group. Similar patterns were observed for groups based on SNP rs7152623-TWBC risk. Furthermore, these associations became more pronounced with increasing age ( p  < 3.33 × 10 -3 )., Conclusion: The longitudinal association of TG and TWBC biomarkers with PWV varied by SNPs rs3742207 and rs7152623 genotype. Further studies are warranted to investigate the function of genetics, lipids, and inflammation biomarkers on PWV change., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors EF, EL declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (© 2024 Asefa, Meirelles, Lakatta, Fiorillo, Scuteri, Cucca, Marongiu, Delitala, Schlessinger and Launer.)

Some people remain healthier throughout life than others but the underlying reasons are poorly understood. Here we hypothesize this advantage is attributable in part to optimal immune resilience (IR), defined as the capacity to preserve and/or rapidly restore immune functions that promote disease resistance (immunocompetence) and control inflammation in infectious diseases as well as other causes of inflammatory stress. We gauge IR levels with two distinct peripheral blood metrics that quantify the balance between (i) CD8 +  and CD4 +  T-cell levels and (ii) gene expression signatures tracking longevity-associated immunocompetence and mortality-associated inflammation. Profiles of IR metrics in ~48,500 individuals collectively indicate that some persons resist degradation of IR both during aging and when challenged with varied inflammatory stressors. With this resistance, preservation of optimal IR tracked (i) a lower risk of HIV acquisition, AIDS development, symptomatic influenza infection, and recurrent skin cancer; (ii) survival during COVID-19 and sepsis; and (iii) longevity. IR degradation is potentially reversible by decreasing inflammatory stress. Overall, we show that optimal IR is a trait observed across the age spectrum, more common in females, and aligned with a specific immunocompetence-inflammation balance linked to favorable immunity-dependent health outcomes. IR metrics and mechanisms have utility both as biomarkers for measuring immune health and for improving health outcomes., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Extracellular vesicles (EVs) mediate cell interactions in biological processes, such as receptor activation or molecule transfer. Estimates of variation by age and sex have been limited by small sample size, and no report has assessed the contribution of genetic factors to levels of EVs. Here, we evaluated blood levels of 25 EV and 3 platelet traits in 974 individuals (933 genotyped) and reported the first genome-wide association study (GWAS) on levels of these traits. EV levels all decreased with age, whereas the trend for their surface markers was more heterogeneous. Platelets and CD31dim platelet EVs significantly increased in females compared to males, although CD31 expression on both platelets and platelet EVs decreased in females. Levels of the other EV subsets were similar between sexes. GWAS revealed three statistically significant genetic signals associated with EV levels in the F10 and GBP1 genes and in the intergenic region between LRIG1 and KBTBD8 . These add to a signal in the 3'UTR of RHOF associated with CD31 expression on platelets that was previously found to be associated with other platelet traits. These findings suggest that EV formation is not a simple, constant adjunct of metabolism but is under both age-related and genetic control that can be independent of the regulation of the levels of the cells from which the EVs derive.

Hypertrophic carotid geometric phenotypes (h-CGP) are predictors of incident cardiovascular disease (CVD). While arterial aging is hypothesized as a contributor to this associated risk, the association of CGPs with chronological age is not clear. In this manuscript we examine whether hypertrophic CGPs represent accelerated biological, rather than chronological, aging by examining their association with carotid-femoral pulse wave velocity (PWV), the hallmark of arterial aging. We analyzed data from 5516 participants of the SardiNIA study with a wide range of age at baseline (20-101 years), and a median follow-up time of 13 years (mean 11.5 years; maximum 17.9 years). Baseline CGPs were defined based on the common carotid lumen diameter, wall thickness, and their ratio. Subject-specific rates of change of PWV, blood pressure parameters, body mass index, glucose, and lipids were estimated using linear mixed effects models. Compared to those with typical(t-) CGP, those with dilated hypertrophy (dh-) CGP had a greater longitudinal increase in PWV; this increase was significantly greater among older individuals and men. The greater PWV longitudinal increase in dh-CGP remained significant after adjusting for baseline values and rates of change of covariates. Dilated hypertrophic CGP is independently associated with accelerated increase in age-associated arterial stiffening over time, with a strong association in men than in women. Future studies are needed to examine if this association mediates the increased risk for CVD observed in individuals with hypertrophic cardiac remodelling and the role of retarding it to reduce this risk. HIGHLIGHTS: • Individuals with dilated hypertrophic geometric phenotypes of the common carotid artery (increased age- and sex-specific wall thickness and lumen diameter) have greater future central arterial stiffening, independently of other determinants of arterial stiffening. • The dilated hypertrophic phenotype group has a greater age-specific arterial dilation, wall thickening, and stiffness (the arterial aging triad). This suggests that this phenotype is a form of accelerated aging that might explain the worse clinic outcomes observed in this group. • Understanding the natural history of the carotid geometric phenotype across the lifespan and the determinants of the deleterious progression towards the dilated hypertrophic phenotype are needed to develop interventions that reduce the adverse clinical outcomes associated with it., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The influence of nutritional factors on frailty syndrome is still poorly understood. Thus, we aimed to confirm cross-sectional associations of diet-related blood biomarker patterns with frailty and pre-frailty statuses in 1271 older adults from four European cohorts. Principal component analysis (PCA) was performed based on plasma levels of α-carotene, β-carotene, lycopene, lutein + zeaxanthin, β-cryptoxanthin, α-tocopherol, γ-tocopherol and retinol. Cross-sectional associations between biomarker patterns and frailty status, according to Fried's frailty criteria, were assessed by using general linear models and multinomial logistic regression models as appropriate with adjustments for the main potential confounders. Robust subjects had higher concentrations of total carotenoids, β-carotene and β-cryptoxanthin than frail and pre-frail subjects and had higher lutein + zeaxanthin concentrations than frail subjects. No associations between 25-Hydroxyvitamin D3 and frailty status were observed. Two distinct biomarker patterns were identified in the PCA results. The principal component 1 (PC1) pattern was characterized by overall higher plasma levels of carotenoids, tocopherols and retinol, and the PC2 pattern was characterized by higher loadings for tocopherols, retinol and lycopene together and lower loadings for other carotenoids. Analyses revealed inverse associations between PC1 and prevalent frailty. Compared to participants in the lowest quartile of PC1, those in the highest quartile were less likely to be frail (odds ratio: 0.45, 95% CI: 0.25-0.80, p = 0.006). In addition, those in the highest quartile of PC2 showed higher odds for prevalent frailty (2.48, 1.28-4.80, p = 0.007) than those in the lowest quartile. Our findings strengthen the results from the first phase of the FRAILOMIC project, indicating carotenoids are suitable components for future biomarker-based frailty indices.

