Protein Profiles Research Articles

Lung proteomic and metabolomic changes induced by carbon black nanoparticles and high humidity in a mouse asthma model.

Allergic asthma is a significant international concern in respiratory health, which can be exacerbated by the increasing levels of non-allergenic pollutants. This rise in airborne pollutants is a primary driver behind the growing prevalence of asthma, posing a health emergency. Additionally, climatic risk factors can contribute to the onset and progression of asthma. Understanding the complex interplay between pollution, climate, and asthma induction is crucial to elucidate how environmental changes intensify asthma. In this study, we investigated the proteomic and metabolomic changes in the lungs of a mouse asthma model following co-exposure to carbon black nanoparticles and high humidity, which represent airborne and climatic factors, respectively. An asthma model was established using ovalbumin, and mice were intratracheally instilled with 15 or 30μg/kg of carbon black and simultaneously exposed to either 70% or 90% relative humidity. Protein and metabolite profiles from the lung were used to analyze the most significantly changed clusters, and potential biomarkers and enriched pathways were identified to dissect the adverse effects of the two risk factors. The lung proteome and metabolome are significantly altered by the co-exposure, with the effects modulated by carbon black concentration and humidity level. This study proposes 10 proteins and 18 metabolites as candidate biomarkers. The significantly enriched KEGG pathways include one protein pathway (primary immunodeficiency) and six metabolic pathways (ABC transporters, nucleotide metabolism, Parkinson's disease, purine metabolism, choline metabolism in cancer, and biosynthesis of cofactors). A joint proteomic and metabolomic analysis identifies five common pathways across both omics, namely, ABC transporters, central carbon metabolism in cancer, EGFR tyrosine kinase inhibitor resistance, glioma, and NF-kappa B signaling pathway, disturbed by the co-exposure. We provide a multi-omic basis for the health risk assessment and management of co-exposures to environmental risk factors.

Styryl dyes for viscosity measurement and detection of pathological processes in mitochondria of living cells using fluorescence lifetime imaging microscopy, a critical study

In the present work, two molecular rotors based on styryl dyes were synthesized and investigated. Both dyes showed significant sensitivity to the viscosity of the medium. Due to their significant positive charge, they efficiently accumulated in mitochondria of living cells. Binding to mitochondria resulted in increased fluorescence intensity and fluorescence lifetime of the dyes, allowing them to be visualized without washing off the unbound dye. We have also shown that the fluorescence lifetime of the studied molecular rotors is determined not only by the viscosity of the medium, but also by interactions with cell components such as proteins and nucleic acids. The present work clearly shows that these interactions do not allow a reliable estimation of viscosity in cell organelles using the synthesized dyes. This compromises the results of previous viscosity studies using molecular rotors at least of the styryl type. Nevertheless, induction of cell apoptosis by benzylviologen led to an increase in brightness and fluorescence lifetime of the dyes, which can be caused by both changes in viscosity and changes in the expression profile of proteins localized in mitochondria and interacting with the dyes. Thus, the obtained styryl dyes, like previously published homologous compounds, cannot be used for viscosity measurements, but can be used for identification of pathological states of the cell.

Triticain alpha represents the major active papain-like cysteine protease in naturally senescing and ozone-treated leaves of wheat

Current background tropospheric ozone (O3) concentrations have significant adverse effects on wheat. O3 generally induces oxidative damages and premature leaf senescence leading to important yield losses. As leaf protein degradation and recycling is involved in both maintaining cell longevity during abiotic stresses and performing efficient nitrogen remobilization during senescence, we aimed to identify proteases involved in acidic endoproteolytic activities during natural and O3-induced leaf senescence in wheat. Field-grown plants of two winter wheat cultivars were exposed to ambient and semi-controlled chronic O3 concentrations, from pre-anthesis to grain harvest. Yield parameters were impacted by the most elevated O3 exposure for both cultivars. At the cellular level, our analysis revealed that both natural leaf senescence and O3 treatments induced a stimulation of acidic (pH 5.5) endoproteolytic activities, mostly due to papain-like cysteine proteases (PLCPs). Identification of active PLCPs using activity-based protein profiling (ABPP) revealed that Triticain α was the major active PLCP in senescing flag leaves and the only PLCP whose abundance increased with O3 stress, a result of positive transcriptional regulation. Our study provides novel insight into the implication of PLCP-mediated proteolysis in O3 sensitivity in a major crop.

Spatially resolved protein profiling in pancreatic adenosquamous carcinoma.

