7196 research outputs found
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Engineered 3D Kidney Glomerular Microtissues to Model Podocyte-Centric Diseases for the Validation of New Drug Targets.
No full textPodocytopathies are a diverse group of kidney diseases characterized by podocyte injury, leading to proteinuria and reduced kidney function. This injury often disrupts cytoskeletal dynamics and cellular adhesion, causing glomerular dysfunction. Current in vitro models fail to accurately mimic the three-dimensional (3D) organization and mechanics of kidney tissue, hindering the understanding of podocyte pathophysiology and therapeutic development. In this study, 3D microtissues are developed that replicate the structure and mechanics of the glomerular filtration barrier, enabling the modeling of chemically and genetically induced podocyte injuries for drug target validation. These microtissues simulate the glomerulus's three-layer structure and hemodynamic mechanical stretch, providing a platform to evaluate relevant mechanobiological signaling pathways and podocyte dynamics. Collective cellular forces are measured to assess podocyte resilience against genetic or chemical injuries. As a proof-of-concept, podocyte injury is modeled through transient receptor potantial canonical 6 (TRPC6) overexpression, a validated target in podocytopathies, and evaluated by the TRPC6 inhibitor SAR7334. The results demonstrated a loss of podocyte contractile forces upon TRPC6 overexpression, with recovery following treatment. This highlights the potential of glomerular microtissues to model podocyte mechano-pathophysiology and serves as a robust platform for screening new therapies
Expanding the repertoire of chemically induced covalent neoantigens
No full textThe ubiquitin-proteasome system generates peptide fragments that are displayed on MHC-I molecules. Recent studies revealed that covalent small molecules can hijack this process, leading to the presentation of hapten-modified peptide fragments, or covalent neoantigens. Here, we report a chemical proteomics platform to systematically investigate this process and harness covalent neoantigens for immune cell recruitment and activation. Using a bioorthogonal cysteine-reactive probe (IA-mTz), we demonstrate that diverse intracellular proteins can be modified, processed, and presented as covalent neoantigens across multiple cell lines. Mass spectrometry confirmed the presence of IA-mTz-modified peptides within the MHC-I immunopeptidome, and structural modeling revealed stable peptide-MHC-I interactions. We further integrated a bioorthogonal strategy that enables immune engagement: IA-mTz-modified cells conjugated with trans-cyclooctene (TCO)-IgG elicited Fc receptor activation in a reporter assay, supporting immune recruitment via antibody-dependent cellular phagocytosis. This approach was extended to additional cysteine-reactive probes and to 2,4-dinitrochlorobenzene (DNCB), a common skin sensitizer, which we show induces covalent neoantigen formation in a cell- and HLA-dependent manner. These findings reveal a previously underappreciated breadth of covalent neoantigen formation and provide a generalizable strategy for immune targeting of covalent neoantigens
The Impact of the Tumor Microenvironment on the Effect of IL-1β Blockade in NSCLC: Biomarker Analyses from CANOPY-1 and CANOPY-N Trials.
No full textPreclinical studies have shown that interleukin (IL)-1β blockade can modulate the tumor microenvironment (TME) to activate antitumor immunity and, in combination with immune checkpoint inhibitors (ICIs), prevent cancer growth. Our study investigates if immune biomarkers in the TME impact outcomes in patients with non-small cell lung cancer (NSCLC) treated with the IL-1β inhibitor canakinumab plus an ICI-based therapy and describes canakinumab effects on the TMEs of these patients.Exploratory analyses were conducted in two prospective trials evaluating canakinumab combined with pembrolizumab-based regimens in patients with NSCLC: CANOPY-1 (first-line setting) and CANOPY-N (neoadjuvant setting). Immunohistochemistry and transcriptomic analyses were performed on baseline tumor samples from CANOPY-1 and immunohistochemistry and multiplex immunofluorescence analyses were performed on baseline and post-treatment tumor samples from CANOPY-N. Associations with clinical outcomes were evaluated.In CANOPY-1, in patients with low levels of T-cell infiltration in the tumor, the addition of canakinumab to a pembrolizumab-based regimen was associated with progression-free and overall survival improvements. Low levels of T-cell infiltration were associated with an immunosuppressive gene expression phenotype, supporting the role of low T-cell infiltration as a surrogate of an overall immunosuppressive TME. In CANOPY-N, a reduction in immunosuppressive cells in the TME was observed following canakinumab and pembrolizumab treatment.Our exploratory biomarker analyses from the CANOPY-1 and CANOPY-N trials suggest that IL-1β blockade may shift the TME towards an immune-activated status and that patients with immunosuppressive TME features could benefit from the addition of canakinumab to an ICI-based treatment
Scientific and Regulatory Policy Committee Points to Consider: Proposal and Recommendations to Reduce Euthanasia of Control Nonhuman Primates in Nonclinical Toxicity Studies