Few genome-wide association studies (GWAS) analyzing genetic regulation of morphological traits of white blood cells have been reported. We carried out a GWAS of 12 morphological traits in 869 individuals from the general population of Sardinia, Italy. These traits, included measures of cell volume, conductivity and light scatter in four white-cell populations (eosinophils, lymphocytes, monocytes, neutrophils). This analysis yielded seven statistically significant signals, four of which were novel (four novel, PRG2, P2RX3, two of CDK6). Five signals were replicated in the independent INTERVAL cohort of 11 822 individuals. The most interesting signal with large effect size on eosinophil scatter (P-value = 8.33 x 10-32, beta = -1.651, se = 0.1351) falls within the innate immunity cluster on chromosome 11, and is located in the PRG2 gene. Computational analyses revealed that a rare, Sardinian-specific PRG2:p.Ser148Pro mutation modifies PRG2 amino acid contacts and protein dynamics in a manner that could possibly explain the changes observed in eosinophil morphology. Our discoveries shed light on genetics of morphological traits. For the first time, we describe such large effect size on eosinophils morphology that is relatively frequent in Sardinian population., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The use of cryopreserved peripheral blood mononuclear cells is common in biological research. It is widely accepted that primary cells are rendered unusable by several freezing cycles, although this practice might be very helpful when the biological material is valuable and its re-collection is impractical. To determine the extent to which primary cells undergoing repeated freezing cycles are comparable to one another and to fresh samples, we evaluated overall lymphocyte viability, their proliferation and cytokine production capabilities, as well as the levels of 27 cell subtypes in ten human peripheral blood mononuclear cells frozen for five years and repeatedly thawed. As expected, we observed a progressive increase in cell death percentages on three rounds of thawing, but the frequency of the main lymphocyte subsets was stable across the three thawings. Nevertheless, we observed a significant reduction of B cell frequency in frozen samples compared to fresh ones. On repeated thawings and subsequent conventional stimulation, lymphocyte proliferation significantly decreased, and IL-10, IL-6, GM-CSF, IFN-gamma, and IL-8 showed a trend to lower values.

Tobacco and alcohol use are heritable behaviours associated with 15% and 5.3% of worldwide deaths, respectively, due largely to broad increased risk for disease and injury 1-4 . These substances are used across the globe, yet genome-wide association studies have focused largely on individuals of European ancestries 5 . Here we leveraged global genetic diversity across 3.4 million individuals from four major clines of global ancestry (approximately 21% non-European) to power the discovery and fine-mapping of genomic loci associated with tobacco and alcohol use, to inform function of these loci via ancestry-aware transcriptome-wide association studies, and to evaluate the genetic architecture and predictive power of polygenic risk within and across populations. We found that increases in sample size and genetic diversity improved locus identification and fine-mapping resolution, and that a large majority of the 3,823 associated variants (from 2,143 loci) showed consistent effect sizes across ancestry dimensions. However, polygenic risk scores developed in one ancestry performed poorly in others, highlighting the continued need to increase sample sizes of diverse ancestries to realize any potential benefit of polygenic prediction., (© 2022. The Author(s).)

Monitoring immune responses to SARS-CoV-2 vaccination and its clinical efficacy over time in Multiple Sclerosis (MS) patients treated with disease-modifying therapies (DMTs) help to establish the optimal strategies to ensure adequate COVID-19 protection without compromising disease control offered by DMTs. Following our previous observations on the humoral response one month after two doses of BNT162b2 vaccine (T1) in MS patients differently treated, here we present a cross-sectional and longitudinal follow-up analysis six months following vaccination (T2, n=662) and one month following the first booster (T3, n=185). Consistent with results at T1, humoral responses were decreased in MS patients treated with fingolimod and anti-CD20 therapies compared with untreated patients also at the time points considered here (T2 and T3). Interestingly, a strong upregulation one month after the booster was observed in patients under every DMTs analyzed, including those treated with fingolimod and anti-CD20 therapies. Although patients taking these latter therapies had a higher rate of COVID-19 infection five months after the first booster, only mild symptoms that did not require hospitalization were reported for all the DMTs analyzed here. Based on these findings we anticipate that additional vaccine booster shots will likely further improve immune responses and COVID-19 protection in MS patients treated with any DMT., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Idda, Pitzalis, Lodde, Loizedda, Frau, Lobina, Zoledziewska, Virdis, Delogu, Marini, Mingoia, Masala, Lorefice, Fronza, Carmagnini, Carta, Pilotto, Castiglia, Chessa, Uzzau, Farina, Solla, Steri, Devoto, Fiorillo, Floris, Zarbo, Cocco and Cucca.)