760 Background: Pancreatic adenosquamous carcinoma (PASC) is a rare histopathological type of pancreatic cancer that is more aggressive than pancreatic ductal adenocarcinoma (PDAC). Although PASC has a unique tumor biology and poorer prognosis, there are no guidelines for differentially treating PASC patients, and its rarity makes it significantly understudied. We used Nanostring GeoMX digital spatial profiling (DSP) to provide a targeted proteomic characterization of key tumor-promoting proteins in PASC and PDAC. Methods: FFPE tissue from PASC (n=10 primary, n= 4 met) and PDAC (n=21 primary, n=5 met) cases were analyzed using the DSP platform. Sections were stained with an 82-protein cocktail of antibodies conjugated to UV-photocleavable DNA barcodes. A pan-cytokeratin morphology marker was used to select tumor regions of interest (ROI) and inferred surrounding stromal (i.e., pan-CK negative) ROIs. UV excitation of the defined ROIs was used to release the DNA barcodes and were quantified on the NanoString nCounter system. For PASC, squamous enriched ROI were inferred from corresponding p40 IHC. Corresponding clinical and mutational status for all cases were abstracted from medical records. Results: TP53 (PDAC, 80%; PASC, 83%) and KRAS (PDAC, 76%; PASC, 100%) were the most prevalent mutations . In comparing the tumor ROIs of PASC and PDAC (all cases), DSP-assessed global expression and activation of EGFR-MAPK signaling is significantly increased in PASC, whereas PI3K-AKT signaling is significantly decreased. Compared to PDAC, EGFR expression and phosphorylation (p-EGFR), as well as p-MEK1, are significantly increased in PASC. PASC showed significantly lower levels of PLCG1 and INPP4B, while the majority of other AKT signaling effectors were unchanged. For DNA damage response signaling, PASC showed significantly higher levels of p-ATM, p-CHK2, and p21, but not p-ATR. Likewise, expression of oncoproteins c-MYC and BCL6 were significantly increased in PASC (vs. PDAC). Conclusions: Given that the normal pancreas lacks squamous cells, their histological presence is a uniquely aberrant feature that can be used to guide deep spatially-resolved interrogation of complex cellular and molecular programs. Our preliminary findings show that regions of squamous cell differentiation within PASC have intrinsically higher levels of active EGFR signaling. With emerging RAS-targeted agents poised to alter the treatment landscape, high EGFR signaling in PASC suggest a possible compensatory resistance mechanism that may require additional targeting. Likewise, reliance on ATM signaling highlights this as preferential target for DDR-directed therapies. Building on these results with additional multiomic assays will inform strategies for improving the management of patients with PASC.

ИССЛЕДОВАНИЕ ВЛИЯНИЯ БЕЛКОВОГО ПРОФИЛЯ НА КРИСТАЛЛООБРАЗОВАНИЕ В МОДЕЛЬНЫХ МОЛОЧНЫХ СИСТЕМАХ С ПРОМЕЖУТОЧНОЙ ВЛАЖНОСТЬЮ

The purpose of research is to study the influence of the protein profile of model dairy systems with intermediate moisture content on the process of crystal formation of milk sugar (lactose). Research objectives: selection of formulation components for compiling a protein profile and study of its influence on the process of formation and growth of lactose crystals in model systems during storage. The objects of research were model systems with targeted adjustment of pH values in each of them, imitating skimmed condensed milk with sugar, in which the complete or partial replacement of the protein phase with whey proteins was carried out through the use of dry whey protein concentrate, dry hydrolyzed whey protein (obtained in native or denaturing conditions), dry cheese whey. Studies of microstructural changes in consistency were carried out using the method for determining the size of lactose crystals according to GOST 29245-91. It has been established that a change in the protein profile towards an increase in the content of native and transformed whey proteins in model systems led to a slowdown in the process of lactose crystal formation. At the same time, hydrolyzed whey proteins had a higher degree of crystal growth restriction. The complete replacement of the protein phase with dry whey protein concentrate and dry cheese whey contributed to the intensive growth of crystals at their initial minimum average size (relative to other samples). However, after 14 days of storage, the average crystal size in this model system was comparable to the rest of the samples (including the control), except for the sample using hydrolyzed whey proteins. Regularities of the influence of the active acidity of the medium on the size of the crystals were also revealed. A decrease in pH from 6.2 to 5.6 leads to the formation of lactose crystals of a smaller average size by 1.9–3.3 times.

CSF cytokine, chemokine and injury biomarker profile of glial fibrillary acidic protein (GFAP) autoimmunity.

Defining the CSF cytokine/chemokine and injury biomarker signature of glial fibrillary acidic protein (GFAP) autoimmunity can inform immunopathogenesis. CSF GFAP-IgG-positive samples (N = 98) were tested for 17 cytokines/chemokines, neurofilament light chain (NfL), and GFAP (ELLA, Bio-Techne). Controls included non-inflammatory (N = 42), AQP4-IgG-positive (N = 83), CNS infections (N = 13), and neurosarcoidosis (N = 32). IL5, IL6, IL10, IL8/CXCL8, CXCL9, CXCL10, CXCL13, BAFF, GM-CSF, IFN-gamma, and TNF-alpha concentrations were higher compared to non-inflammatory controls (P < 0.01). GFAP concentrations were similar to those of AQP4-IgG-positive patients; NfL was higher (P < 0.001) and correlated with MRI changes and outcomes. CSF cytokine/chemokine findings in GFAP autoimmunity correlate with histopathology; GFAP and NfL hold promise as disease biomarkers.

