Tissue Lysates Research Articles

A Method for Fabricating Tissue-Specific Extracellular Matrix Blocks From Decellularized Tissue Powders.

Decellularized tissues retain the extracellular matrix (ECM), shape, and composition that are unique to the source tissue. Previous studies using decellularized tissue lysates and powders have shown that tissue-specific ECM plays a key role in cellular function and wound healing. However, creating decellularized tissues composed of tissue-specific ECM with customizable shapes and structures for use as scaffolding materials remains challenging. In this study, a method for compacting decellularized tissue powder into blocks is developed using cold isostatic pressing (CIP). Custom-shaped ECM blocks and composite ECM blocks are fabricated using silicone molds. Additionally, an ECM block with a two-layer structure is obtained through a two-step CIP process. Cells are observed to infiltrate porous ECM blocks that are created using sodium chloride and transglutaminase. These results highlight the development of an effective method for producing ECM blocks using CIP with customizable shapes, compositions, and structures, making them suitable for use as cell culture scaffolds.

Efficient Biomarker for Immunotherapy: Measuring Broad Clones Effector Tumor Antigen-Specific T Cells in the Blood of Esophageal Cancer Patients.

Cancer is the result of the interactions between tumor cells and tumor-specific immune responses. The current biomarkers detect tumor cells' properties, but accurate measurement of tumor-specific immunity is lacking. Most immunotherapies work by activating new effector tumor antigen-specific T cells (ETASTs) or reactivating pre-existing ETASTs' repertoire. The responses to immunotherapy depend on the increase of ETASTs. The amount of ETASTs, especially in blood, is critical for therapeutic efficacy. Distinguishing ETASTs from other T cells by their structural characteristics is difficult. Therefore, nanoparticles loading whole tumor antigens are utilized to activate broad clones ETASTs pre-existing in peripheral blood, followed by detecting them. Thus, the differences between ETASTs and other T cells are transformed to the differences between activated states and unactivated states. By measuring the markers of activated states and cytotoxic functions, we can distinguish ETASTs from other T cells. Nanoparticles loading mixed multiple allogeneic tumor tissue lysates or mixed multiple tumor cell lines can be utilized as universal nanoparticles to replace nanoparticles loading personalized tumor tissue. ETASTs (TATAN-activated CD8+IFN-γ+) in esophageal cancer patients are more than those in healthy people. Measurement of the ETASTs in the blood of esophageal cancer patients before and after ongoing therapy showed that ETATSs increased in the blood of patients who were responsive to immunotherapy but did not increase in the blood of nonresponders. These illustrated that therapeutic efficacy was positively correlated with the level of ETASTs in PBMC. Altogether, this study provides us a highly accurate and specific biomarker for predicting the therapeutic efficacy of cancer immunotherapy and potentially other therapies, such as radiotherapy.

Abstract 4138661: Targeting Arginase I Alleviates Cardiomyopathy

Introduction: Emerging data have shown that pathological cardiac fibrosis and heart failure is driven by recruitment of CD4 + T cells to injured heart tissue. Systemic deletion of CD4 + T cells or splenectomy has been shown to attenuate CD4 + T cell heart infiltration and inflammation. We engineered cargo-less nanoparticles using a biocompatible and biodegradable polymer that target splenic macrophages to overexpress arginase I, leading to local depletion of L-arginine, an amino acid crucial for CD4 + T cell activation and expansion. Hypothesis: Cargo-less nanoparticles attenuate CD4 + T cell activation, expansion, and heart infiltration in experimental models of cardiomyopathy by regulating cross talk between macrophages and CD4 + T cells via L-arginine depletion in the spleen. Methods: Spleen-targeting, cargo-less nanoparticles were synthesized from poly(L-lactide-co-glycolide) (PLLGA) and administered to murine and human macrophages in vitro. Arginase I expression and activity were assessed via Western blotting and arginase activity assay. Epigenetic modulation was analyzed using Western blotting and CHIP-seq. Conditioned media was collected to culture CFSE-labeled T cells to assess proliferation. The nanoparticles were administered to mouse heart failure models induced by isoproterenol or angiotensin II and phenylephrine. Immune cell composition in mouse spleens and hearts was investigated by flow cytometry. Cardiac function was assessed by echocardiography and heart fibrosis was examined via immunohistochemistry and Western blotting. Results: Primary murine macrophages treated with our nanoparticles exhibited a dose-dependent T cell suppression in vitro. This effect was abrogated when an arginase inhibitor was administered together with the nanoparticles. The nanoparticles upregulated arginase I protein expression and arginase activity by increasing histone lactylation. In vivo testing with mouse heart injury models showed that the nanoparticles restored levels of effector and regulatory T cells in injured mice to non-injured levels. Additionally, the nanoparticles reduced cardiac fibrosis and restored ejection fraction. Furthermore, Western blotting of heart tissue lysate revealed that the nanoparticles downregulated the TGFb and p-SMAD signaling pathway. Conclusion: Collectively, these findings highlight a novel therapeutic strategy for heart failure, addressing the gap of fibrosis treatment by targeting upstream immunological pathways.