Clonal expansions driven by somatic mutations become pervasive across human tissues with age, including in the haematopoietic system, where the phenomenon is termed clonal haematopoiesis 1-4 . The understanding of how and when clonal haematopoiesis develops, the factors that govern its behaviour, how it interacts with ageing and how these variables relate to malignant progression remains limited 5,6 . Here we track 697 clonal haematopoiesis clones from 385 individuals 55 years of age or older over a median of 13 years. We find that 92.4% of clones expanded at a stable exponential rate over the study period, with different mutations driving substantially different growth rates, ranging from 5% (DNMT3A and TP53) to more than 50% per year (SRSF2 P95H ). Growth rates of clones with the same mutation differed by approximately ±5% per year, proportionately affecting slow drivers more substantially. By combining our time-series data with phylogenetic analysis of 1,731 whole-genome sequences of haematopoietic colonies from 7 individuals from an older age group, we reveal distinct patterns of lifelong clonal behaviour. DNMT3A-mutant clones preferentially expanded early in life and displayed slower growth in old age, in the context of an increasingly competitive oligoclonal landscape. By contrast, splicing gene mutations drove expansion only later in life, whereas TET2-mutant clones emerged across all ages. Finally, we show that mutations driving faster clonal growth carry a higher risk of malignant progression. Our findings characterize the lifelong natural history of clonal haematopoiesis and give fundamental insights into the interactions between somatic mutation, ageing and clonal selection., (© 2022. The Author(s).)

Fresh blood immunophenotyping by flow cytometry, based on the reliable simultaneous detection of several markers in a cell, is the method of choice to study the circulating human immune system. Especially in large and multicenter studies, high sample quality is difficult to achieve, and adequate collection and storage of samples with fine-tuned whole blood cryopreservation is mandatory. Here, we compared the quality of immunophenotypic data obtained from fresh blood with those obtained after five cryopreservation methods by quantifying the levels of 41 immune cell populations. They comprised B and T lymphocyte subsets and their maturation stages, as well as monocytes and granulocytes. Three methods used fixative solutions and two other methods used dimethyl sulfoxide solutions to preserve cell viability. The fixative methods prevented detection of markers critical for identification of B and T cell subsets, including CD27, CXCR3, and CCR6. The other two methods permitted reliable discrimination of most immune-cell populations in thawed samples, though some cell frequencies varied compared to the corresponding fresh sample. Of those two methods, the one preserving blood in media containing dimethyl sulfoxide produced results that were most similar to those with fresh samples.

Objectives: Vaccination against COVID-19 is highly recommended to patients affected by multiple sclerosis (MS); however, the impact of MS disease-modifying therapies (DMTs) on the immune response following vaccination has been only partially investigated. Here, we aimed to elucidate the effect of DMTs on the humoral immune response to mRNA-based anti-SARS-CoV-2 vaccines in MS patients., Methods: We obtained sera from 912 Sardinian MS patients and 63 healthy controls 30 days after the second dose of BNT162b2 vaccine and tested them for SARS-CoV-2 response using anti-Spike (S) protein-based serology. Previous SARS-CoV-2 infection was assessed by anti-Nucleocapsid (N) serology. Patients were either untreated or undergoing treatment with a total of 13 different DMTs. Differences between treatment groups comprised of at least 10 patients were assessed by generalized linear mixed-effects model. Demographic and clinical data and smoking status were analyzed as additional factors potentially influencing humoral immunity from COVID-19 vaccine., Results: MS patients treated with natalizumab, teriflunomide, azathioprine, fingolimod, ocrelizumab, and rituximab showed significantly lower humoral responses compared to untreated patients. We did not observe a statistically significant difference in response between patients treated with the other drugs (dimethyl fumarate, interferon, alemtuzumab and glatiramer acetate) and untreated patients. In addition, older age, male sex and active smoking were significantly associated with lower antibody titers against SARS-CoV-2. MS patients previously infected with SARS-CoV-2 had significantly higher humoral responses to vaccine than uninfected patients., Conclusion: Humoral response to BNT162b2 is significantly influenced by the specific DMTs followed by patients, as well as by other factors such as previous SARS-CoV-2 infection, age, sex, and smoking status. These results are important to inform targeted strategies to prevent clinically relevant COVID-19 in MS patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pitzalis, Idda, Lodde, Loizedda, Lobina, Zoledziewska, Virdis, Delogu, Pirinu, Marini, Mingoia, Frau, Lorefice, Fronza, Carmagnini, Carta, Orrù, Uzzau, Solla, Loi, Devoto, Steri, Fiorillo, Floris, Zarbo, Cocco and Cucca.)

