Protein Secretion Profile Research Articles

A new subtype of artificial cell-derived vesicles from dental pulp stem cells with the bioequivalence and higher acquisition efficiency compared to extracellular vesicles.

Extracellular vesicles (EVs) derived from dental pulp stem cells (DPSC) have been shown an excellent efficacy in a variety of disease models. However, current production methods fail to meet the needs of clinical treatment. In this study, we present an innovative approach to substantially enhance the production of 'Artificial Cell-Derived Vesicles (ACDVs)' by extracting and purifying the contents released by the DPSC lysate, namely intracellular vesicles. Comparative analysis was performed between ACDVs and those obtained through ultracentrifugation. The ACDVs extracted from the cell lysate meet the general standard of EVs and have similar protein secretion profile. The new ACDVs also significantly promoted wound healing, increased or decreased collagen regeneration, and reduced the production of inflammatory factors as the EVs. More importantly, the extraction efficiency is improved by 16 times compared with the EVs extracted using ultracentrifuge method. With its impressive attributes, this new subtype of ACDVs emerge as a prospective candidate for the future clinical applications in regenerative medicine.

Secretome of Cancer-Associated Fibroblasts (CAFs) Influences Drug Sensitivity in Cancer Cells.

Resistance is a major problem with effective cancer treatment and the stroma forms a significant portion of the tumor mass but traditional drug screens involve cancer cells alone. Cancer-associated fibroblasts (CAFs) are a major tumor stroma component and its secreted proteins may influence the function of cancer cells. The majority of secretome studies compare different cancer or CAF cell lines exclusively. Here, we present the direct characterization of the secreted protein profiles between CAFs and KRAS mutant-cancer cell lines from colorectal, lung, and pancreatic tissues using multiplexed mass spectrometry. 2573 secreted proteins were annotated, and differential analysis highlighted understudied CAF-enriched secreted proteins, including Wnt family member 5B (WNT5B), in addition to established CAF markers, such as collagens. The functional role of CAF secreted proteins was explored by assessing its effect on the response to 97 anticancer drugs since stromal cells may cause a differing cancer drug response, which may be missed on routine drug screening using cancer cells alone. CAF secreted proteins caused specific effects on each of the cancer cell lines, which highlights the complexity and challenges in cancer treatment and so the importance to consider stromal elements.

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Abstract Inflammation-associated fibroblasts (IAFs) are a cell population that emerges in tissue inflammation. In inflammatory bowel diseases (IBD), IAFs correlate with disease severity and are enriched in colon tissue of patients refractory to anti-TNF therapy. IAFs can amplify tissue inflammation and mediate pro-fibrotic matrix remodeling and therefore represent a novel cellular target for treating inflammatory diseases like IBD. To explore the cellular and molecular pathways that mediate transition to an IAF-like phenotype, we developed a co-culture of human PBMCs and primary colonic fibroblasts to study stromal-immune crosstalk in promoting IAFs. Culturing primed PBMCs with fibroblasts in direct contact drives fibroblast transition to an IAF phenotype through upregulation of inflammatory mediators, evidenced by modulation of gene expression and secreted protein profiles. Single-cell RNAseq revealed IAF development is influenced by PBMC priming conditions. Exposure to LPS-primed PBMCs promoted a matrix-remodeling IAF phenotype while PHA-primed PBMCs promoted an immunomodulatory IAF. IAF activation can be dampened by inhibitors, including TNF and JAK inhibitors. This co-culture system can therefore be used to define mechanisms mediating IAF transition and identify IAF states that can be tailored to accentuate pathways for focused interrogation. We aim to leverage this system to identify novel targets and test prospective therapies to modulate IAF transition and downstream activity.

Using Macrophage Polarization in Human Platelet Lysate to Test the Immunomodulatory Potential of Cells for Clinical Use.