No full textNonhuman primates (NHPs) have been and remain a highly valuable animal model with an essential role in translational research and pharmaceutical drug development. Based on current regulatory guidelines, the nonclinical safety of novel therapeutics should be evaluated in relevant nonclinical species, which commonly includes the NHP for biotherapeutics. Given the practical and ethical limitations on availability and/or use of NHPs and in line with the widely accepted guiding “3 Rs” principles, many approaches have been considered to optimize toxicity study designs to meaningfully reduce the use of NHPs. Standard general toxicity studies usually include four groups of equal size, including one group of vehicle control animals. Here we describe an approach to achieve an overall significant reduction in control animal use, while also resolving many of the issues that may limit application of fully virtual control animals. We propose in GLP-compliant safety studies to maintain concurrent control group animals for the in-life phase of the studies, but to limit euthanasia to a subset of control animals. The non-terminated control animals can then be returned to the facility colony for reuse in subsequent studies. The proposed study design could lead to a 15 to 20% reduction in NHP usage. The scientific, logistical, and animal welfare considerations associated with such an approach and suggested solutions are discussed in detail
2024 White Paper on Recent Issues in Bioanalysis: Three Way-Cross Validation; Urine Clinical Analysis; Automated Methods; Regulatory Queries on Plasma Protein Binding; Automated Biospecimen Management; ELN Migration; Ultra-Sensitivity Mass Spectrometry (Part 1A - Recommendations on Advanced Strategies for Mass Spectrometry Assays, Chromatography, Sample Preparation and BMV/Regulated Bioanalysis Part 1B - Regulatory Agencies' Inputs on Regulated Bioanalysis/BMV).
No full textThe 18th Workshop on Recent Issues in Bioanalysis (18th WRIB) took place in San Antonio, TX, USA on May 6-10, 2024. Over 1100 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 18th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.Moreover, in-depth workshops on "IVDR Implementation in EU & Changes for LDT in the US" and on "Harmonization of Vaccine Clinical Assays Validation" were the special features of the 18th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues.This 2024 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2024 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication (Part 1) covers in Part 1A the Recommendations on Mass Spectrometry Assays and Regulated Bioanalysis/BMV and in Part 1B the Regulatory Inputs on these topics. Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) and Part 2 (Biomarkers/BAV, IVD/CDx, LBA and Cell-Based Assays) are published in volume 17 of Bioanalysis, issues 3 and 4 (2025), respectively
Designing and developing a prescription digital therapeutic for at home heart rate variability biofeedback to support and enhance patient outcomes in post-traumatic stress disorder treatment
No full textPost-traumatic stress disorder (PTSD) is a psychiatric condition producing considerable distress, dysfunction, and impairment in affected individuals. While various forms of psychotherapy are commonly utilized in PTSD treatment, the known neurological pathologies associated with PTSD are insufficiently addressed by these conventional approaches. Heart rate variability biofeedback (HRV-BFB) is a promising tool for correcting autonomic dysfunction in PTSD, with subsequent changes in clinically significant outcome measures. This paper outlines a systematic approach for the development, distribution, and implementation of a prescription at-home HRV-BFB digital therapeutic. We provide recommendations for evidence-generation strategies and propose appropriate regulatory pathways within existing frameworks. Widespread access to HRV-BFB could potentially reduce the distress, disability, and healthcare burden associated with PTSD. Promoting HRV-BFB as a primary intervention could also serve to reduce the stigma associated with ‘mental’ illness and increase health literacy regarding the neuroimmune impacts of psychosocial factors. These processes might in turn improve treatment-seeking, adherence, and supported self-management of these conditions
Complete loss of SLC30A8 in humans improves glucose metabolism and beta cell function.
No full textGenetic association studies have demonstrated that partial loss of SLC30A8 Function protects against type 2 diabetes in humans. We investigated the impact of complete loss of SLC30A8 Function on type 2 diabetes risk and related phenotypes in humans.The Pakistan Genome Resource (PGR), a biobank comprising whole-exome and whole-genome sequences of 145,037 participants, was analysed for phenotypic associations with SLC30A8 loss-of-function (LoF) variants. To follow up on the observations in the PGR, we conducted recall-by-genotype analyses of SLC30A8 LoF heterozygotes and homozygotes, as well as their participating family members, using OGTTs.We identified 18 SLC30A8 knockouts, including homozygotes for a variant enriched in South Asians (Gln174Ter), and 1024 heterozygotes for LoF variants. Type 2 diabetes risk was lower in SLC30A8 LoF heterozygotes and homozygotes relative to non-carriers, and the protective effect strengthens in a gene dose-dependent manner (ORadditive=0.62; 95% CI 0.53, 0.72; p=1.1×10-9; ORrecessive=0.34; 95% CI 0.12, 0.93; p=0.04). OGTTs in recall-by-genotype studies showed a gene dose-dependent reduction in glucose levels, coupled with elevated insulin.The corrected insulin response, disposition index and insulin sensitivity index in LoF heterozygotes and homozygotes indicated higher glucose-stimulated insulin secretion with preserved beta cell function that was independent of BMI. These data suggest that therapeutic inhibition of SLC30A8, up to and including complete knockout, may treat type 2 diabetes safely and effectively
Predictors of response and rational combinations for the novel MCL-1 inhibitor MIK665 in acute myeloid leukemia.
