Biology subjects

Clemente, F.

Publications and source records attributed to Clemente, F..

4 recordsLinked to original sources

bioRxiv2026

A generalizable normalization framework to decouple protocol and instrument effects: Application to high-sensitivity proteomics multicentric study (PME13)

Multicenter studies are essential for benchmarking analytical workflows, yet their interpretation is often confounded by the combined effects of experimental protocols and instrumentation. To address this challenge, we introduce a simple normalization-based analytical framework, the recovery metric ({rho}), designed to decouple protocol driven effects from instrument dependent variability. We applied this framework to the 13th Proteomics Multicentric Experiment (PME13), a large multicentric proteomics dataset generated across 27 laboratories using high sensitivity workflows and varying sample preparation protocols. By leveraging a common digested reference sample, {rho} enables direct cross-comparison of all datasets on a unified scale, effectively minimizing instrument-related biases. Using this approach, we demonstrate that apparent instrument dependent trends are largely removed when evaluated through {rho}, revealing consistent protocol driven effects across laboratories. Statistical modeling identified key variables influencing {rho}, including sample input amount, reduction and alkylation, and the use of n-dodecyl-{beta}-D-maltoside (DDM). While DDM was associated with improved {rho}, reduction and alkylation and additional handling steps led to reduced performance, particularly at low input levels. We further highlight practical considerations for the application of ratio based normalization, including the occurrence of values exceeding theoretical bounds, which reflect deviations from underlying assumptions and require appropriate filtering. Overall, this work establishes a generalizable analytical strategy for disentangling confounding factors in multicentric datasets and provides practical guidelines for optimizing high sensitivity proteomics (HSP) workflows. The proposed framework is broadly applicable to other analytical fields where cross laboratory comparability is required.

bioinformatics↗

bioRxiv2024

From high protection to lethal effect: diverse outcomes of immunization against invasive candidiasis with different Candida albicans extracellular vesicles

Extracellular vesicles (EVs) from Candida albicans can elicit immune responses, positioning them as promising acellular vaccine candidates. We characterized EVs from an avirulent C. albicans cell wall mutant (ecm33{Delta}) and evaluated their protective potential against invasive candidiasis. EVs from the yeast (YEVs) and hyphal (HEVs) forms of the SC5314 wild-type strain were also tested, yielding high survival rates with SC5314 YEVs (91%) and ecm33 YEVs immunization (64%). Surprisingly, HEV immunization showed a dual effect, resulting in 36% protection but also causing premature death in some mice. Proteomic analyses revealed distinct profiles among the top 100 proteins in the different EVs which may explain these effects: a shared core of 50 immunogenic proteins such as Pgk1, Cdc19, and Fba1; unique, relevant immunogenic proteins in SC5314 YEVs, and proteins linked to pathogenesis, like Ece1 in SC5314 HEVs. Sera from SC5314 YEVs-immunized mice showed the highest IgG2a titers and moderate IL-17, IFN-{gamma}, and TNF- levels, indicating the importance of both humoral and cellular responses for protection. These findings highlight the distinct immunogenic properties of C. albicans EVs, suggesting their potential in acellular vaccine development while emphasizing the need to carefully evaluate pathogenic risks associated with certain EVs.

microbiology↗

bioRxiv2024

Autosomal Dominant-Hyper-IgE Syndrome patients contain pre-Th17-cells that are activated by opportunistic pathogens to produce IL-10

BackgroundAutosomal Dominant-Hyper-IgE Syndrome (AD-HIES) is caused by dominant-negative (DN) STAT3 mutations and characterized by high IgE levels, a lack of Th17-cells and recurrent infections with extracellular pathogens. We previously identified an enigmatic population of IL-10 producing CCR6+B-helper T-cells and investigated here their relationship to Th17-cells and STAT3 signalling requirements. MethodsHuman blood lymphocytes were analysed by multiparametric flow cytometry in healthy donors and AD-HIES patients. Analysis was performed by conventional gating or with bioinformatic tools. FACS-purified T-cell subsets were activated in vitro and Th17 differentiation assessed. T-cell antigen specificities were assessed by activation with heat-killed pathogens or antigenic peptide pools. B helper capacities were determined according to antibody secretion in B-T co-cultures by ELISA. ResultsCCR6+Th-cells that lacked subset-defining differentiation markers were mostly non-polarised central memory T-cells (TCM) that produced IL-10 and expressed ROR{gamma}t. They were pre-committed to a Th17 fate, since TCR stimulation in the absence of polarising cytokines induced efficient Th17 differentiation. The latter was promoted by an autocrine loop of STAT3-activating cytokines. CCR6+Th-cells were reduced in patients with DN-STAT3 mutations but contained activated CCR6+TCM that produced IL-10 and responded vigorously to AD-HIES-associated pathogens. These residual CCR6+Th-cells provided B-cell help for IgG and IgE production. ConclusionsTh17 differentiation in AD-HIES patients was not completely impaired but arrested at an intermediate stage of IL-10 producing "pre-Th17"-cells. Surprisingly, DN-STAT3 mutations did not inhibit IL-10 production by CD4+T-cells. Pre-Th17-cells were activated by AD-HIES-associated pathogens and possessed B-helper functions, suggesting that they are not protective but promote aberrant IgE production.

immunology↗

bioRxiv2024

Regulatory T-cells in multiple sclerosis are activated by Epstein-Barr Virus and produce IL-10 in the central nervous system

Regulatory T-cells (Tregs) maintain immune homeostasis, but antigens activating adaptive Tregs in human pathologies are ill-defined. EOMES+type-1 regulatory (Tr1)-like T-cells had a dysregulated homeostasis in multiple sclerosis (MS), which was related to their activation in the central nervous system (CNS). EOMES+Tr1-like cells were enriched and clonally expanded in patients cerebrospinal fluid (CSF) and were the major IL-10-producing T-cell subset in the CNS. Regulatory T-cells from PwMS produced IL-10 and IFN-{gamma} with antigens derived from Epstein-Barr Virus (EBV), but not from myelin. EOMES+Tr1-like cells responded selectively to the latency-associated antigen EBNA1, whereas FOXP3+Tregs and Th1-cells responded also to lytic EBV antigens. EBNA1-specific EOMES+Tr1-like cells were present in patients carrying the HLA-DRB1*15:01 risk allele, were associated with anti-EBNA1 IgG and disappeared upon therapeutic B-cell depletion. IL-10+EOMES+Tr1-like were present in MS brain lesions, and some were in the vicinity of EBV-infected B-cells and CD8+T-cells. Notably, EOMES+Tr1-like cells suppressed CD8+T-cell activation by EBV-infected B-cells. We propose that the insufficient protective functions of Tregs in MS are due to their aberrant anti-viral specificities that promote immune escape of a disease-associated virus.

immunology↗