Macrophage-based co-cultures are used to test the immunomodulatory function of candidate cells for clinical use. This study aimed to characterize a macrophage polarization model using human platelet lysate (hPL) as a GMP-compliant alternative to Fetal Bovine Serum (FBS). Primary human monocytes were differentiated into unpolarized (M0) or polarized (M1, M2a, and M2c) macrophages in an hPL- or FBS-based medium. The protein secretion profiles and expression of phenotypic markers (CD80 for M1, CD206 for M2a, and CD163 for M2c) were analyzed. Subsequently, chondrocytes were tested in an hPL-based co-culture model to assess their immunomodulatory function in view of their possible use in patients with osteoarthritis. The results showed similar marker regulation between hPL and FBS cultures, but lower basal levels of CD206 and CD163 in hPL-cultured macrophages. Functional co-culture experiments with chondrocytes revealed increased CD206 expression both in hPL and in FBS, indicating an interaction between macrophages and chondrocytes. While markers in FBS-cultured macrophages were confirmed in hPL-cultured cells, the interpretation of marker modulation in immunomodulatory assays with hPL-based cultures should be carried out cautiously due to the observed differences in the basal marker levels for CD206 and CD163. This research underscores the utility of hPL as a GMP-compliant alternative to FBS for macrophage-based co-cultures and highlights the importance of understanding marker expressions in different culture conditions.

Changes in saliva protein profile throughout Rhipicephalus microplus blood feeding

BackgroundWhen feeding on a vertebrate host, ticks secrete saliva, which is a complex mixture of proteins, lipids, and other molecules. Tick saliva assists the vector in modulating host hemostasis, immunity, and tissue repair mechanisms. While helping the vector to feed, its saliva modifies the site where pathogens are inoculated and often facilitates the infection process. The objective of this study is to uncover the variation in protein composition of Rhipicephalus microplus saliva during blood feeding.MethodsTicks were fed on calves, and adult females were collected, weighed, and divided in nine weight groups, representing the slow and rapid feeding phases of blood feeding. Tick saliva was collected, and mass spectrometry analyses were used to identify differentially secreted proteins. Bioinformatic tools were employed to predict the structural and functional features of the salivary proteins. Reciprocal best hit analyses were used to identify conserved families of salivary proteins secreted by other tick species.ResultsChanges in the protein secretion profiles of R. microplus adult female saliva during the blood feeding were observed, characterizing the phenomenon known as “sialome switching.” This observation validates the idea that the switch in protein expression may serve as a mechanism for evading host responses against tick feeding. Cattle tick saliva is predominantly rich in heme-binding proteins, secreted conserved proteins, lipocalins, and protease inhibitors, many of which are conserved and present in the saliva of other tick species. Additionally, another remarkable observation was the identification of host-derived proteins as a component of tick saliva.ConclusionsOverall, this study brings new insights to understanding the dynamics of the proteomic profile of tick saliva, which is an important component of tick feeding biology. The results presented here, along with the disclosed sequences, contribute to our understanding of tick feeding biology and might aid in the identification of new targets for the development of novel anti-tick methods.Graphical

Biological properties and characterization of several variations of a clinical human plasma-based skin substitute model and its manufacturing process.

Human plasma is a natural biomaterial that due to their protein composition is widely used for the development of clinical products, especially in the field of dermatology. In this context, this biomaterial has been used as a scaffold alone or combined with others for the development of cellular human plasma-based skin substitutes (HPSSs). Herein, the biological properties (cell viability, cell metabolic activity, protein secretion profile and histology) of several variations of a clinical HPSS model, regarding the biomaterial composition (alone or combined with six secondary biomaterials - serine, fibronectin, collagen, two types of laminins and hyaluronic acid), the cellular structure (trilayer, bilayer, monolayer and control without cells) and their skin tissue of origin (abdominal or foreskin cells) and the manufacturing process [effect of partial dehydration process in cell viability and comparison between submerged (SUB) and air/liquid interface (ALI) methodologies] have been evaluated and compared. Results reveal that the use of human plasma as a main biomaterial determines the in vitro properties, rather than the secondary biomaterials added. Moreover, the characteristics are similar regardless of the skin cells used (from abdomen or foreskin). However, the manufacture of more complex cellular substitutes (trilayer and bilayer) has been demonstrated to be better in terms of cell viability, metabolic activity and wound healing protein secretion (bFGF, EGF, VEGF-A, CCL5) than monolayer HPSSs, especially when ALI culture methodology is applied. Moreover, the application of the dehydration, although required to achieve an appropriate clinical structure, reduce cell viability in all cases. These data indicate that this HPSS model is robust and reliable and that the several subtypes here analysed could be promising clinical approaches depending on the target dermatological disease.