Increased blood lipid levels are heritable risk factors of cardiovascular disease with varied prevalence worldwide owing to different dietary patterns and medication use 1 . Despite advances in prevention and treatment, in particular through reducing low-density lipoprotein cholesterol levels 2 , heart disease remains the leading cause of death worldwide 3 . Genome-wideassociation studies (GWAS) of blood lipid levels have led to important biological and clinical insights, as well as new drug targets, for cardiovascular disease. However, most previous GWAS 4-23 have been conducted in European ancestry populations and may have missed genetic variants that contribute to lipid-level variation in other ancestry groups. These include differences in allele frequencies, effect sizes and linkage-disequilibrium patterns 24 . Here we conduct a multi-ancestry, genome-wide genetic discovery meta-analysis of lipid levels in approximately 1.65 million individuals, including 350,000 of non-European ancestries. We quantify the gain in studying non-European ancestries and provide evidence to support the expansion of recruitment of additional ancestries, even with relatively small sample sizes. We find that increasing diversity rather than studying additional individuals of European ancestry results in substantial improvements in fine-mapping functional variants and portability of polygenic prediction (evaluated in approximately 295,000 individuals from 7 ancestry groupings). Modest gains in the number of discovered loci and ancestry-specific variants were also achieved. As GWAS expand emphasis beyond the identification of genes and fundamental biology towards the use of genetic variants for preventive and precision medicine 25 , we anticipate that increased diversity of participants will lead to more accurate and equitable 26 application of polygenic scores in clinical practice., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

In tropical regions, heavy precipitations may lead to catastrophic flooding due to the degradation of catchments and the expansion of settlements in flood prone zones. In the current situation, where information on rainfall and exposed assets is either scant, or requires significant time to be collected, pluvial flood risk assessments are conducted using participatory tools, without any scientific support. Another option is to use satellite precipitation products, digital terrain models and satellite images at high to moderate-resolution. However, these datasets do not reach the required accuracy at the local scale. Consequently, the potential damages and the evaluation component of risk assessment are often missing. Risk evaluation is pivotal for informed decision-making, with regards to the choice of treating or accepting the risk, implementing more effective measures, and for determining the safest areas for development. We proposed an improved method for assessing the risk of pluvial floods, which merges local and scientific knowledge and is consistent with the ISO 31010 standard. The method was successfully applied in five rural settlements in Niger and can be replicated in areas where information is scarce., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

In recent years, systematic genome-wide association studies of quantitative immune cell traits, represented by circulating levels of cell subtypes established by flow cytometry, have revealed numerous association signals, a large fraction of which overlap perfectly with genetic signals associated with autoimmune diseases. By identifying further overlaps with association signals influencing gene expression and cell surface protein levels, it has also been possible, in several cases, to identify causal genes and infer candidate proteins affecting immune cell traits linked to autoimmune disease risk. Overall, these results provide a more detailed picture of how genetic variation affects the human immune system and autoimmune disease risk. They also highlight druggable proteins in the pathogenesis of autoimmune diseases; predict the efficacy and side effects of existing therapies; provide new indications for use for some of them; and optimize the research and development of new, more effective and safer treatments for autoimmune diseases. Here we review the genetic-driven approach that couples systematic multi-parametric flow cytometry with high-resolution genetics and transcriptomics to identify endophenotypes of autoimmune diseases for the development of new therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Orrù, Steri, Cucca and Fiorillo.)

Phenotype-specific omic expression patterns in people with frailty could provide invaluable insight into the underlying multi-systemic pathological processes and targets for intervention. Classical approaches to frailty have not considered the potential for different frailty phenotypes. We characterized associations between frailty (with/without disability) and sets of omic factors (genomic, proteomic, and metabolomic) plus markers measured in routine geriatric care. This study was a prevalent case control using stored biospecimens (urine, whole blood, cells, plasma, and serum) from 1522 individuals (identified as robust (R), pre-frail (P), or frail (F)] from the Toledo Study of Healthy Aging (R=178/P=184/F=109), 3 City Bordeaux (111/269/100), Aging Multidisciplinary Investigation (157/79/54) and InCHIANTI (106/98/77) cohorts. The analysis included over 35,000 omic and routine laboratory variables from robust and frail or pre-frail (with/without disability) individuals using a machine learning framework. We identified three protective biomarkers, vitamin D3 (OR: 0.81 [95% CI: 0.68-0.98]), lutein zeaxanthin (OR: 0.82 [95% CI: 0.70-0.97]), and miRNA125b-5p (OR: 0.73, [95% CI: 0.56-0.97]) and one risk biomarker, cardiac troponin T (OR: 1.25 [95% CI: 1.23-1.27]). Excluding individuals with a disability, one protective biomarker was identified, miR125b-5p (OR: 0.85, [95% CI: 0.81-0.88]). Three risks of frailty biomarkers were detected: pro-BNP (OR: 1.47 [95% CI: 1.27-1.7]), cardiac troponin T (OR: 1.29 [95% CI: 1.21-1.38]), and sRAGE (OR: 1.26 [95% CI: 1.01-1.57]). Three key frailty biomarkers demonstrated a statistical association with frailty (oxidative stress, vitamin D, and cardiovascular system) with relationship patterns differing depending on the presence or absence of a disability.