Locally increased dysferlin expression improves left-ventricular function by stabilizing membrane nanodomains of cardiomyocytes in the myocardial infarction border zone

Abstract Introduction Dysferlin, a transmembrane protein with multiple Ca2+-binding C2-domains, is expressed in the sarcolemma and vesicle membranes of striated muscle cells. Upon Ca2+ entry, Dysferlin is thought to mediate vesicle fusion and membrane repair events in order to rapidly seal sarcolemmal lesions. Dysferlin mutations are associated with progressive muscular dystrophies and dilated cardiomyopathy. However, the precise role of Dysferlin in protection of membrane nanodomains in ventricular myocytes (VMs) after myocardial infarction (MI) remains elusive. Purpose We hypothesized that Dysferlin is crucial to prevent VMs from loss-of-function and cell death by stabilizing membrane nanodomains like the transverse-axial tubule (TAT) system and the intercalated disc (ICD) cell-cell contact sites post-MI. Methods and Results 1 week post-MI induced by LAD artery ligation, investigator-blinded echocardiography revealed increased infarct sizes and decreased left-ventricular (LV) ejection fraction of Dysferlin-knockout (KO) vs. WT mice (mean±SEM: 21±2 vs. 28±2%, n=20/30 mice, P<0.05). Immunoblotting of WT LV tissue lysates showed an upregulated Dysferlin expression to 148% compared to sham-operated controls. Importantly, immunohistology identified a maximal Dysferlin expression of 230% in the MI border zone that faces a high ischemic and mechanical stress. Hence, we used data-independent mass spectrometry (DIA-MS) to analyse the proteomic profile of the infarction zone, border zone and remote zone from WT vs. KO hearts 1 week post-MI (each n = 5 mice). Overall, we reproducibly quantified 4360 proteins across all LV samples, identifying local genotype-specific proteotypes post-MI. In WT vs. KO samples, DIA-MS revealed 725 differentially abundant proteins in the zone pairwise comparison, highlighting a vital role of Dysferlin for LV remodelling post-MI. In addition, superresolution stimulated emission depletion (STED) microscopy was applied to study the subcellular localization and function of Dysferlin in the MI border zone. Here, we observed VMs with disrupted TAT endomembrane networks and significantly extended ICD regions. Interestingly, STED imaging revealed Dysferlin clusters that highly decorated residual TAT structures and interdigitated ICD membrane folds in the MI border zone. DIA-MS analysis of anti-Dysferlin co-imunoprecipitation experiments from WT vs. KO LV tissues characterized the cardiac interactome of Dysferlin, confirming interactions with proteins of the TAT system and the gap junction hemichannel Connexin-43. Conclusions Our data demonstrate Dysferlin as an endogenous membrane repair protein necessary to stabilize sarcolemmal nanodomains in the MI border zone. Dysferlin not only prevents from loss of TAT structures, but also compensates for increased mechanical stress at the ICDs. Hence, Dysferlin emerges as a novel therapeutic target to control membrane integrity in VMs to prevent from loss-of-function and cell death post-MI.

Integration of four-dimensional proteomics and network pharmacology to reveal molecular mechanisms of multi-components multi-targets effects of Sini decoction on myocardial infarction

Sini Decoction (SND) has been proven to be an effective formula to alleviate cardiac injury of myocardial infarction (MI). However, the potential mechanism of SND remains unclear. In this study, the MI rat model was established by ligating the left anterior descending coronary artery. A total of 17 SND-distributed components in heart were identified by using ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOFMS). The combination of four-dimensional (4D) proteomics and network pharmacology was employed to find the potential targets for therapeutic intervention, and molecular docking and cellular thermal shift assay (CETSA) were used to reveal the interactions between the potential targets and the potential active components distributed in heart of SND. 33 SND-effected proteins were identified by 4D proteomics, which was involved in carbon metabolism, fatty acid metabolism, valine, leucine and isoleucine degradation, tricarboxylic acid (TCA) cycle and PPAR signaling pathway. 17 potential SND-targeted direct proteins were screened by comparing SND-effected proteins generated from 4D proteomics with the MI-related proteins obtained from disease database. The potential relationships between 17 components and 17 potential SND-targeted direct proteins were established by molecular docking analysis, in which songorine, benzoylhypaconine, hypaconine, formononetin, and liquiritigenin could be bound to the surrounding amino acid residues in the binding pocket of Mtor, Parp1, Acadm, Crat, and Aldh2. Then, CETSA analysis further confirmed that songorine and benzoylhypaconine could increase the heat stability of Mtor and Parp1 in cardiac tissue lysate, respectively, which suggested that there existed direct interactions between songorine and Mtor, and benzoylhypaconine and Parp1. In summary, this work concluded that SND produced cardioprotective effects mainly through preserving energy metabolism, also demonstrated that the combination of 4D proteomics and network pharmacology was a promising tool for uncovering the molecular mechanisms of multi-components multi-targets effects of TCM.

Lack of Cannabinoid Type 2 Promoter Activity in Normal or Injured Kidneys Using a Cnr2-GFP Reporter Mouse.