Cross-sectional network analysis of plasma proteins/metabolites correlated with pathogenesis and therapeutic response in acute promyelocytic leukemia.

The treatment of PML/RARA+ acute promyelocytic leukemia (APL) with all-trans-retinoic acid and arsenic trioxide (ATRA/ATO) has been recognized as a model for translational medicine research. Though an altered microenvironment is a general cancer hallmark, how APL blasts shape their plasma composition is poorly understood. Here, we reported a cross-sectional correlation network to interpret multilayered datasets on clinical parameters, proteomes, and metabolomes of paired plasma samples from patients with APL before or after ATRA/ATO induction therapy. Our study revealed the two prominent features of the APL plasma, suggesting a possible involvement of APL blasts in modulating plasma composition. One was characterized by altered secretory protein and metabolite profiles correlating with heightened proliferation and energy consumption in APL blasts, and the other featured APL plasma-enriched proteins or enzymes catalyzing plasma-altered metabolites that were potential trans-regulatory targets of PML/RARA. Furthermore, results indicated heightened interferon-gamma signaling characterizing a tumor-suppressing function of the immune system at the first hematological complete remission stage, which likely resulted from therapy-induced cell death or senescence and ensuing supraphysiological levels of intracellular proteins. Overall, our work sheds new light on the pathophysiology and treatment of APL and provides an information-rich reference data cohort for the exploratory and translational study of leukemia microenvironment.

3D model of the bronchial epithelial barrier to study repeated exposure to xenobiotics: Application to silver nanoparticles

There is still a lack of in vitro human models to evaluate the chronic toxicity of drugs and environmental pollutants. Here, we used a 3D model of the human bronchial epithelium to assess repeated exposures to xenobiotics. The Calu-3 human bronchial cell line was exposed to silver nanoparticles (AgNP) 5 times during 12 days, at the air-liquid interface, to mimic single and repeated exposure to inhaled particles. Repeated exposures induced a stronger induction of the metal stress response and a steady oxidative stress over time. A sustained translocation of silver was observed after each exposure without any loss of the epithelial barrier integrity. The proteomic analysis of the mucus revealed changes in the secreted protein profiles associated with the epithelial immune response after repeated exposures only. These results demonstrate that advanced in vitro models are efficient to investigate the adaptive response of human cells submitted to repeated xenobiotic exposures.

Substrate stiffness can affect the crosstalk between adipose derived mesenchymal stem cells and macrophages in bone tissue engineering.

Purpose: This study aimed to explore the effect of biomaterials with different stiffness on Adipose Derived Mesenchymal Stem Cells (ADSC)-macrophage crosstalk in bone tissue engineering and its role in bone repair. Methods: Biomaterials with Young's modulus of 64 and 0.2kPa were selected, and the crosstalk between ADSCs and macrophages was investigated by means of conditioned medium treatment and cell co-culture, respectively. Polymerase chain reaction (PCR) and flow cytometry were used to evaluate the polarization of macrophages. Alkaline phosphatase (ALP) and alizarin red staining (ARS) solutions were used to evaluate the osteogenic differentiation of ADSCs. Transwell assay was used to evaluate the chemotaxis of ADSCs and macrophages. Moreover, mass spectrometry proteomics was used to analyze the secreted protein profile of ADSCs of different substrates and macrophages in different polarization states. Results: On exploring the influence of biomaterials on macrophages from ADSCs on different substrates, we found that CD163 and CD206 expression levels in macrophages were significantly higher in the 64-kPa group than in the 0.2-kPa group in conditioned medium treatment and cell co-culture. Flow cytometry showed that more cells became CD163+ or CD206+ cells in the 64-kPa group under conditioned medium treatment or cell co-culture. The Transwell assay showed that more macrophages migrated to the lower chamber in the 64-kPa group. The proteomic analysis found that ADSCs in the 64-kPa group secreted more immunomodulatory proteins, such as LBP and RBP4, to improve the repair microenvironment. On exploring the influence of biomaterials on ADSCs from macrophages in different polarization states, we found that ALP and ARS levels in ADSCs were significantly higher in the M2 group than in the other three groups (NC, M0, and M1 groups) in both conditioned medium treatment and cell co-culture. The Transwell assay showed that more ADSCs migrated to the lower chamber in the M2 group. The proteomic analysis found that M2 macrophages secreted more extracellular remodeling proteins, such as LRP1, to promote bone repair. Conclusion: In bone tissue engineering, the stiffness of repair biomaterials can affect the crosstalk between ADSCs and macrophages, thereby regulating local repair immunity and affecting bone repair.

