Jocelyne Demengeot

This article is dedicated to elucidating and showcasing the concept of a unified Core Facility for laboratory animals within a research institute specialized in basic biology and biomedical research. In many research centres, animal facilities operate as autonomous entities. Here, we discuss that the centralization of all animal model units within a consolidated organizational framework offers a multitude of benefits in terms of communication with a variety of institutional stakeholders, including the Direction Board, Operational Logistics (Maintenance, Lab Operations and Safety Units), Procurement and Accounting Offices, Research Funding Affairs, Institutional Communication, and IT Units. This integrated approach facilitates the implementation of consistent policies and service pricing strategies. Moreover, it promotes staff flexibility across species, allows for responsiveness to evolving research dynamics, emergence of new scientific areas and infrastructure challenges. This concept also inspires technical advancement within the animal facilities, supports training in Laboratory Animal Science, stimulates the standardization of animal welfare practices, and instils a culture of care transversal to all animal models, ultimately enhancing overall animal welfare. This strategy facilitated the integration of non-vertebrate animals and plant models into the Core Facility. Despite significant differences from vertebrate models, this expansion presented advantages, such as incorporation of specialized staff into a larger organizational structure, offering them new opportunities for skill development and enhancing the overall flexibility of the Core Facility’s operations.

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten–eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.

Background: Early-onset Type 1 diabetes (EOT1D) is considered a disease subtype with distinctive immunological and clinical features. While both Human Leukocyte Antigen (HLA) and non-HLA variants contribute to age at T1D diagnosis, detailed analyses of EOT1D-specific genetic determinants are still lacking. This study scrutinized the involvement of the HLA class II locus in EOT1D genetic control. Methods: We conducted genetic association and regularized logistic regression analyses to evaluate genotypic, haplotypic and allelic variants in DRB1 , DQA1 and DQB1 genes in children with EOT1D (diagnosed at ≤5 years of age; n=97), individuals with later-onset disease (LaOT1D; diagnosed 8-30 years of age; n=96) and nondiabetic control subjects (n=169), in the Portuguese population. Findings: Analysis of EOT1D and LaOT1D unrelated patients in comparison with controls, revealed the rare DRB1*04:08-DQ8 haplotype is specifically associated with EOT1D (corrected p-value=1.4×10 ^-5 ) and represents the major discriminative HLA class II genetic factor. Allelic association further indicated the DRB1*04:08 allele is a distinctive EOT1D susceptibility factor (corrected p-value=7.0×10 ^-7 ). Conversely, the classical T1D risk allele DRB1*04:05 was absent in EOT1D children while was associated with LaOT1D (corrected p-value=1.4×10 ^-2 ). Interpretation: This study uncovered that EOT1D holds a distinctive spectrum of HLA class II susceptibility loci , which includes risk factors overlapping with LaOT1D and discriminative genetic configurations. These findings warrant replication studies in larger multicentric settings and may impact target screening strategies and follow-up of young children with high T1D genetic risk. Funding: European Foundation for the Study of Diabetes, Maratona da Saúde and Fundação para a Ciência e a Tecnologia.

Background: Infection due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with clinical features of diverse severity. Few studies investigated the severity and mortality predictors of coronavirus disease 2019 (COVID-19) in Africa. Herein, we investigated the clinical features of severity and mortality among COVID-19 patients in Luanda, Angola. Methods: This multicenter cohort study involved 101 COVID-19 patients, between December 2020 and April 2021, with clinical and laboratory data collected. Analysis was done using independent-sample t-tests and Chi-square tests. The results were deemed significant when p < 0.05. Results: The mean age of patients was 51 years (ranging from 18 to 80 years) and 60.4% were male. Fever (46%), cough (47%), gastrointestinal symptoms (26.7%), and asthenia (26.7%), were the most common symptoms. About 64.4% of the patients presented coexistent disorders, including hypertension (42%), diabetes (17%), and chronic renal diseases (6%). About 23% were non-severe, 77% were severe, and 10% died during hospitalization. Variations in the concentration of neutrophil, urea, creatinine, c-reactive protein, sodium, creatine kinase, and chloride were independently associated with severity and/or mortality (p < 0.05). Conclusion: Several factors contributed to the severity and mortality among COVID-19 patients in Angola. Further studies related to clinical features should be carried out to help clinical decision-making and follow-up of COVID-19 patients in Angola.

Cellular adoptive transfer and mixed bone marrow chimera are cornerstone experimental tools for immuno-biology. Here we describe protocols for adoptive transfer and bone marrow chimera to address the effect of a specific mutation on T regulatory cell (Treg) function and differentiation, respectively. Treg function can be quantitatively measured by analyzing the expansion of conventional CD4 T cells and their differentiation into helper cells. The quantitative measure of Treg differentiation is addressed by analyzing the number and phenotype of Foxp3-expressing cells. The use of congenic markers is instrumental for these approaches. Key words Regulatory T cells Bone Marrow Differentiation Adoptive transfer Function Mice Proliferation Chimera Homeostasis Disease

While mRNA vaccines are administrated worldwide in an effort to contain the COVID-19 pandemic, the heterogeneity of the humoral immune response they induce at the population scale remains unclear. Here, in a prospective, longitudinal, cohort-study, including 1245 hospital care workers and 146 nursing home residents scheduled for BNT162b2 vaccination, together covering adult ages from 19 to 99 years, we analyse seroconversion to SARS-CoV-2 spike protein and amount of spike-specific IgG, IgM and IgA before vaccination, and 3-5 weeks after each dose. We show that immunogenicity after a single vaccine dose is biased to IgG, heterogeneous and reduced with increasing age. The second vaccine dose normalizes IgG seroconversion in all age strata. These findings indicate two dose mRNA vaccines is required to reach population scale humoral immunity. The results advocate for the interval between the two doses not to be extended, and for serological monitoring of elderly and immunosuppressed vaccinees.