Introduction: Although cannabinoid type 2 (CB2) receptor activity is known to promote diverse biological functions in the kidney, published data regarding CB2 receptor protein levels and cellular distribution within the kidney is inconsistent. The goal of the present study was to investigate the changes of CB2 in the kidney obtained from mice exposed to various forms of kidney injury using a genetic mouse model expressing green fluorescent protein (GFP) driven by the endogenous cannabinoid receptor 2 (Cnr2) promoter. Materials and Methods: Kidney injury was established in a genetic mouse model expressing green fluorescent protein (GFP) driven by the endogenous Cnr2 promoter. Kidney injury was initiated by either treatment with different chemicals [cisplatin or lipopolysaccharide (LPS)] or by unilateral ureteral obstruction (UUO). Changes in the detection of GFP were used as a proxy for CB2 levels and localization. Histological changes due to the injury stimuli were observed by time-related, morphological changes in kidney cytoarchitecture and blood parameters, such as serum creatinine levels. Cnr2 mRNA levels were detected by reverse transcription coupled to polymerase chain reaction (RT-PCR) while protein changes in the tissue lysates were measured by Western blot analysis. Cellular localization of GFP was detected by fluorescent microscopy. Results: Our data demonstrated that there was no band or a minimally detectable band for GFP using kidney lysates from vehicle- or cisplatin-treated mice. A similar lack of GFP was detected in the UUO kidney versus the contralateral control kidney. This is consistent with the low, albeit detectable levels of Cnr2 mRNA in the kidney samples from control or cisplatin treatment. In frozen kidney sections from vehicle and cisplatin-treated mice, GFP fluorescence was not detectable in tubular epithelia, glomeruli or blood vessels in the cortex. Instead, GFP was detected in rare cells within the interstitial space. A second chemical injury model using LPS found a similar lack of GFP protein levels and an absence of legitimate GFP fluorescence in the main cell types within the kidney. Conclusion: These findings suggest that Cnr2 promoter activity is minimally active in normal or injured kidneys, and that pharmacological manipulation of CB2 receptors may be associated with receptors being expressed in cells recruited to the kidney.

Intestinal Epithelial PTPN2 Limits Pathobiont Colonization by Immune-Directed Antimicrobial Responses.

Loss of activity of the inflammatory bowel disease (IBD) susceptibility gene, protein tyrosine phosphatase non-receptor type 2 (PTPN2), is associated with altered microbiome composition in both human subjects and mice. Further, expansion of the bacterial pathobiont, adherent-invasive E. coli (AIEC), is strongly linked to IBD pathogenesis. The mechanism by which intestinal epithelial cells (IEC) maintain equilibrium between commensal microbiota and immune cells to restrict invading pathobionts is poorly understood. Here, we investigated the role of IEC-specific PTPN2 in regulating AIEC colonization. Tamoxifen-inducible, intestinal epithelial cell-specific Ptpn2 knockout mice (Ptpn2 ΔIEC) and control Ptpn2 fl/fl mice were infected with either non-invasive E. coli K12, or fluorescent-tagged mAIEC (mAIECred) for four consecutive days or administered PBS. Subsequently, bacterial colonization in mouse tissues was quantified. mRNA and protein expression were assayed in intestinal epithelial cells (IECs) or whole tissue lysates by PCR and Western blot. Tissue cytokine expression was determined by ELISA. Intestinal barrier function was determined by in vivo administration of 4 kDa FITC-dextran (FD4) or 70kDa Rhodamine-B dextran (RD70) fluorescent probes. Confocal microscopy was used to determine the localization of tight-junction proteins. Ptpn2 ΔIEC mice exhibited increased mAIECred - but not K12 - bacterial load in the distal colon compared to infected Ptpn2 fl/fl mice. The higher susceptibility to mAIECred infection was associated with altered levels of antimicrobial peptide (AMPs). Ileal RNA expression of the alpha-defensin AMPs, Defa5 and Defa6, as well as MMP7, was significantly lower in Ptpn2 ΔIEC vs. Ptpn2 fl/fl mice, after mAIECred but not K12 infection. Further, we observed increased tight junction-regulated permeability determined by elevated in vivo FD4 but not RD70 permeability in Ptpn2 ΔIEC-K12 mice compared to their respective controls. This effect was further exacerbated in Ptpn2 ΔIEC mAIEC-infected mice. Further, Ptpn2 ΔIEC mice displayed lower IL-22, IL-6, IL-17A cytokine expression post mAIEC infection compared to Ptpn2 fl/fl controls. Recombinant IL-22 reversed the FD4 permeability defect and reduced bacterial burden in Ptpn2 ΔIEC mice post mAIEC challenge. Our findings highlight that intestinal epithelial PTPN2 is crucial for mucosal immunity and gut homeostasis by promoting anti-bacterial defense mechanisms involving coordinated epithelial-immune responses to restrict pathobiont colonization.