Proteomic analysis of Trichoderma harzianum secretome and their role in the biosynthesis of zinc/iron oxide nanoparticles

The fungal green synthesis of nanoparticles (NPs) has gained great interest since it is a cost-effective and easy handling method. The process is simple because fungi secrete metabolites and proteins capable of reducing metal salts in aqueous solution, however the mechanism remains largely unknown. The aim of this study was to analyze the secretome of a Trichoderma harzianum strain during the mycobiosynthesis process of zinc and iron nanoparticles. Different profiles of proteins secreted by the fungus grown in the culture media or in the aqueous filtrate were observed through SDS‒PAGE and LC‒MS/MS analysis identifying 99 and 304 proteins, respectively. Particularly, in the aqueous filtrate proteins of metabolic processes and stress response mainly oxidoreductases, were identified. Successfully, ZnO and FeO NPs were synthesized and characterized by transmission electron microscopy, energy dispersive X-ray spectroscopy, dynamic light scattering, thermogravimetric, and FTIR analysis. FTIR revealed organic compounds in nanoparticles acting as probably capping agents. This research is the first report in which a proteomic analysis identifies multiple enzymes involved in the biogenic process of NP biosynthesis from T. harzianum, and its role is clearly demonstrated by the formation of zincite and magnetite nanoparticles.

Blastocystis ST1: Protein Profile and Specific Serum Immunoglobulin in Irritable Bowel Syndrome (IBS) Patients.

Blastocystis sp. is a common enteric human parasite, which recently has been linked to gastrointestinal disorders i.e. Irritable Bowel Syndrome (IBS) and symptomatic patients (non IBS). Analyzing antibodies level in these patients could help in differential diagnosis. The current study aimed to identify the protein profile of the Blastocystis ST1 (Syrian strain: OR537347) lysates and to investigate the differences in IgG serum immunoglobulin between patients with IBS and non IBS. Twenty two IBS (Rome III) and nineteen patients suffering from different gastrointestinal disorders (non IBS), positive for Blastocystis were enrolled in this study. SDS-PAGE was used to identify the protein profile of the Blastocystis ST1 lysates and immunoblotting using sera from patients was used for reactivity compared to known Blastocystis protein targets. The crude protein profile of Blastocystis ST1 showed 24 protein bands ranged between 10 and 130kDa. Western blot demonstrated that the proteins (27-29);32;(39-42);(50-51) kDa had similar immunogenicity characteristic in IBS and non IBS patients while the proteins (60-95kDa) only interacted with IBS patients' sera. Our results highlighted the importance of Blastocystis proteins 60-95kDa (probably a metalloproteases) in IBS patients compared to non IBS, suggesting that these metalloproteases may be important Blastocystis antigens and can be used as a serologic test tool or as a biomarker for differential diagnosis.

ChIP-mini: a low-input ChIP-exo protocol for elucidating DNA-binding protein dynamics in intracellular pathogens.

Genome-wide identification of binding profiles for DNA-binding proteins from the limited number of intracellular pathogens in infection studies is crucial for understanding virulence and cellular processes but remains challenging, as the current ChIP-exo is designed for high-input bacterial cells (>1010). Here, we developed an optimized ChIP-mini method, a low-input ChIP-exo utilizing a 5,000-fold reduced number of initial bacterial cells and an analysis pipeline, to identify genome-wide binding dynamics of DNA-binding proteins in host-infected pathogens. Applying ChIP-mini to intracellular Salmonella Typhimurium, we identified 642 and 1,837 binding sites of H-NS and RpoD, respectively, elucidating changes in their binding position and binding intensity during infection. Post-infection, we observed 21 significant reductions in H-NS binding at intergenic regions, exposing the promoter region of virulence genes, such as those in Salmonella pathogenicity islands-2, 3 and effectors. Furthermore, we revealed the crucial phenomenon that novel and significantly increased RpoD bindings were found within regions exhibiting diminished H-NS binding, thereby facilitating substantial upregulation of virulence genes. These findings markedly enhance our understanding of how H-NS and RpoD simultaneously coordinate the transcription initiation of virulence genes within macrophages. Collectively, this work demonstrates a broadly adaptable tool that will enable the elucidation of DNA-binding protein dynamics in diverse intracellular pathogens during infection.

Ailanthone induces triple-negative breast cancer cells death involving the inhibition of OTUB1-mediated ERRα deubiquitylation.