Background: Patients on hemodialysis (HD) are at higher risk for COVID-19, overall are poor responders to vaccines, and were prioritized in the Portuguese vaccination campaign.Objective: This work aimed at evaluating in HD patients the immunogenicity of BTN162b2 after the two doses induction phase, the persistence of specific antibodies along time, and factors predicting these outcomes.Methods: We performed a prospective, 6-month long longitudinal cohort analysis of 156 HD patients scheduled to receive BTN162b2. ELISA quantified anti-spike IgG, IgM, and IgA levels in sera were collected every 3 weeks during the induction phase (t0 before vaccine; t1, d21 post first dose; and t2 d21 post second dose), and every 3–4 months during the waning phase (t3, d140, and t4, d180 post first dose). The age-matched control cohort was similarly analyzed from t0 to t2.Results: Upon exclusion of participants identified as previously exposed to SARS-CoV-2, seroconversion at t1 was lower in patients than controls (29 and 50%, respectively, p = 0.0014), while the second vaccine dose served as a boost in both cohorts (91 and 95% positivity, respectively, at t2, p = 0.2463). Lower response in patients than controls at t1 was a singularity of the participants ≤ 70 years (p = 2.01 × 10⁻⁰⁵), associated with immunosuppressive therapies (p = 0.013), but not with lack of responsiveness to hepatitis B. Anti-spike IgG, IgM, and IgA levels decreased at t3, with IgG levels further waning at t4 and resulting in >30% seronegativity. Anti-spike IgG levels at t1 and t4 were correlated (ρ = 0.65, p < 2.2 × 10⁻¹⁶).Conclusions: While most HD patients seroconvert upon 2 doses of BNT162b2 vaccination, anti-spike antibodies levels wane over the following 4 months, leading to early seroreversion in a sizeable fraction of the patients. These findings warrant close monitoring of COVID-19 infection in vaccinated HD patients, and advocate for further studies following reinforced vaccination schedules.

ETV6-RUNX1 and RUNX1 directly promote RAG1 expression. ETV6-RUNX1 and RUNX1 preferentially bind to the −1200 bp enhancer of RAG1 and the −80 bp promoter of RAG1 gene respectively, and compete for these bindings. ETV6-RUNX1 and RUNX1 induce an excessive RAG recombinase activity. ETV6-RUNX1 participates directly in two events of the multi-hit ALL leukemogenesis: as an initiating event and as an activator of RAG1 expression.

Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, imposing the need to monitor SARS-CoV2 evolution and dynamics in the population. Determining signatures in SARS-CoV-2 that render the virus resistant to neutralizing antibodies is critical. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations in the spike protein, including all defining mutations in the variants of concern, to identify the effect of single and synergic amino acid substitutions in promoting immune escape. We confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations.In silicoanalysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and with mutations able to increase binding to ACE2, such as N501Y. Our analysis of synergic mutations provides a signature for hotspots for immune evasion and for targets of therapies, vaccines and diagnostics.

Background and objectives Patients undergoing hemodialysis are at higher risk of developing severe complications upon SARS-CoV-2 infection and were prioritized in the Portuguese vaccination campaign. Immunogenicity of COVID-19 vaccines in hemodialyzed patients was not addressed by the phase 3 clinical trials leading to their emergency approval. Design, setting, participants, and measurements We performed a prospective, longitudinal, cohort analysis of 156 hemodialyzed patients and 143 age-matched controls scheduled for BTN162b2 vaccine. Excluded from analysis were five patients previously diagnosed with SARS-CoV-2, three sero-positive for anti-SARS-CoV-2 N, two dropouts and two deaths. ELISA was used to quantify anti-full-length Spike IgG, IgM and IgA levels in sera collected at day of the first vaccine dose (t0); 3 weeks later (day of the second dose, t1); and 3 weeks after the second inoculation (t2). Results Seroconversion after the first vaccine dose (t1) was remarkably low in patients, with positivity for anti-spike IgG, IgM and IgA antibodies of 29.4%, 12% and 41%, respectively. The second vaccine dose raised seroconversion to 90.9% and 83.9% for IgG and IgA, respectively, while IgM positivity remained unchanged. At t1 the anti-Spike IgG level was significantly lower in patients with ages below 70 years when compared to age-matched controls, showing a profile similar to aged individuals (above 70 years). Immunosuppression was associated with lower antibody responses along the vaccine schedule (p=0.005 at t1; p=0.008 at t2). Noteworthy, previous unresponsiveness to hepatitis B vaccination (75/129, 58% of patients negative for anti-HBs antibodies) did not correlate with humoral unresponsiveness to BTN162b2. Other clinical and laboratory parameters had marginal correlations with response to vaccination. Conclusions The large majority of hemodialyzed patients showed IgG seroconversion upon BNT162b2 mRNA vaccination but a sizable proportion of patients presented poor responses. These results support further investigation into the relationship between vaccination, serologic response and host protection.