1583-P: Dynamic Tracking and Identification of Liver-Specific Secretory Proteins in the Circulation of Live Mice

Secretory proteins are an essential component of interorgan communication networks that regulate animal physiology. Current approaches for identifying secretory proteins from specific cell and tissue types are largely limited to in vitro or ex vivo models which often fail to recapitulate in vivo biology. As such, there is mounting interest in developing in vivo analytical tools that can provide accurate information on the origin, identity, and spatiotemporal dynamics of secretory proteins. Here, we describe iSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which selectively labels proteins that transit through the classical secretory pathway via catalytic actions of Sec61b-TurboID, a proximity labeling enzyme anchored in the ER lumen. To validate iSLET in a whole-body system, we express iSLET in the mouse liver and demonstrate efficient labeling of liver secretory proteins which could be tracked and identified within circulating blood plasma. Furthermore, proteomic analysis of the labeled liver secretome enriched from liver iSLET mouse plasma is highly consistent with previous reports of liver secretory protein profiles. Taken together, iSLET is a versatile and powerful tool for studying spatiotemporal dynamics of secretory proteins, a valuable class of biomarkers and therapeutic targets. Disclosure K.Kim: None. J.Suh: None. Funding National Research Foundation of Korea (2018R1A2A3075389, 2016M3A9B6902871, 2017K1A1A2013124, 2021R1A2C2007573, 2020R1C1C1013927); KAIST Key Research Institutes Project; Bio and Medical Technology Development Program (2019M3E5D3073104); Ministry of Science

Effects of adipocyte-conditioned cell culture media on S1P treatment of human triple-negative breast cancer cells.

Sphingosine-1-phosphate (S1P) is a potent sphingolipid metabolite that regulates a wide range of biological functions such as cell proliferation, cell apoptosis and angiogenesis. Its cellular level is elevated in breast cancer, which, in turn, would promote cancer cell proliferation, survival, growth and metastasis. However, the cellular concentration of S1P is normally in the low nanomolar range, and our previous studies showed that S1P selectively induced apoptosis of breast cancer cells at high concentrations (high nanomolar to low micromolar). Thus, local administration of high-concentration S1P alone or in combination of chemotherapy agents could be used to treat breast cancer. The breast mainly consists of mammary gland and connective tissue stroma (adipose), which are dynamically interacting each other. Thus, in the current study, we evaluated how normal adipocyte-conditioned cell culture media (AD-CM) and cancer-associated adipocyte-conditioned cell culture media (CAA-CM) would affect high-concentration S1P treatment of triple-negative breast cancer (TNBC) cells. Both AD-CM and CAA-CM may suppress the anti-proliferative effect and reduce nuclear alteration/apoptosis caused by high-concentration S1P. This implicates that adipose tissue is likely to be detrimental to local high-concentration S1P treatment of TNBC. Because the interstitial concentration of S1P is about 10 times higher than its cellular level, we undertook a secretome analysis to understand how S1P would affect the secreted protein profile of differentiated SGBS adipocytes. At 100 nM S1P treatment, we identified 36 upregulated and 21 downregulated secretome genes. Most of these genes are involved in multiple biological processes. Further studies are warranted to identify the most important secretome targets of S1P in adipocytes and illustrate the mechanism on how these target proteins affect S1P treatment of TNBC.