Triple-negative breast cancer (TNBC) remains the most aggressive subtype of breast cancer, and effective therapeutic strategies are needed. Estrogen-related receptor alpha (ERRα) is considered a promising target for managing TNBC. Here, we aimed to screen natural products to find downregulator of ERRα and elucidate its mechanism of action. TNBC cells (MDA-MB-231, MDA-MB-468, MDA-MB-453, and BT-549) were used for in vitro studies, and a subcutaneous MDA-MB-231 tumor model was created for in vivo studies. Immunofluorescence assessed protein distribution, while competitive activity-based protein profiling identified potential target proteins. Co-immunoprecipitation detected protein interactions and modifications, and a luciferase reporter assay evaluated ERRα transcriptional activity. The natural product Ailanthone (AIL) effectively induced cell death in TNBC cells by reducing the protein level of ERRα. The mechanism of action involved AIL promoting the degradation of ERRα through the ubiquitin-proteasome system, consequently reducing its transcriptional activity. The competitive-ABPP method mapped the profile of target proteins for AIL, and OTU domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) was identified as a pivotal target of AIL in regulating ERRα protein levels. OTUB1 was validated as a novel deubiquitinating enzyme for ERRα, with its C91 residue being crucial for this deubiquitination process. AIL was found to inhibit the enzyme activity of OTUB1 by interacting with the C91 residue and disrupt the interaction between OTUB1 and ERRα, ultimately leading to the inhibition of ERRα. AIL is a promising downregulator of ERRα, and the mechanism of this downregulation has been elucidated. Additionally, a new regulatory relationship between ERRα and OTUB1 is identified. The research presented in this article is anticipated to yield potential lead compounds for ERRα regulatory agents and to stimulate the development of novel therapeutic strategies designed to modulate ERRα activity for the treatment of TNBC.

Renal implications of systemic lupus erythematosus pathogenesis and treatment

Lupus nephritis (LN) is a severe complication of systemic lupus erythematosus (SLE) that significantly contributes to morbidity and mortality. Its pathogenesis involves complex interactions between immune dysregulation, genetic susceptibility, and environmental factors, resulting in immune complex deposition and subsequent renal inflammation. Advances in understanding these mechanisms have highlighted the role of B cells, cytokines, and the complement system in disease progression. Current therapeutic strategies rely on immunosuppressants such as glucocorticoids, cyclophosphamide, and mycophenolate mofetil, which have proven effective in managing renal inflammation but carry risks of significant adverse effects. Biologic agents targeting B cells and complement components, including belimumab and eculizumab, have demonstrated promise in improving outcomes and reducing disease activity in refractory cases. The discovery of novel biomarkers is transforming the diagnosis and management of LN. Urinary markers like MCP-1 and complement activation products offer non-invasive tools for monitoring disease activity and predicting relapses. Molecular studies have identified microRNAs and genetic variants as potential indicators of disease susceptibility and therapeutic response. Advances in metabolomics and proteomics have revealed metabolic and protein profiles unique to LN, offering insights into disease mechanisms and new targets for intervention. Emerging technologies such as artificial intelligence are revolutionizing the analysis of complex biomarker data, enabling personalized treatment approaches. These advances highlight the importance of integrating multidisciplinary research efforts to optimize patient care. While challenges remain, including the heterogeneity of LN and disparities in access to care, ongoing research is paving the way for improved therapeutic options and outcomes. Precision medicine, driven by biomarker discovery and innovative therapies, holds the potential to transform the landscape of LN management, offering hope for better renal preservation and quality of life for affected individuals.

Functional heterogeneity of mesenchymal stem cells and their therapeutic potential in the K18-hACE2 mouse model of SARS-CoV-2 infection

BackgroundDespite many years of investigation into mesenchymal stem cells (MSCs) and their potential for treating inflammatory conditions such as COVID-19, clinical outcomes remain variable due to factors like donor variability, different tissue sources, and diversity within MSC populations. Variations in MSCs’ secretory and proliferation profiles, and their proteomic and transcriptional characteristics significantly influence their therapeutic potency, highlighting the need for enhanced characterization methods to better predict their efficacy. This study aimed to evaluate the biological characteristics of MSCs from different tissue origins, selecting the most promising line for further validation in a K18-hACE2 mouse model of SARS-CoV-2 infection.MethodsWe studied nine MSC lines sourced from either bone marrow (hBMMSC), dental pulp (hDPMSC), or umbilical cord tissue (hUCMSC). The cells were assessed for their proliferative capacity, immunophenotype, trilineage differentiation, proteomic profile, and in vitro immunomodulatory potential by co-culture with activated lymphocytes. The most promising MSC line was selected for further experimental validation using the K18-hACE2 mouse model of SARS-CoV-2 infection.ResultsThe analyzed cells met the minimum criteria for defining MSCs, including the expression of surface molecules and differentiation capacity, showing genetic stability and proliferative potential. Proteomic analysis revealed distinct protein profiles that correlate with the tissue origin of MSCs. The immunomodulatory response exhibited variability, lacking a discernible pattern associated with their origin. In co-culture assays with lymphocytes activated with anti-CD3/CD28 beads, all MSC lines demonstrated the ability to inhibit TNF-α, to induce TGF-β and Indoleamine 2,3-dioxygenase (IDO), with varying degrees of inhibition observed for IFN-γ and IL-6, or induction of IL-10 expression. A module of proteins was found to statistically correlate with the potency of IL-6 modulation, leading to the selection of one of the hUCMSCs as the most promising line. Administration of hUCMSC to SARS-CoV-2-infected K18 mice expressing hACE2 was effective in improving lung histology and modulating of a panel of cytokines.ConclusionsOur study assessed MSCs derived from various tissues, uncovering significant variability in their characteristics and immunomodulatory capacities. Particularly, hUCMSCs demonstrated potential in mitigating lung pathology in a SARS-CoV-2 infection model, suggesting their promising therapeutic efficacy.

Salivary proteomic analysis in patients with type 2 diabetes mellitus and periodontitis.