No full textDespite promising anti-leukemic activity of MCL-1 inhibitors in preclinical studies of acute myeloid leukemia (AML), clinical progress has been hindered by limited knowledge of target patient subgroups. To stratify patients for MCL-1 inhibitor treatment, we evaluated the sensitivity of 42 primary AML samples to MCL-1 inhibitor MIK665 (S64315) and analyzed their molecular profiles. We observed that MIK665-sensitive samples had a more differentiated phenotype, whereas resistant samples displayed higher levels of ABCB1 (MDR1) and the anti-apoptotic protein BCL-XL. Moreover, ABCB1 expression had good predictive performance in identifying resistant samples. To induce sensitivity, we treated MIK665-resistant samples with ABCB1 inhibitors elacridar or tariquidar, BCL-XL inhibitor A1331852, or BCL-2 inhibitor venetoclax in combination with MIK665. The combination of MIK665 with each of elacridar, tariquidar, or venetoclax effectively eliminated AML blasts compared to the agents alone, while the combination with A1331852 showed limited efficacy for this patient subgroup. Additionally, the combination of MIK665 with venetoclax restored sensitivity in samples with primary venetoclax resistance. Overall, this study indicates that elevated ABCB1 expression is a potentially targetable resistance mechanism in the context of MIK665 resistance, and that a combination of MIK665 with venetoclax may be effective for overcoming resistance to either MCL-1 or BCL-2 inhibition
Nonproliferative and Proliferative Lesions of the Rat and Mouse Female Reproductive System: Revised INHAND Terms for Ovarian Sex Cord/Stromal Lesions.
No full textThe nomenclature for the female reproductive system was originally published in 2014. After 10 years of practical use, the scientific community requested from the organ working group (OWG) a review of the terminology and criteria for diagnosing ovarian sex cord/stromal lesions. As a result, OWG proposes the use of "sex cord/stromal" as the base terminology for hyperplasia and tumors to better reflect their origin from the sex cord/stroma and make the terminology internally consistent. When no predominant cell type is present, these lesions should then be designated as mixed cell type (e.g., "hyperplasia, sex cord/stromal, mixed," or "tumor, sex cord/stromal, mixed, benign"). When a clear, predominate cell type is present, the diagnosis should indicate that cell type (e.g., "sex cord/stromal, granulosa cell" or "sex cord/stromal, theca cell"). In the case of tumors, benign or malignant would be applied as appropriate. With these diagnostic revisions, the OWG for the female reproductive system attempts to provide clarification and refinement of criteria to be used for sex cord/stromal lesions
Beyond Liquid Chromatography: Cyclic Ion Mobility Spectrometry for Phosphorothioate Diastereomer Separation in siRNA
No full textPhosphorothioate (PS) modifications in small interfering RNA (siRNA) moieties enhance stability and therapeutic efficacy but introduce diastereomeric heterogeneity, complicating structural characterization. Conventional chromatographic methods, such as ion-pair reversed-phase liquid chromatography, provide limited resolution of complex stereoisomer systems, necessitating alternative analytical approaches. In this work, we systematically evaluate cyclic ion mobility spectrometry (cIMS) for the separation and identification of PS diastereomers by investigating oligonucleotide systems with varying chain length and PS linkage patterns that mimic the metabolic diversity in siRNA therapeutics. Our results demonstrate that cIMS effectively separates diastereomers in short (5-mer) to medium-length (9-mer) oligonucleotides, with separation efficiency influenced by charge state and salt adduction. Higher charge states enhance resolution by narrowing conformational distributions and enabling increased numbers of cIMS passes, while sodium and potassium adducts improve separation by promoting distinct gas-phase conformers. However, as system size increases (15-mer), the relative influence of terminal diastereomers on the overall structure diminishes, leading to a reduced separation efficiency and ultimately preventing the resolution of long-chain siRNAs. Comparisons between experimental and computationally predicted collisional cross sections (CCS) underscore both the potential and limitations of CCS-based diastereomer assignment, emphasizing the need for refined computational models or orthogonal validation methods. These findings highlight cIMS as a powerful and complementary tool for the structural characterization of stereochemically complex oligonucleotide therapeutics, providing valuable insights for siRNA drug development