Applying single-cell highly multiplexed secretome proteomics to characterize immunotherapeutic products and predict clinical responses.

Genetically and phenotypically identical immune cell populations can be highly heterogenous in terms of their immune functions and protein secretion profiles. The microfluidic chip-based single-cell highly multiplexed secretome proteomics enables characterization of cellular heterogeneity of immune responses at different cellular and molecular layers. Increasing evidence has demonstrated that polyfunctional T cells that simultaneously produce 2+ proteins per cell at the single-cell level are key effector cells that contribute to the development of potent and durable cellular immunity against pathogens and cancers. The functional proteomic technology offers a wide spectrum of cellular function assessment and can uniquely define highly polyfunctional cell subsets with cytokine signatures from live individual cells. This high-dimensional single-cell analysis provides deep dissection into functional heterogeneity and helps identify predictive biomarkers and potential correlates that are crucial for immunotherapeutic product design optimization and personalized immunotherapy development to achieve better clinical outcomes.

P054 Secreted inflammatory protein profiles following treatment of ex-vivo human inflammatory bowel disease explants with licenced biologic therapies

Abstract Background Patient-derived inflammatory bowel disease (IBD) ex-plants have potential for biomarker and therapy discovery. Infliximab (IFX), ustekinumab (USTK) and vedolizumab (VDZ) are biologic therapies licenced for the induction and maintenance of remission in ulcerative colitis (UC) and Crohn’s disease (CD). All are efficacious in IBD despite differing mechanisms of action. Treatment of IBD explants ex-vivo with licensed biologic therapies may result in further insights into their mechanism of action. We aimed to evaluate the effect of IFX, USTK and VDZ on inflammatory protein secretion profiles in ex-vivo human IBD ex-plants. Methods Patients with IBD, undergoing endoscopy, were prospectively recruited. Biopsies were collected from the sigmoid and IBD ex-plants generated as per previously described methods. IBD explants were then co-cultured for 24 hours with an IgG control vehicle, IFX, USTK and VDZ. After 24 hours, tissue conditioned media (TCM) from IBD explants was collected. TCM secreted inflammatory protein profiles were quantified using 54 V-plex ELISA (Meso Scale Diagnostics, USA). Secreted inflammatory protein profiles were compared between IgG vehicle (control) and USTK, IFX, VDZ treated ex-plants. All continuous variables are presented as median [interquartile range (IQR)]. P values < 0.05 were considered significant in analyses. Results 37 patients with IBD were included (51% CD, 49% UC); age (median, [IQR]) 41[33-53] years, 49% male; disease duration (median, [IQR]) 9 [5-14] years; 51% of patients were anti-TNF naïve. TNF-a secretion was significantly lower in IBD explants treated with IFX compared with IgG control, p=0.006. IL-23 secretion was significantly lower in IBD explants treated with USTK compared with IgG control, p=0.043. Biologic therapy ex-vivo resulted in significant differences in angiogenesis and vascular injury panel biomarkers compared to control; CRP, VEGF-A and SAA. Pro-inflammatory cytokines and chemokines were also significantly decreased with ex-vivo biologic therapy compared to control; IL-2, IL-10, IL-6, MCP-1, IL-1a, TNF-a and MDC. Three Th17 panel proteins were significantly decreased with ex-vivo biologic therapy compared to control; IL-22, IL-17A, IL17A Gen B. Conclusion The statistically significant decrease in both TNF-a and IL-23 secretion with ex vivo IFX and USTK treatment respectively demonstrates that the IBD explant model recapitulates expected IBD disease biology and therapy responses. These data demonstrate that treatment with licenced biologic therapies ex-vivo reduces multiple pro-inflammatory cytokines, chemokines and secreted proteins in IBD explants. Further study is required to completely understand the effects of licenced biologic therapies on the IBD tissue microenvironment.

Infertile human endometrial organoid apical protein secretions are dysregulated and impair trophoblast progenitor cell adhesion.