A mouse model of overexpression of human IL-6 by tissue-resident macrophages in the context of LPS-induced inflammation

Chronic inflammation caused by overexpression of IL-6 underlies a number of pathological conditions Mouse models of systemic chronic inflammation with overexpression of human IL-6 (hIL-6) are in demand not only in the context of studying the molecular mechanisms of inflammation, but also in assessing the effectiveness of clinically approved or newly developed IL-6 inhibitors. One experimental approach in addressing such models in mice relies on the induction of systemic acute inflammation in response to lipopolysaccharide (LPS) administration. This work describes mice with tamoxifen-dependent overexpression of human IL-6 in CX3CR1+ myeloid cells in the context of systemic inflammation induced by LPS administration. Our study demonstrates that the highest expression of the transgene carrying IL6 was observed in the heart, while high production of this cytokine was detected in the blood serum. In response to LPS administration, the production of hIL-6 in the blood increased in transgenic mice, while the production of mIL-6 also increased and was comparable to that in wild-type mice. The consequences of high systemic production of hIL-6, which in our model originates from CX3CR1+ tissue-resident macrophages, were noticeable even in the organs in which these cells are not present. Thus, significant amounts of hIL-6 were detected in tissue lysates of the lungs of transgenic mice after LPS administration. Evaluation of the expression of genes encoding cytokines and markers of tissue remodeling upon injury using quantitative real-time PCR showed significant changes in their expression in the context of LPS-induced systemic inflammation. Thus, this work demonstrates the feasibility of using a mouse model with tamoxifen-dependent transgene activation in CX3CR1+ tissue-resident macrophages to study the effects of systemic overexpression of IL-6 and pharmacological blockade of this cytokine with clinically approved or newly developed inhibitors in the context of experimentally induced diseases.

Edwardsiella tarda induces airways inflammation and production of autoantibodies against lung tissues through regulation of the IL-33-ST2 axis.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent chronic respiratory disease characterised by irreversible airways obstruction associated with chronic airways inflammation and remodelling, while the pathogenesis and the mechanistic differences between patients remain to be fully elucidated. We previously reported that alarmin cytokine IL-33 may contribute to the production of autoantibodies against respiratory epithelial cells. Here we expand the hypothesis that pulmonary autoimmune responses induced by airway microbiota also contribute to the progression of COPD. We focused on Edwardsiella tarda which we detected uniquely in the induced sputum of patients with acute exacerbations of COPD. Pernasal challenge of the airways of WT mice with supernatants of cultured E. tarda induced marked, elevated expression of IL-33 in the lung tissues. Immunisation of animals with supernatants of cultured E. tarda resulted in significantly elevated airways inflammation, the formation of tertiary lymphatic structures and significantly elevated proportions of T follicular helper T cells in the lung tissue and mediastinal lymph nodes. Interestingly, such challenge also induced production of IgG autoantibodies directed against lung tissue lysate, alveolar epithelial cell proteins and elastin fragment, while putrescine, one of metabolites generated by the bacterium, might play an important role in the autoantibody production. Furthermore, all of these effects were partly but significantly abrogated in mice with deletion of the IL-33 receptor ST2. Collectively, these data support the hypothesis that COPD is progressed at least partly by airways microbiota such as E. tarda initiating autoimmune attack of the airways epithelium mediated at least partly through the IL-33-ST2 axis.

Screening and identification of peptidyl arginine deiminase 4 inhibitors from herbal plants extracts and purified natural products by a trypsin assisted sensitive immunoassay based on streptavidin magnetic beads

Peptidyl arginine deiminase 4 (PAD4) plays a critical role in many autoimmune diseases including rheumatoid arthritis. Herein, a trypsin assisted highly immunoassay method was established to determine PAD4 activity and screen potent inhibitors from herbal plants extracts and purified natural products. The method was applied to determine endogenous PAD4 activity in both cell and tissue lysates, as well as the inhibitory effects of 20 herbal plants and 50 purified natural products. The Cinnamomi ramulus extract showed strongest inhibitory potency with IC50 value lower than 5 μg/mL. Meanwhile, pyrroloquinoline quinone (PQQ), widely used as a dietary supplement, was discovered as a promising PAD4 inhibitor with an IC50 value lower than 4 μM. The inhibition kinetic analysis, drug affinity response target stability (DARTS) and molecular docking were performed to confirm the interaction between PQQ and PAD4. This method has great potential for researchers to monitor activities and discover potential inhibitors of PAD4.

Identification of iodothyronines in plant tissues

It has become widespread knowledge that many signaling molecules are common to organisms of different groups. This is likely to be valid for such important metabolism regulators as iodothyronines. A number of studies have confirmed the presence of thyroid hormone activity in compounds of plant origin. However, these studies do not explain whether the compounds under consideration are iodine derivatives of thyronine, similar to animal and human thyroid hormones, or whether they are mimetics of thyroid hormones. In this work, we aim to verify the presence of iodothyronine analogs with different degrees of iodization in plant tissues. We also aim to determine iodine concentrations in plant tissue lysates and to compare them with the theoretically calculated values in order to test the assumption about the identity of their structure to human thyroid hormones. It was shown that tetraiodothyronine (T4) and triiodothyronine (T3) analogs are simultaneously present in potato tubers and wheat leaves. In potato tubers at dormancy, the concentration of T4 was 118 ± 16 nmol/L (n = 15), while the concentration of T3 in the same samples was 4.01 ± 0.96 nmol/L. T4 and T3 concentrations in wheat leaf lysates were 60.24 ± 79 and 6.76 nmol/L (n = 15), respectively. According to the results of inductively coupled plasma mass spectrometry, the studied samples contain iodine in the amounts consistent with the assumption about the presence of tetraiodinated tyronine derivatives.