This study aimed to compare the salivary protein profile in individuals with Type 2 Diabetes Mellitus (DM2) and periodontitis and their respective controls. Eighty participants were included in the study. The four groups were formed by individuals with DM2 and periodontitis (DM2 + P, n = 20), DM2 without periodontitis (DM2, n = 20), periodontitis without DM2 (P, n = 20) and individuals without periodontitis and without DM2 (H, n = 20). Periodontal clinical examinations were performed and unstimulated saliva was collected. Proteomic analysis was performed by shotgun mass spectrometry. The results were obtained by searching the Homo sapiens database of the UniProt catalog. A total of 220 proteins were identified in saliva samples. In the comparison between DM2 + P and DM2 groups, 27 proteins were up-regulated [e.g. S100-A8 was 6 times up-regulated (humoral immune response pathway)]. The DM2 + P and P groups had 26 up-regulated proteins [e.g. Immunoglobulin lambda constant 7 more than 2 times up-regulated (complement activation pathway)]. The non-DM2 groups (P and H) presented 22 up-regulated proteins [e.g. Glyceraldehyde-3-phosphate dehydrogenase more than 2 times up-regulated (Peptidyl-cysteine S-nitrosylation pathway)]. The groups without periodontitis (DM2 and H) showed 23 were up-regulated proteins [e.g. Hemoglobin subunit alpha that was more than 10 times up-regulated (cellular oxidant detoxification pathway)]. The presence of DM2 and periodontitis significantly impacts the salivary proteome. Our proteomic analysis demonstrated that changes in the S100 family proteins (S100A8 and S100 A9) are highly related to the presence of DM2 and periodontitis. Diabetes Mellitus (DM) and periodontitis are highly prevalent chronic diseases that present a wide variety of signs and symptoms. They present a bidirectional relationship, where patients with DM have a higher prevalence and severity of periodontitis, and patients with periodontitis have a higher prevalence of DM, worse glycemic control, and more diabetic complications. Diagnosing periodontitis requires specific clinical examinations, which require a highly trained operator. In this study, we used high throughput proteomics in order to evaluate non-invasive biomarkers for periodontitis in type 2 DM subjects. The results can contribute to earlier, more accurate, and less costly diagnosis of periodontitis in diabetic subjects, enabling better diabetes control.

P0046 Proteomic Profiles Associated with Residual Ultrastructural Anomalies in the Epithelium of IBD patients in Endoscopic Remission

Abstract Background Increased permeability, mucosal barrier and epithelial anomalies (crypt distortion, wider crypt lumen, epithelial gaps) as well as altered tight junctions have been described in active ulcerative colitis (UC) and Crohn’s Disease (CD). Probe based Confocal laser endomicroscopy (pCLE) is able to monitor epithelial anomalies in vivo1. This study aimed at comparing the proteomic profiles of the colonic epithelium associated with pCLE anomalies in UC, CD and non-IBD subjects. Methods Biopsies were collected in IBDs in endoscopic remission (n=37) and non-IBD control (CT) (n=10) undergoing colonoscopy with pCLE as previously described1. Biopsy sampled in the right colon and the rectum were analysed by mass spectrometry label free proteomics, with enrichment of epithelial cells using laser capture microdissection2. Comparisons of epithelial protein profiles, in presence (1) or absence (0) of fluorescein leaks were obtained in the colon and rectum, in CD, UC and non-IBD-CT. Significant proteins obtained were confronted and the entire protein profiles were evaluated using Gene Ontology functional annotations. Results Colon and rectum samples analyses provided 3227 and 3303 protein identifications (≥50% occurrence in minimum 1 group). Unique proteins (≥50% occurrence in only 1 group) were identified in the colon (n=40) and rectum (n=18). Differential analyses (0vs1) in the colon highlighted 165 and 152 significant proteins in CD and UC respectively, from which 13 were in common. In non-IBD CT (0vs1) 88 proteins were discriminant, with 9 and 6 being shared with CD and UC respectively. In the rectum, 74 and 172 discriminant proteins were obtained in CD and UC, from which 16 were also found significant in colon CD and UC protein lists(0vs1). DAZAP, STK4, LAMC1 and PDCD10 showed similar distribution tendencies in both segments in CD0vs1. While HNRPH1 and 2, VASP, TMX1 and PREP distributions were akin in UC0vs1 in both segments (Fig.1.A). GO analyses highlighted more numerous proteins with a decrease when leaks were observed in IBDs than in non-IBD-CT (Fig.2. Right arm of volcano plots). These proteins are associated with junction proteins, matrix and cytoskeleton, proteasome, protease, peptidase functions, or are involved in apoptosis and innate immune controls and morphogenesis. STK4 product, a key regulator of the HIPPO and MAPK pathways, showed a drop (0&amp;gt;1) in IBDs and non-IBD CT with leaks (Fig.1.B). Conclusion This proteomic study reveals proteins with perturbed epithelial distributions associated with pCLE fluorescein leaks. Functional analyses are required to disclose and understand our findings and might confirm very early epithelial key players involved in IBD epithelial homeostasis loss. References 1Jean-Philippe Loly, Sophie Vieujean, Catherine Reenaers, Catherine Van Kemseke, Laurence Seidel ScD, Edouard Louis, Joan Somja In-Depth Assessment of Endoscopic Remission in Inflammatory Bowel Disease Treated by Anti-TNF or Vedolizumab Inflamm Bowel Dis. 2024 Feb 1;30(2):240-246. doi: 10.1093/ibd/izad066. DOI: 10.1093/ibd/izad066 2.Angela-Maria Merli, Sophie Vieujean, Charlotte Massot, Noella Blétard, Florence Quesada Calvo, Dominique Baiwir, Gabriel Mazzucchelli, Laurence Servais, Odile Wéra, Cécile Oury, Laurence de Leval, Christine Sempoux, Roberto Manzini, Sena Bluemel, Michael Scharl, Gerhard Rogler, Edwin De Pauw, C Coimbra Marques, Arnaud Colard, Anne Vijverman, Philippe Delvenne, Edouard Louis, Marie-Alice Meuwis Solute carrier family 12 member 2 as a proteomic and histological biomarker of dysplasia and neoplasia in ulcerative colitis J Crohns Colitis 2020 Sep 13:jjaa168. doi: 10.1093/ecco-jcc/jjaa168.