Background: Thyroid hormone modulation of cardiovascular function has been associated with cardiovascular disease. Recent evidence suggests that free thyroxine (FT4) levels are associated with an increase in systemic arterial stiffness, but little is known about the effects of FT4 at the local level of the common carotid artery. β-stiffness index is a local elastic parameter usually determined by carotid ultrasound imaging., Methods: We conducted a cross-sectional analysis in the ProgeNIA cohort, including 4846 subjects across a broad age range. For the purpose of this study, we excluded subjects with increased thyrotropin (TSH) levels and those treated with levothyroxine or thyrostatic. We assessed β stiffness, strain, wall-lumen ratio, carotid cross-sectional area (CSA), and stress and flow in the right common carotid artery. We tested whether FT4, heart rate, and their interactions were associated with carotid parameters., Results: FT4 was positively and independently associated with β stiffness index (β = 0.026, p = 0.041), and had a negative association with strain (β = -0.025, p = 0.009). After adding heart rate and the interaction between FT4 and heart rate to the model, FT4 was still associated with the β stiffness index (β = 0.186, p = 0.06), heart rate was positively associated with the stiffness index (β = 0.389, p < 0.001) as well as their interaction (β = 0.271, p = 0.007)., Conclusion: This study suggests that higher FT4 levels increase arterial stiffness at the common carotid level, consistent with a detrimental effect on elastic arteries. The effect of FT4 is likely to be primarily attributable to its effect on heart rate.

Aims: Previous cross-sectional observation identified arterial aging, indexed as pulse-wave velocity (PWV), as a key determinant of the simultaneous multiple organ damage (heart, carotid artery, and kidney). The aim of the present cohort study is to investigate trajectories of repeated measures of PWV and traditional CV risk factors in subjects who eventually presented clinical evidence of multiple organ damage in the SardiNIA study., Methods and Results: Organ damage was measured in the heart (left ventricular hypertrophy, LVH), the common carotid artery (intima-media thickness > 0.9 mm and/or plaque), and the kidney (eGFR < 60 ml/min/1.73 m 2 ) of 2130 men and women of a broad age range participating the SardiNIA study. SHATS was defined as the simultaneous occurrence of all the three-organ damages. Trajectory in traditional CV risk factors and PWV was analyzed retrospectively (four observations over 9 years) according to the number of organ damage (from 0 to 3). Compared to subjects with no organ damage, after controlling for traditional CV risk factors, each 1 m/s increase in baseline PWV was accompanied by a 93% higher odds of developing SHATS; and each 1 cm/s (0.01 m/s) annual increase in PWV by a 31% greater odds of developing SHATS., Conclusions: Arterial stiffness, a proxy of arterial aging that can be measured clinically as PWV, is an integrated predictive marker of multiple age-associated organ damage recognized as clinical diseases.

Background: The relationship of kidney size to ageing, kidney function and kidney disease risk factors is not fully understood., Methods: Ultrasound length and parenchymal kidney volume were determined from a population-based sample of 3972 Sardinians (age range 18-100 years). We then identified the subset of 2256 'healthy' subjects to define age- and sex-specific reference ranges (2.5-97.5 percentile) of kidney volume. Logistic regression (accounting for family clustering) was used to identify the clinical characteristics associated with abnormally large kidneys or abnormally small kidneys., Results: In the healthy subset, kidney volume and length increased up to the fourth to fifth decade of life followed by a progressive decrease in men, whereas there was a gradual kidney volume decrease throughout the lifespan of women. In the whole sample, independent predictors of lower kidney volume (<2.5 percentile for age and sex) were male sex, low body mass index, short height, low waist:hip ratio and high serum creatinine (SCr); the independent predictors of larger kidney volume (>97.5 percentile for age and sex) were younger age, female sex, diabetes, obesity, high height, high waist:hip ratio and lower SCr. Estimated heritability for kidney volume was 15%, and for length 27%; kidney volume correlated strongly with birthweight., Conclusions: Overall, in a general healthy population, kidney measures declined with age differently in men and women. The determinants of kidney parenchymal volume include genetic factors and modifiable clinical factors., (Published by Oxford University Press on behalf of ERA-EDTA 2018. This work is written by US Government employees and is in the public domain in the US.)

In systemic lupus erythematosus (SLE), perturbed immunoregulation underpins a pathogenic imbalance between regulatory and effector CD4 + T-cell activity. However, to date, the characterization of the CD4 + regulatory T cell (Treg) compartment in SLE has yielded conflicting results. Here we show that patients have an increased frequency of CD4 + FOXP3 + cells in circulation owing to a specific expansion of thymically-derived FOXP3 + HELIOS + Tregs with a demethylated FOXP3 Treg-specific demethylated region. We found that the Treg expansion was strongly associated with markers of recent immune activation, including PD-1, plasma concentrations of IL-2 and the type I interferon biomarker soluble SIGLEC-1. Since the expression of the negative T-cell signaling molecule PTPN22 is increased and a marker of poor prognosis in SLE, we tested the influence of its missense risk allele Trp 620 (rs2476601C>T) on Treg frequency. Trp 620 was reproducibly associated with increased frequencies of thymically-derived Tregs in blood, and increased PD-1 expression on both Tregs and effector T cells (Teffs). Our results support the hypothesis that FOXP3 + Tregs are increased in SLE patients as a consequence of a compensatory mechanism in an attempt to regulate pathogenic autoreactive Teff activity. We suggest that restoration of IL-2-mediated homeostatic regulation of FOXP3 + Tregs by IL-2 administration could prevent disease flares rather than treating at the height of a disease flare. Moreover, stimulation of PD-1 with specific agonists, perhaps in combination with low-dose IL-2, could be an effective therapeutic strategy in autoimmune disease and in other immune disorders., (Copyright © 2019 Ferreira, Castro Dopico, Oliveira, Rainbow, Yang, Trzupek, Todd, McNeill, Steri, Orrù, Fiorillo, Crouch, Pekalski, Cucca, Tree, Vyse, Wicker and Todd.)