Embryo implantation failure leads to infertility. As an important approach to regulate implantation, endometrial epithelial cells produce and secrete factors apically into the uterine cavity in the receptive phase to prepare the initial blastocyst adhesion and implantation. Organoids were recently developed from human endometrial epithelium with similar apical-basal polarity compared to endometrial gland making it an ideal model to study endometrial epithelial secretions. Endometrial organoids were established using endometrial biopsies from women with primary infertility and normal fertility. Fertile and infertile organoids were treated with hormones to model receptive phase of the endometrial epithelium and intra-organoid fluid (IOF) was collected to compare the apical protein secretion profile and function on trophoblast cell adhesion. Our data show that infertile organoids were dysregulated in their response to estrogen and progesterone treatment. Proteomic analysis of organoid apical secretions identified 150 dysregulated proteins between fertile and infertile groups (>1.5-fold change). Trophoblast progenitor spheroids (blastocyst surrogates) treated with infertile organoid apical secretions significantly compromised their adhesion to organoid epithelial cell monolayers compared to fertile group (P < 0.0001). This study revealed that endometrial organoid apical secretions alter trophoblast cell adhesiveness relative to fertility status of women. It paves the way to determine the molecular mechanisms by which endometrial epithelial apical released factors regulate blastocyst initial attachment and implantation.

Identification of Biochemical Differences in White and Brown Adipocytes Using FTIR Spectroscopy

This study was conducted to investigate the developmental characteristics of adipocytes and to identify selectively white and brown adipocytes through Fourier transform infrared (FTIR) spectroscopy. For the developmental characterization of adipocytes, cells and conditioned media of white and brown adipocytes were respectively collected and analyzed. A higher amide I/amide II ratio was observed in the conditioned medium of brown adipocyte than in that of white adipocyte, indicating differences in secretory protein profiles. In contrast, an amide I/amide II ratio was higher in white adipocytes than in brown adipocytes, and mature adipocytes have higher lipid amounts than pre-adipocytes. Lipid acyl chain length was the longest in white adipocytes. These differences suggested that FTIR spectroscopy can be used to characterize developmental stages and/or types of adipocytes. To identify the possibility of selectively classifying adipose-derived stem cells, FTIR spectroscopy spectra were obtained in cells before/after white/brown adipocyte differentiation using FTIR spectroscopy and then analyzed by the principal component analysis method. All data indicated that the discrimination between adipocytes was possible in the analysis of the infrared spectroscopy spectrum by the principal component analysis technique. This study suggested the possibility of FTIR spectroscopy as a new type of cell sorting system without tagging.

Association Between Ex Vivo Human Ulcerative Colitis Explant Protein Secretion Profiles and Disease Behaviour

BackgroundThe clinical course of ulcerative colitis (UC) is variable. There is an unmet clinical need for biomarkers of UC disease behaviour. We aimed to evaluate the association between ex vivo human UC explant conditioned media (explant-CM) secreted protein profiles and UC disease behaviour.MethodsUC patients undergoing endoscopy were prospectively recruited. Endoscopic biopsies were collected and explant-CM generated. Association between explant-CM protein secretion profiles and disease progression was evaluated. Disease progression was defined as the requirement for corticosteroid therapy, UC-related hospitalisation, UC-related surgery or the introduction of a new immunomodulatory agent. Association between explant-CM secreted protein profiles and anti-TNF failure status was also evaluated. p values < 0.05 were considered significant in analyses.ResultsTwenty-four UC patients were included (age [median, range]) 55 [21–72] years; 50% female. Disease progression during follow-up occurred in twelve (50%) patients. Multivariate analysis, including endoscopic remission status, demonstrated reduced IL-2 secretion to be independently associated with UC disease progression, p = 0.01. In univariate analysis, anti-TNF failure status was associated with significantly increased IL-17A/F (p = 0.015) and IL-12 / IL-23p40 (p = 0.044) concentrations. In multivariate analysis, there was a trend towards an association between IL-17A/F and anti-TNF failure status (p = 0.069); FLT-1 was demonstrated to be independently associated with anti-TNF failure status (p = 0.016).ConclusionReduced explant-CM secreted IL-2 is associated with UC disease progression. Increased secretion of IL-23 pathway-associated cytokines was observed in anti-TNF failure status consistent with previous reports. Ex vivo human UC explants, generated from endoscopic biopsies, have potential as precision medicine tools in inflammatory bowel disease.