Innovative Ricin Toxin Detection: Unraveling Apurinic/Apyrimidinic Lyase Activity and Developing Fluorescence Sensors.

Ricin toxin (RT) is a potential bioterrorism agent because of its high potency, extremely small lethal dose, ease of preparation, and notable stability. Therefore, a portable method is urgently required to efficiently detect and determine the presence of toxicity of RT and evaluate its potency for public health monitoring and counter-bioterrorism responses. Currently, enzyme-based assays for detecting RT mainly focus on its N-glycosidase activity. In this study, we demonstrated that RT exhibits apurinic/apyrimidinic (AP) lyase activity using several methods. Characterization of the enzyme reaction and kinetics revealed that AP lyase activity is optimal at 59 °C and pH 4.0. This activity is highly pH-sensitive, remaining active between pH 3.0 and pH 4.6. Furthermore, we developed a portable fluorescence-based lateral flow assay (FLFA) that detects RT much faster than existing assays based on its N-glycosidase activity. Moreover, this assay can efficiently detect RT at nanogram levels from complex matrix samples within 1.5 h while simultaneously determining its biological activity. In conclusion, the discovery of the AP lyase activity of RT and the development of FLFA represent novel approaches for studying the enzymatic profiles of other ribosome-inactivating proteins.

P1011 Correlations of clinical characteristics and serum proteins with drug levels: A multicentre cohort study of patients with inflammatory bowel disease starting biologics

Abstract Background Inter-individual differences in drug clearance are common in inflammatory bowel disease (IBD) patients treated with biologics. Associations between inflammatory markers, clinical features, and drug levels may indicate that the inflammatory immune response influences the pharmacokinetics of biologics. We assessed the impact of inflammatory and immunity-related proteins on drug levels in IBD. Methods Serum samples from 144 IBD patients initiating biologics in a prospective multicentre cohort were analysed using AFIAS-3 (Boditech Med Inc. Korea). The primary outcome was drug levels after the end of induction treatment (post-induction), analysed as continuous variables or categorised into high or low groups based on the median. Correlations were assessed using the Pearson’s correlation coefficients with false discovery rate (FDR) adjustment. Predictive capacity of clinical and protein data was evaluated with regularised linear regression models. Analyses were stratified by treatment (infliximab, adalimumab, vedolizumab) and diagnosis (Crohn’s disease (CD), ulcerative colitis (UC)). Results Patient demographics and clinical characteristics are provided in Table 1. Median (interquartile range, IQR) post-induction infliximab levels were 5.3 (2.2-9.5) µg/ml in patients with CD (n=34) and 3.8 (1.7-5.6) µg/ml in UC (n=33). The corresponding levels were 12.5 (7.0-16.1) µg/ml (n=36) and 8.8 (5.7-12.7) µg/ml (n=13) for adalimumab, 13.9 (6.4-24.4) µg/ml (n=19) and 17.7 (9.8-22.6) µg/ml (n=9) for vedolizumab. Principal component analyses revealed potential baseline protein profile differences between patients with high vs low post-induction levels for infliximab (p=0.09) and adalimumab (p=0.07) in CD, but no differences for vedolizumab (p=0.78) or UC. Individual protein analyses indicated that patients with higher drug levels post-induction seemed to display lower baseline estimates of inflammatory proteins compared to those with lower drug levels. However, statistical significance after correction for multiple comparisons was only observed for CDCP1 in adalimumab-treated CD patients (PFDR=0.04) (Figure 1). Additionally, baseline Flt31 (r=-0.57, PFDR=0.03) and Wisp1 (r=-0.61, PFDR=0.02) correlated with post-induction s-adalimumab levels in CD, while Mcp3 (r=-0.89, PFDR=0.04) and Frgamma (r=-0.87, PFDR=0.05) correlated in UC. Integrating baseline protein data in prediction models for post-induction drug levels did not improve their accuracy compared to models based on only clinical and biochemical variables. Conclusion Inflammatory protein levels may influence post-induction drug levels in IBD, particularly in adalimumab treated CD patients. This insight could aid in optimising personalised treatment.