Metabolic syndrome (MS) increases cardiovascular risk. The role of thyroid hormone on components of MS is unclear. We analyzed a sample of 4733 euthyroid subjects from SardiNIA study. In female thyrotropin (TSH) was significantly and positively associated with triglycerides (Standardized regression coefficients ( β ) = 0.081, p < 0.001). Free thyroxine (FT4) was positively associated with HDL ( β = 0.056, p < 0.01), systolic blood pressure (SBP) ( β = 0.059, p < 0.001), diastolic blood pressure (DBP) ( β = 0.044, p < 0.01), and fasting glucose ( β = 0.046, p < 0.01). Conversely, FT4 showed a negative association with waist circumference ( β = -0.052, p < 0.001). In TSH was positively associated with triglycerides ( β = 0.111, p = <0.001) and FT4 showed a positive association with DBP ( β = 0.51, p < 0.01). The addition of leptin and adiponectin to the regression models did not substantially change the impact of thyroid hormones on components of MS. Our data suggest that, even within the euthyroid range, excess of truncal adipose tissue is associated with variations in FT4. Leptin and adiponectin exert an additive effect rather than a causal effect. Additional studies should be performed to determine the clinical significance of this finding.

Purpose: Blood vessels of the retina provide an easily-accessible, representative window into the condition of microvasculature. We investigated how retinal vessel structure captured in fundus photographs changes with age, and how this may reflect features related to patient health, including blood pressure., Results: We used two approaches. In the first approach, we segmented the retinal vasculature from fundus photographs and then we correlated 25 parameterized aspects ("traits")-comprising 15 measures of tortuosity, 7 fractal ranges of self-similarity, and 3 measures of junction numbers-with participant age and blood pressure. In the second approach, we examined entire fundus photographs with a set of algorithmic CHARM features. We studied 2,280 Sardinians, ages 20-28, and an U.S. based population from the AREDS study in 1,178 participants, ages 59-84. Three traits (relating to tortuosity, vessel bifurcation number, and vessel endpoint number) showed significant changes with age in both cohorts, and one additional trait (relating to fractal number) showed a correlation in the Sardinian cohort only. When using second approach, we found significant correlations of particular CHARM features with age and blood pressure, which were stronger than those detected when using parameterized traits, reflecting a greater signal from the entire photographs than was captured in the segmented microvasculature., Conclusions: These findings demonstrate that automated quantitative image analysis of fundus images can reveal general measures of patient health status., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Ilya G. Goldberg is employed by Mindshare Medical, Inc. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

The impact of the parental origin of associated alleles in GWAS has been largely ignored. Yet sequence variants could affect traits differently depending on whether they are inherited from the mother or the father, as in imprinted regions, where identical inherited DNA sequences can have different effects based on the parental origin. To explore parent-of-origin effects (POEs), we studied 21 quantitative phenotypes in a large Hutterite pedigree to identify variants with single parent (maternal-only or paternal-only) effects, and then variants with opposite parental effects. Here we show that POEs, which can be opposite in direction, are relatively common in humans, have potentially important clinical effects, and will be missed in traditional GWAS. We identified POEs with 11 phenotypes, most of which are risk factors for cardiovascular disease. Many of the loci identified are characteristic of imprinted regions and are associated with the expression of nearby genes., Competing Interests: The authors declare no competing interests.

Objective: Cytokines release by adipocytes could interact with TSH secretion. We evaluated the relationship between adipocytokines and TSH. We further tested for association of cytokines and thyroid autoimmunity., Methods: We conducted a cross-sectional study in a community-based sample including 5385 individuals (2964 female) with TSH within the reference range. Subjects who reported taking thyroid medications or drugs that alter thyroid function were excluded. TSH, FT4, adiponectin, leptin, antibody against thyroperoxidase and against thyroglobulin were measured. Linear and logistic regression models were used to test for association., Results: Females had higher adiponectin and leptin level and increased frequency of thyroid antibodies. In multiple regression analysis TSH was directly associated with leptin (β = 0.003, p = 0.001) and the presence of circulating antibody against thyroperoxidase (β = 0.315, p < 0.001), but negatively associated with age (β = -0.012, p < 0.001) and FT4 (β = -0.359, p < 0.001). Adiponectin, the presence of antibody against thyroglobulin and smoking habit were not associated with TSH levels (p = 0.223, p = 0.174 and p = 0.788, respectively). Logistic regression analysis revealed that higher adiponectin levels were associated with thyroid autoimmunity., Conclusions: Leptin is positively associated with TSH levels in euthyroid individuals, suggesting an effect of the adipokine on TSH secretion. Our results support the hypothesis that the leptin and pituitary-thyroid axis might interact in the context of energy homeostasis. The effect of adiponectin on thyroid autoimmunity will require more studies., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Thyroid dysfunction is an important public health problem, which affects 10% of the general population and increases the risk of cardiovascular morbidity and mortality. Many aspects of thyroid hormone regulation have only partly been elucidated, including its transport, metabolism, and genetic determinants. Here we report a large meta-analysis of genome-wide association studies for thyroid function and dysfunction, testing 8 million genetic variants in up to 72,167 individuals. One-hundred-and-nine independent genetic variants are associated with these traits. A genetic risk score, calculated to assess their combined effects on clinical end points, shows significant associations with increased risk of both overt (Graves' disease) and subclinical thyroid disease, as well as clinical complications. By functional follow-up on selected signals, we identify a novel thyroid hormone transporter (SLC17A4) and a metabolizing enzyme (AADAT). Together, these results provide new knowledge about thyroid hormone physiology and disease, opening new possibilities for therapeutic targets.