Sensitization of Europium Oxide Nanoparticles Enhances Signal-to-Noise over Autofluorescence with Time-Gated Luminescence Detection.

Clinical translation of nanoparticle-based therapeutics has been limited, and a lack of preclinical delivery characterization is partly to blame, limiting our understanding of the mechanisms of failure. The improvement of the preclinical delivery assessment requires nanoparticles with higher detectability. This work focused on the exploration of several aromatic carboxylic ligands (terephthalic acid, quinaldic acid, and kynurenic acid) for the sensitization of europium oxide nanoparticles with a long emission lifetime to overcome cellular autofluorescence, a key confounder of detection in luminescence-based bioimaging. A facile one-pot synthesis and ligand exchange process generated and sensitized ultrasmall Eu2O3 cores. As reflected in the emission spectra and lifetimes, ligand binding yielded unique coordination environments around Eu3+. Then, the efficacy of sensitization was tested against the autofluorescence provided by tissue lysate. Normal (simultaneous excite-read) measurements showed integrated signal improvements over autofluorescence of 2.2-, 3.9-, and 14.0-fold for EuTA, EuQA, and EuKA, respectively. In time-gated mode, the improvements over autofluorescence were more dramatic with fold differences of 75-, 89-, and 108-fold for EuTA, EuQA, and EuKA, respectively. The investigation of novel sensitizers expands the breadth of the field of sensitized lanthanide oxide nanoparticles, and the signal enhancement observed with sensitization and time-gating supports the utility of the generated samples for future bioimaging applications.

Online Mixed-Bed Ion Exchange Chromatography for Native Top-Down Proteomics of Complex Mixtures.

Native top-down mass spectrometry (nTDMS) allows characterization of protein structure and noncovalent interactions with simultaneous sequence mapping and proteoform characterization. The majority of nTDMS studies utilize purified recombinant proteins, with significant challenges hindering application to endogenous systems. To perform native top-down proteomics (nTDP), where endogenous proteins from complex biological systems are analyzed by nTDMS, it is essential to separate proteins under nondenaturing conditions. However, it remains difficult to achieve high resolution with MS-compatible online chromatography while preserving protein tertiary structure and noncovalent interactions. Herein, we report the use of online mixed-bed ion exchange chromatography (IEC) to enable separation of endogenous proteins from complex mixtures under nondenaturing conditions, preserving noncovalent interactions for nTDP analysis. We have successfully detected large proteins (>146 kDa) and identified endogenous metal-binding and oligomeric protein complexes in human heart tissue lysate. The use of a mixed-bed stationary phase allowed retention and elution of proteins over a wide range of isoelectric points without altering the sample or mobile phase pH. Overall, our method provides a simple online IEC-MS platform that can effectively separate proteins from complex mixtures under nondenaturing conditions and preserve higher-order structure for nTDP applications.

Pharmacokinetic analysis of OMO-103 in patient tissues with advanced solid tumors.

e15168 Background: MYC is a pleiotropic transcription factor aberrantly expressed in up to 70% of human cancers. OMO-103 (Omomyc) is a 91-amino-acids-long polypeptide that derives from the B-HLH-LZ domain of human MYC, containing four amino acid substitutions. It is capable of forming homos and heterodimers with MYC and with MAX and blunt MYC activity by preventing its access to target DNA sequences (E-boxes) and replacing it by inactive complexes. Previous in vivo preclinical experiments showed that Omomyc blocks MYC-driven transcription and proliferation, induces apoptosis and recruitment of intratumoral immune infiltrates, significantly abrogating tumor progression and reducing tumor grading. Here, we present PK results from the first-in-human trial (NCT04808362) of OMO-103 in patients with advanced solid tumors. Methods: Patient eligibility The study included histologically or cytologically proven advanced solid tumors for which there was no curative therapy. Patients had to demonstrate measurable disease according to RECIST v.1.1 criteria as demonstrated by CT/magnetic resonance imaging and documented progression on or following the last line of therapy. Description and dose selection OMO-103 was administered by weekly I.V. infusion over 30–45 min, one treatment cycle corresponding to three infusions. Patients were treated until progression. The starting dose of 0.48 mg kg−1 was determined based on nonclinical toxicology and efficacy studies (anticipated pharmacologically active dose) and subsequent conversion to a human equivalent dose. Pharmacokinetic assays A targeted mass spectrometry (MS) assay for the absolute quantification of 4 Omomyc proteotypic peptides was developed to quantify functional OMO-103 in tumor tissue. LLOQ and linear range of the panel was determined by spiking in dilution series of recombinant OMO-103 protein into tumor tissue lysates. Finally, in the phase I trial, we used the panel for the quantification of OMO-103 in FFPE tumor biopsies from 12 patients who received i.v. injection of OMO-103. Results: MS Data show that 2 h after i.v. administration, higher concentrations of the drug are reached in the tumors compared with serum and persist there with at least one order of magnitude higher concentration than in serum 72 h after administration in preclinical models. Functional Omomyc protein could be quantified in all 12 clinical tumor biopsies from treated patients and PK results confirmed previous observations in preclinical models. where Omomyc exhibits higher tissue concentration and prolonged residency compared to plasma. Conclusions: We demonstrate the utility of targeted MS assay for precise quantification of therapeutic (mini)proteins in clinical tissue samples. Thanks to its multiplexing capability, up to 60 peptides (epitopes) can be monitored simultaneously which enables high sequence coverage and profiling of additional PD or other supporting biomarkers. Clinical trial information: NCT04808362 .