Circulating proteomic profiles are associated with type 2 diabetes in Asian populations - a longitudinal study.

Type 2 diabetes (T2D) is a major global concern, with Asia at its epicenter in recent years. Proteins, products of gene transcription, serve as dynamic biomarkers for pinpointing perturbed pathways in disease development. Previous T2D proteomic association studies primarily focused on European populations. The aim of this study was to investigate the relationship between plasma proteins and the incidence of T2D in Asian participants. We examined the association of 4,775 plasma proteins with incident T2D in a Singapore multi-ethnic cohort of 1,659 Asian participants (539 cases and 1,120 controls) using logistic regression. We used two-sample Mendelian randomization and colocalization analysis to evaluate the causal relationship between proteins and T2D. Our analysis revealed 522 proteins that were associated with incident T2D after adjusting for age, sex, and ethnicity, and 17 proteins that remained significantly associated after adjusting for other T2D risk factors such as fasting glucose, waist circumference, and triglycerides. Among the 522 proteins associated with incident T2D, the change in 205 plasma proteins, observed in parallel with the development of T2D at baseline and six-years follow-up, were further associated with incident T2D. The associated proteins showed enrichment in neuron generation, glycosaminoglycan binding, and insulin-like growth factor binding. Two-sample Mendelian randomization analysis suggested three plasma proteins, GSTA1, INHBC, and FGL1, play causal roles in the development of T2D, with colocalization evidence supporting GSTA1 and INHBC. Our findings reveal plasma protein profiles linked to the onset of T2D in Asian populations, offering insights into the biological mechanisms of T2D development.

P0156 Plasma immune protein profiles in therapy-naïve ulcerative colitis relate to extent of affected tissue and density of intestinal infiltration, differentiating patients with discreet disease courses

Abstract Background Ulcerative colitis (UC) is a chronic intestinal inflammation driven by inflammatory memory T helper cell (ThM) reactivation. Heterogeneity in clinical disease and variability in response to treatment require new strategies targeting the patient’s underlying immune disease. As inflammation in UC only affects the intestinal mucosa, it is debated whether peripheral blood immune responses could reliably reflect ongoing intestinal immune disease. Methods To unequivocally establish this, we performed a uniquely deep immune characterization of therapy-naïve pediatric UC patients (n=34) and controls (n=55) by measuring 92 plasma immune proteins, circulating ThM subpopulations, intestinal mRNA expression, and intestinal immunohistochemistry. Extensive clinical follow-up was used to relate immune responses to clinical disease severity and therapy response. Results At diagnosis, 23 plasma proteins were differentially abundant in UC compared to controls. Increased protein concentrations mainly related to the IL-17 pathway (IL-17, IL-8, CCL20), tissue remodelling (MMP-10, MMP1, HGF), acute phase response (IL-6, OSM) and chemokinesis (CXCL9, CXCL10, CCL3). Interestingly, intestinal mRNA expression of highly abundant plasma proteins similarly increased, whereas no proportional changes in circulating ThM were detected. Unbiased hierarchical clustering of plasma immune profiles yielded two clusters of UC patients; UC_A with significantly increased concentrations of 17 proteins, many of which Th17/neutrophil-related, versus UC_B with lower concentrations. Notable increases in UC_A were: IL- 17A; OSM, a neutrophil product previously associated with anti-TNF therapy resistance; CXCL9 and CXCL11, IFN-g-induced chemokines. Strikingly, this UC_A immune profile strongly related to increased size of the affected intestinal surface and degree of infiltrating IL-17A+ cells and number of neutrophils in the biopsies. Clinically, UC_A patients had higher clinico-pathological parameters, more severe intestinal disease at endoscopy and on histology , and required earlier treatment intensification during 1.5 year follow-up, compared to UC_B patients. Conclusion These data firmly establish that, even though inflammation in UC only affects the intestinal mucosa, peripheral blood immune protein responses reliably reflect the degree of ongoing intestinal immune disease in UC. Implementing immune profiling in routine clinical characterization at diagnosis improves identification of patients who will develop a severe disease course allowing tailored treatment with earlier start of more intensive immunomodulatory treatment to treat to target and avoid tissue damage.