CD4 + regulatory T cells have been intensively studied during aging, but little is still known about age-related changes of other regulatory T cell subsets. It was, therefore, the goal of the present study to analyze CD8 + human leukocyte antigen-antigen D related (HLADR) + T cells in old age, a cell population reported to have suppressive activity and to be connected to specific genetic variants. We demonstrate a strong increase in the number of CD8 + HLADR + T cells with age in a cohort of female Sardinians as well as in elderly male and female persons from Austria. We also show that CD8 + HLADR + T cells lack classical activation molecules, such as CD69 and CD25, but contain increased numbers of checkpoint inhibitory molecules, such as cytotoxic T lymphocyte-associated antigen 4, T cell immunoglobulin and mucin protein-3, LAG-3, and PD-1, when compared with their HLADR - counterparts. They also have the capacity to inhibit the proliferation of autologous peripheral blood mononuclear cells. This suppressive activity is, however, decreased when CD8 + HLADR + T cells from elderly persons are analyzed. In accordance with this finding, CD8 + HLADR + T cells from persons of old age contain lower percentages of checkpoint inhibitory molecules than young controls. We conclude that in spite of high abundance of a CD8 + regulatory T cell subset in old age its expression of checkpoint inhibitory molecules and its suppressive function on a per cell basis are reduced. Reduction of suppressive capacity may support uncontrolled subclinical inflammatory processes referred to as "inflamm-aging."

Background: The study of the gut microbiota (GM) is rapidly moving towards its functional characterization by means of shotgun meta-omics. In this context, there is still no consensus on which microbial functions are consistently and constitutively expressed in the human gut in physiological conditions. Here, we selected a cohort of 15 healthy subjects from a native and highly monitored Sardinian population and analyzed their GMs using shotgun metaproteomics, with the aim of investigating GM functions actually expressed in a healthy human population. In addition, shotgun metagenomics was employed to reveal GM functional potential and to compare metagenome and metaproteome profiles in a combined taxonomic and functional fashion., Results: Metagenomic and metaproteomic data concerning the taxonomic structure of the GM under study were globally comparable. On the contrary, a considerable divergence between genetic potential and functional activity of the human healthy GM was observed, with the metaproteome displaying a higher plasticity, compared to the lower inter-individual variability of metagenome profiles. The taxon-specific contribution to functional activities and metabolic tasks was also examined, giving insights into the peculiar role of several GM members in carbohydrate metabolism (including polysaccharide degradation, glycan transport, glycolysis, and short-chain fatty acid production). Noteworthy, Firmicutes-driven butyrogenesis (mainly due to Faecalibacterium spp.) was shown to be the metabolic activity with the highest expression rate and the lowest inter-individual variability in the study cohort, in line with the previously reported importance of the biosynthesis of this microbial product for the gut homeostasis., Conclusions: Our results provide detailed and taxon-specific information regarding functions and pathways actively working in a healthy GM. The reported discrepancy between expressed functions and functional potential suggests that caution should be used before drawing functional conclusions from metagenomic data, further supporting metaproteomics as a fundamental approach to characterize the human GM metabolic functions and activities.

Genome-wide association studies have previously identified 23 genetic loci associated with circulating fibrinogen concentration. These studies used HapMap imputation and did not examine the X-chromosome. 1000 Genomes imputation provides better coverage of uncommon variants, and includes indels. We conducted a genome-wide association analysis of 34 studies imputed to the 1000 Genomes Project reference panel and including ∼120 000 participants of European ancestry (95 806 participants with data on the X-chromosome). Approximately 10.7 million single-nucleotide polymorphisms and 1.2 million indels were examined. We identified 41 genome-wide significant fibrinogen loci; of which, 18 were newly identified. There were no genome-wide significant signals on the X-chromosome. The lead variants of five significant loci were indels. We further identified six additional independent signals, including three rare variants, at two previously characterized loci: FGB and IRF1. Together the 41 loci explain 3% of the variance in plasma fibrinogen concentration., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A C1858T (R620W) variation in the PTPN22 gene encoding the tyrosine phosphatase LYP is a major risk factor for human autoimmunity. LYP is a known negative regulator of signaling through the T cell receptor (TCR), and murine Ptpn22 plays a role in thymic selection. However, the mechanism of action of the R620W variant in autoimmunity remains unclear. One model holds that LYP-W620 is a gain-of-function phosphatase that causes alterations in thymic negative selection and/or thymic output of regulatory T cells (Treg) through inhibition of thymic TCR signaling. To test this model, we generated mice in which the human LYP-W620 variant or its phosphatase-inactive mutant are expressed in developing thymocytes under control of the proximal Lck promoter. We found that LYP-W620 expression results in diminished thymocyte TCR signaling, thus modeling a "gain-of-function" of LYP at the signaling level. However, LYP-W620 transgenic mice display no alterations of thymic negative selection and no anomalies in thymic output of CD4(+)Foxp3(+) Treg were detected in these mice. Lck promoter-directed expression of the human transgene also causes no alteration in thymic repertoire or increase in disease severity in a model of rheumatoid arthritis, which depends on skewed thymic selection of CD4(+) T cells. Our data suggest that a gain-of-function of LYP is unlikely to increase risk of autoimmunity through alterations of thymic selection and that LYP likely acts in the periphery perhaps selectively in regulatory T cells or in another cell type to increase risk of autoimmunity.