P084 Secreted inflammatory protein profiles following treatment of ex-vivo human Ulcerative Colitis and Crohn’s Disease explants with licenced biologic therapies

Abstract Background Patient-derived inflammatory bowel disease (IBD) ex-plants have potential for biomarker and therapy discovery. Treatment of IBD ex-plants ex-vivo with licensed biologic therapies may result in further insights into their mechanism of action. Methods We aimed to evaluate the effect of Infliximab (IFX), Ustekinumab (USTK) and Vedolizumab (VDZ) on inflammatory protein secretion profiles in ex-vivo human IBD ex-plants. Patients with IBD, undergoing endoscopy, were prospectively recruited. Endoscopic biopsies were collected from the sigmoid colon and IBD ex-plants generated as per previously described methods. IBD ex-plants were then co-cultured for 24 hours with an IgG control vehicle, IFX, USTK and VDZ. After 24 hours, tissue conditioned media (TCM) from IBD ex-plants was collected. TCM secreted inflammatory protein profiles were quantified using 54 V-plex ELISA (Meso Scale Diagnostics, USA). Secreted inflammatory protein profiles were compared between IgG vehicle (control) and USTK, IFX, VDZ treated ex-plants. All continuous variables are presented as median [interquartile range (IQR)]. P values &amp;lt; 0.05 were considered significant in analyses. Results 18 patients with Ulcerative Colitis (UC) and 19 patients with Crohn’s Disease (CD) were included; age (median, [IQR]) 41[33–53], years, 49% male; disease duration (median, [IQR]) 9 [5–14] years; 54% of patients were anti-TNF naïve. IL-22 secretion was significantly lower in UC explants treated with IFX compared with IgG control; 4.5 [3.1 – 39.9] versus 17.5 [10.3 – 98.3] pg/ml per ug of protein respectively, p=0.004. IL-22 secretion was significantly lower in UC explants treated with USTK compared with IgG control; 7.8 [1.9 – 26.5] versus 27.9 [10.3 – 110.7] pg/ml per ug of protein respectively, p=0.004. IL-22 secretion was significantly lower in UC ex-plants treated with VDZ compared with IgG control; 10.6 [3.2 – 35.7] versus 17.5 [10.2 – 73.5] pg/ml per ug of protein respectively, p=0.01. For CD explants, IL-22 secretion was significantly lower in USTK compared with IgG control; 24.4 [17.6 – 57.7] versus 211.7 [75.9 – 588.1] pg/ml per ug of protein respectively, p &amp;lt; 0.001. In UC and CD explants, IL-23 secretion was significantly decreased comparing USTK with IgG control treatments, p &amp;lt; 0.004 for all comparisons. Conclusion IBD ex-plant IL-22 secretion is reduced following biologic therapy particularly in patients with UC. IL-22 is a pleotropic cytokine considered an essential factor in intestinal immune-epithelial cross talk and is capable of both pro- and anti-inflammatory functions depending on tissue microenvironment including cytokine milieu. Further evaluation of the contribution of IL-22 to IBD disease biology is warranted.

P078 Vedolizumab Treatment of ex-vivo Human Ulcerative Colitis (UC) Explants Results in Altered Inflammatory Protein Secretion Profiles