P0817 Large-scale clustering of longitudinal faecal calprotectin and C-reactive protein profiles in Scottish and Danish inflammatory bowel disease

Abstract Background Despite major advances in our understanding of the molecular underpinning of IBD, we remain challenged in deciphering the observed disease heterogeneity. Attempts to characterise longitudinal disease behaviour have either been restricted to symptom-based profiling - as exemplified in the IBSEN cohorts - or reliance on stochastic endpoints of disease “progression” such as hospitalisation, surgery and fibrosis. Here we present a novel method to examine IBD disease behaviour by modelling longitudinal inflammatory patterns in two large, well-characterised cohorts. Methods We conducted a retrospective study in two population cohorts, the Lothian IBD Registry (LIBDR),1 based in Scotland, and IBD patients described in national Danish registries.2,3 Using latent class mixed models,4 we independently clustered subjects by their FC and CRP profiles across 7 and 5 year post-diagnosis for the Lothian and Danish data respectively. The number of assumed clusters for the Danish models were dictated by the number chosen for the LIBDR data. Montreal classification was available for the LIBDR, and prescribing data were available for both cohorts. Results 1036 LIBDR subjects (544 CD, 492 UC/IBDU) and 7880 Danish subjects (3931 CD, 3949 UC) were included in the FC analysis. In the LIBDR, 10545 (median 9 per subject, IQR 6–13) FC observations were available, with 67986 observations available in the Danish registry (7, 4-11). For the CRP analysis, 1838 (805 CD, 1033 UC/IBDU) LIBDR subjects and 10041 (4415 CD, 5626) Danish subjects were included, with 49364 (median 20, IQR 10-36) and 241689 (19, 9-32) CRP observations respectively. When modelling the FC data, we found eight clusters (Figure 1), with the major clusters (FC1-4,7,8) replicating in the Danish data (Figure 2). The longitudinal trajectories characterising the clusters appear to reflect commonly reported clinical behaviours (e.g rapid remitters, delayed remitters, relapsing remitters and non-remitters). No association was found between ileal vs a colonic disease and cluster assignment. The use and timing of advanced therapies differed by cluster with rapid remitters more likely to receive therapy earlier in the disease course. However, prescribing trends described only a proportion of cluster assignments. When modelling the CRP data, we again found eight clusters, although there was broadly poor agreement between FC and CRP clusters. The follow-up required for reliable cluster assignments depended on the shape of the trajectories. Conclusion Distinct patterns of inflammatory behaviour over time are evident in patients with IBD. These data pave the way for a deeper understanding of disease heterogeneity in IBD and enhanced patient stratification in the clinic.

P0044 What are the similarities between primary immunodeficiency states and ulcerative colitis: damage to the microbiome, impaired mucosal secretion of proteins and short-chain fatty acids, or something else?

Abstract Background Primary immunodeficiency (PID), common variable immunodeficiency (CVID) are primary immunodeficiencies with disorder of the adaptive immune system, organ-specific pathology, as well as defects in the gut microbiome [1]. In patients with ulcerative colitis (UC) during relapse, similar changes. Methods The aim of the research is a comprehensive study of the structure of gut microbiota and mucosal protein profile (MPP) in patients with PID, CVID and UC in different phases of the disease course. The research included 19 patients with PID, 28 with CVID and 32 with UC (22 with relapse and 10 with persistent remission of the disease), control group - 20 healthy volunteers. Estimation of gut microbiota was performed by microbacteriological seeding faeces (MSF). Content of short-chain fatty acids (SCFA) in the feces as determined by gas-chromatography-mass spectrometry (GC-MS). Mucosal protein profile was based on isoelectric focusing techniques (SDS-PAGE, 2DGE). Mass spectrograms were obtained using MALDI-TOF-MS/MS (Bruker, USA). Molecular interaction and functional characteristics of proteins were studied using the STRING 10.0 databases. Results SCFA production showed a 6-, 9- and 11-fold decrease in propionic and butyric acids, mainly in patients with PID and UC: 0.2 + 0.1, 0.14 + 0.03 and 0.04 + 0.02, 0.08 + 0.02 mg/g, respectively (tab. 1). MSF showed a decrease in titers of E. coli, bifido- and lactobacilli on average 4.6 + 0.8 Lg. The conditionally pathogenic microbiota was represented by E. coli, hemolytic strains (n=34,104/g), Proteus spp. (n=48,105/g), Staphylococcus spp. (n=29,105–6/g), Candida alb. / glabrata (n=55,104–6/g) and Clostridium spp. (n = 38,104/g). Results of MPP (detection rate of 75% or more) of the colon mucosa in patients with PID and CVID were detected: 1, 2, 4 okkludin, kininogen 1, interleukin-1B, interleukin 8, B2-glycoprotein, heat shock protein 27, in patients with CVID - translational elongation factor, apolipoprotein C-III. In patients with UC - NF-kB, alipoprotein C-III, TNF-α, heat shock protein 27, interleukin-2 and 8 were presented. MPP patients with UC (stable remission phase): a-enolase, b-defensin-1, cathepsin D, prohibitin (tab. 2). Conclusion 1. In patients with PID, CVID and UC during relapse, markers of damage to the intestinal microbiome, a decrease in the production of SCFA and proteins characteristic of inflammation, apoptosis, proliferation, as well as proteins reflecting the activity of the autoimmune process, were identified. 2. During the period of drug remission in patients with UC, PID and CVID, specific components of altered MPP disappear, and a tendency towards restoration of the gut microbiota and SCFA production is recorded. References Jørgensen SF, Trøseid M, Kummen M, et al. Altered gut microbiota profile in common variable immunodeficiency associates with levels of lipopolysaccharide and markers of systemic immune activation. Mucosal Immunol. 2016; 9:1455-1465.