The biodistribution of polystyrene nanoparticles administered intravenously in the chicken embryo

Nanoplastics can cause severe malformations in chicken embryos. To improve our understanding of the toxicity of nanoplastics to embryos, we have studied their biodistribution in living chicken embryos. We injected the embryos in the vitelline vein at stages 18–19. We injected polystyrene nanoparticles (PS-NPs) tagged with europium- or fluorescence. Their biodistribution was tracked using inductively-coupled plasma mass spectrometry on tissue lysates, paraffin histology, and vibratome sections analysed by machine learning algorithms. PS-NPs were found at high levels in the heart, liver and kidneys. Furthermore, PS-NPs crossed the endocardium of the heart at sites of epithelial-mesenchymal transformation; they also crossed the liver endothelium. Finally, we detected PS-NPs in the allantoic fluid, consistent with their being excreted by the kidneys. Our study shows the power of the chicken embryo model for analysing the biodistribution of nanoplastics in embryos. Such experiments are difficult or impossible in mammalian embryos. These findings are a major advance in our understanding of the biodistribution and tissue-specific accumulation of PS-NPs in developing animals.

Differentiation of bone marrow mesenchymal stem cells into Leydig-like cells with testicular extract liquid in vitro.

Differentiation of Leydig cells plays a key role in male reproductive function. Bone marrow mesenchymal stem cells (BMSCs) have emerged as a potential cell source for generating Leydig-like cells due to their multipotent differentiation capacity and accessibility. This study aimed to investigate the morphological and genetic expression changes of BMSCs during differentiation into Leydig-like cells. Testicular extract liquid, which simulates the microenvironment in vivo, induced the third passage BMSCs differentiated into Leydig-like cells. Changes in cell morphology were observed by microscopy, the formation of lipid droplets of androgen precursor was identified by Oil Red Staining, and the expression of testicular specific genes 3β-HSD and SF-1 in testicular stromal cells was detected by RT-qPCR. BMSCs isolated from the bone marrow of Sprague-Dawley (SD) rats were cultured for 3 generations and identified as qualified BMSCs in terms of morphology and cell surface markers. After 14 days of induction with testicular tissue lysate, lipid droplets appeared in the cytoplasm of P3 BMSCs by Oil Red O staining. RT-qPCR detection was performed on BMSCs on the 3rd, 7th, 14th, and 21st day after induction. Relative expression levels of 3β-HSD mRNA significantly increased after 14 days of induction, while the relative expression of SF-1 mRNA increased after 14 days of induction but was not significant. BMSCs can differentiate into testicular interstitial cells with reserve androgen precursor lipid droplets after induction by testicular tissue lysate. The differentiation ability of BMSCs provides the potential to reconstruct the testicular microenvironment and is expected to fundamentally improve testicular function and provide new treatment options for abnormal spermatogenesis diseases.

The IBD Risk gene, PTPN2, Is Required for Commensal-associated Th17 Responses to Protect Against Pathobiont Colonization