Abstract Background Patient-derived inflammatory bowel disease (IBD) ex-plants have potential for biomarker and therapy discovery. Vedolizumab (VDZ) is a monoclonal antibody targeting a4b47 integrin whose mechanism of action is the reduction of inflammatory immune cell trafficking to the intestinal tract. The association between VDZ exposure and treatment response is unclear and appears insufficiently explained by serum levels. For this reason it is hypothesised that VDZ may also have effects at the tissue level. Methods We aimed to evaluate the effect of VDZ on inflammatory protein secretion profiles in ex-vivo human ulcerative colitis ex-plants (UC ex-plants). Patients with UC, undergoing endoscopy, were prospectively recruited. Endoscopic biopsies were collected and UC ex-plants generated as per previously described methods. UC ex-plants were then co-cultured for 24 hours with an IgG control vehicle or VDZ. After 24 hours tissue conditioned media (TCM) from UC ex-plants was collected. TCM secreted protein profiles were quantified using 54 V-plex ELISA (Meso Scale Diagnostics, USA). Secreted cytokine profile were compared between IgG vehicle (control) and VDZ treated ex-plants. P values &amp;lt; 0.05 were considered significant in analyses. Results Thirteen patients with UC were included; age (mean, [range]) 45.8 years [30–78], 54% male; disease duration (mean, [range]) 8 [1–24] years; 62% of patients were anti-TNF naïve. Baseline total Mayo score (median [range]) was 6 [0–9]; endoscopic Mayo score (median [range]) was 2 [0–3]. Comparing VDZ with control treatment, 5 of 54 ex-plant secreted proteins differed significantly. GMCSF, IL-16, IL-22, IL-23 and sVCAM-1 secretion were significantly decreased comparing VDZ and control treatments, p &amp;lt; 0.03 for all comparisons. Conclusion The predominant mechanism of action of VDZ is the blocking of gut homing pro-inflammatory immune cell trafficking from the peripheral circulation to the intestinal tract. These data demonstrated that VDZ reduces pro-inflammatory protein secretions from UC ex-plants suggesting an additional local effect of this therapy. Further evaluation is required to determine whether a component of VDZ therapeutic effect occurs at the tissue level.

Reformulating the Hydrolytic Enzyme Cocktail of Trichoderma reesei by Combining XYR1 Overexpression and Elimination of Four Major Cellulases to Improve Saccharification of Corn Fiber.

The industrial fungus Trichoderma reesei has an outstanding capability of secreting an enzyme cocktail comprising multiple plant biomass-degrading enzymes. Herein, the overexpression of XYR1, the master transactivator controlling (hemi)cellulase gene expression, was performed in T. reesei lacking four main cellulase-encoding genes. The resultant strain Δ4celOExyr1 was able to produce a dramatically different profile of secretory proteins on soluble glucose or lactose compared with that of the wild-type T. reesei. The Δ4celOExyr1 secretome included cellulases EGIII and BGLI as well as several hemicellulases and nonhydrolytic cellulose degradation-associated proteins that are not preferentially induced in the wild-type T. reesei strain. Δ4celOExyr1 produced a significant amount of α-arabinofuranosidase I on lactose, and the crude enzyme cocktail of Δ4celOExyr1 not only released a considerable quantity of glucose but also exhibited remarkable performance in the hydrolytic release of xylose, arabinose, and mannose from un-pretreated corn fiber. These results showed that the engineered T. reesei strain holds great potential for improving the saccharification efficiency of the hemicellulosic constituents within corn fiber.

Dynamic tracking and identification of tissue-specific secretory proteins in the circulation of live mice

Secretory proteins are an essential component of interorgan communication networks that regulate animal physiology. Current approaches for identifying secretory proteins from specific cell and tissue types are largely limited to in vitro or ex vivo models which often fail to recapitulate in vivo biology. As such, there is mounting interest in developing in vivo analytical tools that can provide accurate information on the origin, identity, and spatiotemporal dynamics of secretory proteins. Here, we describe iSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which selectively labels proteins that transit through the classical secretory pathway via catalytic actions of Sec61b-TurboID, a proximity labeling enzyme anchored in the ER lumen. To validate iSLET in a whole-body system, we express iSLET in the mouse liver and demonstrate efficient labeling of liver secretory proteins which could be tracked and identified within circulating blood plasma. Furthermore, proteomic analysis of the labeled liver secretome enriched from liver iSLET mouse plasma is highly consistent with previous reports of liver secretory protein profiles. Taken together, iSLET is a versatile and powerful tool for studying spatiotemporal dynamics of secretory proteins, a valuable class of biomarkers and therapeutic targets.