The complex network of specialized cells and molecules in the immune system has evolved to defend against pathogens, but inadvertent immune system attacks on "self" result in autoimmune disease. Both genetic regulation of immune cell levels and their relationships with autoimmunity are largely undetermined. Here, we report genetic contributions to quantitative levels of 95 cell types encompassing 272 immune traits, in a cohort of 1,629 individuals from four clustered Sardinian villages. We first estimated trait heritability, showing that it can be substantial, accounting for up to 87% of the variance (mean 41%). Next, by assessing ∼8.2 million variants that we identified and confirmed in an extended set of 2,870 individuals, 23 independent variants at 13 loci associated with at least one trait. Notably, variants at three loci (HLA, IL2RA, and SH2B3/ATXN2) overlap with known autoimmune disease associations. These results connect specific cellular phenotypes to specific genetic variants, helping to explicate their involvement in disease., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A missense C1858T single nucleotide polymorphism in the PTPN22 gene recently emerged as a major risk factor for human autoimmunity. PTPN22 encodes the lymphoid tyrosine phosphatase (LYP), which forms a complex with the kinase Csk and is a critical negative regulator of signaling through the T cell receptor. The C1858T single nucleotide polymorphism results in the LYP-R620W variation within the LYP-Csk interaction motif. LYP-W620 exhibits a greatly reduced interaction with Csk and is a gain-of-function inhibitor of signaling. Here we show that LYP constitutively interacts with its substrate Lck in a Csk-dependent manner. T cell receptor-induced phosphorylation of LYP by Lck on an inhibitory tyrosine residue releases tonic inhibition of signaling by LYP. The R620W variation disrupts the interaction between Lck and LYP, leading to reduced phosphorylation of LYP, which ultimately contributes to gain-of-function inhibition of T cell signaling.

The AIRE protein plays a remarkable role as a regulator of central tolerance by controlling the promiscuous expression of tissue-specific antigens in thymic medullary epithelial cells. Defects in the AIRE gene cause the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, a rare disease frequent in Iranian Jews, Finns, and Sardinian population. To this day, the precise function of the AIRE protein in regulating transcription and its interacting proteins has yet to be entirely clarified. The knowledge of novel AIRE interactors and their precise role will improve our knowledge of its biological activity and address some of the foremost autoimmunity-related questions. In this study, we have used a yeast two-hybrid system to identify AIRE-interacting proteins. This approach led us to the discovery of a new AIRE-interacting protein called DAXX. The protein is known to be a multifunctional adaptor with functions both in apoptosis and in transcription regulation pathways. The interaction between AIRE and DAXX has been validated by in vivo coimmunoprecipitation analysis and colocalization study in mammalian cells. The interaction has been further confirmed by showing in transactivation assays that DAXX exerts a strong repressive role on the transcriptional activity of AIRE.

The sperm antigen fertilin alpha/beta and the integrin complex alpha6beta1 present on the oolemma are two of the most promising candidates to mediate gamete interaction. During growth, the plasma membrane of both hamster and mouse zona-free oocytes acquires the capacity to fuse with acrosome-reacted sperm when oocytes reach the size of 25-30 microm in diameter, suggesting changes in the membrane molecular composition. The present study has two aims: to determine the timing of (1) gene expression of alpha6 and beta1 integrins and (2) localization of these integrin subunits on the plasma membrane in primordial germ cells and in oocytes during oogenesis. We found that both alpha6 and beta1 genes are expressed in female germ cells during all the stages of development analyzed, from 10.5 to 18.5 d.p. c., during oocyte growth, and in ovulated eggs. The alternatively spliced isoform alpha6B is expressed from 10.5 d.p.c., whereas alpha6A begins to be expressed at 12.5 d.p.c., suggesting a different role for the two variants. In situ immunodetection of alpha6 or beta1 shows a ring of fluorescence on the female germ cell plasma membrane for both integrins at 10.5 d.p.c., then the fluorescent signal becomes undetectable at 12.5 d.p.c. to reappear again, this time with a patchy distribution, at 18.5 d.p.c. This pattern of localization is maintained in oocytes isolated from newborn individuals and only when oocytes during growth reach the size of about 25-30 microm in diameter does the fluorescence become homogenous all around the whole oocyte surface. These data, although not conclusive, support the hypothesis of an involvement of alpha6 and beta1 integrins in sperm-egg fusion., (Copyright 1998 Academic Press.)