Background/Aim: Inflammatory bowel disease (IBD) involves multiple factors including genetic susceptibilities, intestinal permeability, microbiome dysbiosis, and expansion of intestinal pathobionts such as adherent-invasive E. coli (AIEC). Mice lacking the IBD risk gene, Ptpn2, exhibit expansion of murine adherent-invasive Escherichia coli ( mAIEC strain, UCR-PP2). Internal transcribed spacer analysis of luminal and mucosal-associated bacteria also indicated a decrease in the protective commensal bacteria, segmented filamentous bacteria (SFB). SFB adherence to intestinal epithelial cells (IEC) induces Th17 cell secretion of IL-17 and IL-22 to promote mucosal defense. The goal of this study was to determine if constitutive whole-body, or IEC-specific Ptpn2 loss in mice modulates SFB abundance, adherence, and downstream production of protective Th17 cytokine in mice. Methods: All mice were housed in specific pathogen free (SPF) vivarium. Distal ileum (2-3 cm) was collected from whole body Ptpn2-wild-type (WT), heterozygous (Het) and Ptpn2 constitutive knockout (KO) mice [Balb/c]; or tamoxifen (TMX)-induced IEC specific Ptpn2 knockout mice ( Ptpn2ΔIEC) and TMX-treated control littermates ( Ptpn2fl/fl) [C57Bl/6]. Tissues were fixed and observed using the Hitachi TM4000 Scanning Electron Microscope (SEM) to visualize SFB. qPCR was used to quantify relative abundance of SFB in the Ileum tissue obtained from Ptpn2ΔIEC and Ptpn2fl/fl mice. Confirmed SFB-free C57Bl/6 mice (10 weeks old; JAX labs; Stock# 000664) were infected for four consecutive days by 109 mAIEC bacteria in PBS. Cytokine protein levels were quantified by Luminex Multiplex Array of whole tissue lysates from cecum, proximal and distal colon regions from 18-21day old WT, HET or KO mice. Immune cells were isolated from the cecum, colon, lamina propria, the mesenteric lymph nodes and the spleen of WT, HET and KO mice and analyzed by flow cytometry for IL-22+, and IL-17+ T cells. Results: SFB adherence to IECs in Ptpn2 constitutive KO mice was visibly decreased by SEM compared to WT and Ptpn2 Het littermates. SEM showed a dramatic decrease in SFB abundance in Ptpn2ΔIEC mice compared to control littermates ( Ptpn2fl/fl). This was confirmed by PCR analysis showing a significant decrease in SFB in Ptpn2ΔIEC mice (p<0.05; n=8). The absence of SFB permitted mAIEC to colonize and cause mild disease in SFB-free mice (JAX; p<0.01; n=8). SFB loss in whole-body Ptpn2-KO mice correlated with a decrease in IL-17+ CD4+ cells in the colon compared to Ptpn2-Het (p<0.001; n=7) and Ptpn2-WT littermates (p<0.05; n=5). Ptpn2-KO mice exhibited higher IL-22 levels in the distal colon (p<0.01; n=3), and increased IL-22+CD4+ T-cells in the cecum, mesenteric lymph nodes and spleen compared to Ptpn2-WT and Het littermates (p<0.001). Conclusion: Loss of the IBD risk gene Ptpn2 in mice, either constitutively or in IECs, reduces the abundance of SFB a commensal inducer of Th17 responses. Consistent with reduced SFB, IL-17+CD4+ T cells were reduced in whole-body Ptpn2-KO mice indicating a loss of SFB-induced Th17 maturation. Despite reduced IL-17, we observed increased IL-22 expression in Ptpn2-KO mice suggesting that loss of PTPN2 provokes a compensatory SFB/IL-17-independent increase in IL-22 production. These findings identify how loss of PTPN2 activity disrupts homeostatic commensal-mucosal immune crosstalk to facilitate IBD-relevant pathobiont colonization. This study was supported by grants NIH-2R01-DK091281, 1R01AI153314-01, R21AI152017, 1R01DK130373, and R01AI165490 (D.F.M.) from the National Institutes of Health (NIH). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Altered lactate/pyruvate ratio may be responsible for aging-associated intestinal barrier dysfunction in male rats

Some evidence points to a link between aging-related increased intestinal permeability and mitochondrial dysfunction in in-vivo models. Several studies have also demonstrated age-related accumulation of the of specific deletion 4834-bp of “common” mitochondrial DNA (mtDNA) in various rat tissues and suggest that this deletion may disrupt mitochondrial metabolism. The present study aimed to investigate possible associations among the mitochondrial DNA (mtDNA) common deletion, mitochondrial function, intestinal permeability, and aging in rats. The study was performed on the intestinal tissue from (24 months) and young (4 months) rats. mtDNA4834 deletion, mtDNA copy number, mitochondrial membrane potential, and ATP, lactate and pyruvate levels were analyzed in tissue samples. Zonulin and intestinal fatty acid-binding protein (I-FABP) levels were also evaluated in serum. Serum zonulin and I-FABP levels were significantly higher in 24-month-old rats than 4-month-old rats (p = 0.04, p = 0.026, respectively). There is not significant difference in mtDNA4834 copy levels was observed between the old and young intestinal tissues (p > 0.05). The intestinal mitochondrial DNA copy number was similar between the two age groups (p > 0.05). No significant difference was observed in ATP levels in the intestinal tissue lysates between old and young rats (p > 0.05). ATP levels in isolated mitochondria from both groups were also similar. Analysis of MMP using JC-10 in intestinal tissue mitochondria showed that mitochondrial membrane potentials (red/green ratios) were similar between the two age groups (p > 0.05). Pyruvate tended to be higher in the 24-month-old rat group and the L/P ratio was found to be approximately threefold lower in the intestinal tissue of the older rats compared to the younger rats (p < 0.002). The tissue lactate/pyruvate ratio (L/P) was three times lower in old rats than in young rats. Additionally, there were significant negative correlations between intestinal permeability parameters and L/P ratios. The intestinal tissues of aged rats are not prone to accumulate mtDNA common deletion, we suggest that this mutation does not explain the age-related increase in intestinal permeability. It seems to be more likely that altered glycolytic capacity could be a link to increased intestinal permeability with age. This observation strengthens assertions that the balance between glycolysis and mitochondrial metabolism may play a critical role in intestinal barrier functions.

Impaired local Vitamin D3 metabolism contributes to IL-36g overproduction in epithelial cells in chronic rhinosinusitis with nasal polyps.

Vitamin D (VD) possesses immunomodulatory properties, but its role in chronic rhinosinusitis with nasal polyps (CRSwNP) remains poorly studied. Herein, we aim to explore the regulation and function of VD3 in CRSwNP. 25-hydroxyvitamin D3 (25VD3) levels in serum and tissue lysates were detected by ELISA. The expression of VD receptor (VDR) and cytochrome P450 family 27 subfamily B member 1 (CYP27B1), the enzyme that converts 25VD3 to the active 1,25-hydroxyvitamin D3 (1,25VD3), and their expression regulation in human nasal epithelial cells (HNECs) were studied by RT-PCR, western blotting, immunofluorescence, and flow cytometry. RNA sequencing was performed to identify genes regulated by 1,25VD3 in HNECs. HNECs and polyp tissue explants were treated with 1,25VD3, 25VD3, and dexamethasone. 25VD3 levels in serum and nasal tissue lysates were decreased in patients with eosinophilic and noneosinophilic CRSwNP than control subjects. The expression of VDR and CYP27B1 were reduced in eosinophilic and noneosinophilic CRSwNP, particularly in nasal epithelial cells. VDR and CYP27B1 expression in HNECs were downregulated by interferon y and poly (I:C). Polyp-derived epithelial cells demonstrated an impaired ability to convert 25VD3 to 1,25VD3 than control tissues. 1,25VD3 and 25VD3 suppressed IL-36y production in HNECs and polyp tissues, and the effect of 25VD3 was abolished by siCYP27B1 treatment. Tissue 25VD3 levels negatively correlated with IL-36y expression and neutrophilic inflammation in CRSwNP. Reduced systemic 25VD3 level, local 1,25VD3 generation and VDR expression result in impaired VD3 signaling activation in nasal epithelial cells, thereby exaggerating IL-36y production and neutrophilic inflammation in CRSwNP.

Abstract 2055: Delivery of oligonucleotide therapeutic drugs: Universal detection reagents specific for nucleic acid modifications

Abstract Nucleic acid therapies, including oligonucleotide therapeutics (ONTs) and vaccines, have increased in development, respectively, since their inception as an emerging drug modality and after the first mRNA COVID-19 vaccine was granted EUA approval in 2020. RNA-based therapies, such as antisense oligonucleotides (ASO), RNAi, aptamers, and mRNA, are designed to modulate protein synthesis in patients and have applications in oncology, gene therapy, and infectious disease prevention. Strategies for ONT drug delivery have advanced over recent years with the goal of overcoming limitations when targeting extrahepatic tissues. Improving the “drug-likeness” of ONTs is facilitated by chemical modifications to the sugar-phosphate backbone and/or nitrogenous bases. Modifications are designed to increase ONT drug stability, uptake, and efficacy, but may increase the likelihood of toxicity. First generation chemical modifications include phosphorothioate (PS) modification of the phosphate portion of the backbone, whereas second generation modifications include adding substituent groups to the 2’ position of the ribose portion of the backbone, specifically 2’-O-methyl (OME), 2’-O-methoxy-ethyl (MOE), and 2’-fluoro modifications of RNA. Third generation modifications include peptide nucleic acids (PNA), locked nucleic acids (LNA), morpholino phosphoroamidate (PMO) modifications, and others. We have developed, optimized, and validated a library of monoclonal antibody reagents that detect these chemical modifications independent of nucleic acid composition, structure, strandedness, configuration, or platform. These “universal detection reagents” can be used in (1) immunohistochemistry (IHC) to determine biodistribution, (2) immunofluorescence (IF) to determine intracellular localization, (3) ELISA to determine anti-drug antibody binding, immunogenicity, and drug ranking, (4) immunoprecipitation to determine protein binding in serum and/or tissue lysates, (5) in vivo cell culture studies to determine potency, and (6) other immunoassays. While in situ hybridization (ISH) is an established method for the detection and localization of ONTs in cells and tissues, this method has limitations that include lack of utility (as unique probes must be designed individually for each ONT), high costs in time and materials to produce probes, the inability of ISH to bind and detect short ONT drug sequences, and difficulties encountered when repeated sequences are present within the ONT sequence. Here we report differential sensitivity and specificity of panels of antibodies specific for PS and MOE modifications. We demonstrate the performance of these antibodies by various in vitro assays that show the utility of these specialty reagents to facilitate the collection of analytical data to support ADMET studies useful to ensure ONT approval by regulatory agencies. Citation Format: Todd Giardiello, Amy Anderson, Renee Salvo, Carl Ascoli. Delivery of oligonucleotide therapeutic drugs: Universal detection reagents specific for nucleic acid modifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2055.