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Long-Term Human Immune Reconstitution, T-Cell Development, and Immune Reactivity in Mice Lacking the Murine Major Histocompatibility Complex: Validation with Cellular and Gene Expression Profiles.
Get PDFBackground: Humanized mice transplanted with CD34+ hematopoietic cells (HPCs) are broadly used to study human immune responses and infections in vivo and for testing therapies pre-clinically. However, until now, it was not clear whether interactions between the mouse major histocompatibility complexes (MHCs) and/or the human leukocyte antigens (HLAs) were necessary for human T-cell development and immune reactivity. Methods: We evaluated the long-term (20- week) human hematopoiesis and human T-cell development in NOD Scid Gamma (NSG) mice lacking the expression of MHC class I and II (NSG-DKO). Triplicate experiments were performed with HPCs obtained from three donors, and humanization was confirmed in the reference strain NOD Rag Gamma (NRG). Further, we tested whether humanized NSG-DKO mice would respond to a lentiviral vector (LV) systemic delivery of HLA-A*02:01, HLA-DRB1*04:01, human GM-CSF/IFN-α, and the human cytomegalovirus gB antigen. Results: Human immune reconstitution was detectable in peripheral blood from 8 to 20 weeks after the transplantation of NSG-DKO. Human single positive CD4+ and CD8+ T-cells were detectable in lymphatic tissues (thymus, bone marrow, and spleen). LV delivery harnessed the detection of lymphocyte subsets in bone marrow (αβ and γδ T-cells and NK cells) and the expression of HLA-DR. Furthermore, RNA sequencing showed that LV delivery increased the expression of different human reactome pathways, such as defense responses to other organisms and viruses. Conclusions: Human T-cell development and reactivity are independent of the expression of murine MHCs in humanized mice. Therefore, humanized NSG-DKO is a promising new model for studying human immune responses, as it abrogates the xenograft mouse MHC interference
ARID1A-BAF coordinates ZIC2 genomic occupancy for epithelial-to-mesenchymal transition in cranial neural crest specification.
Get PDFThe BAF chromatin remodeler regulates lineage commitment including cranial neural crest cell (CNCC) specification. Variants in BAF subunits cause Coffin-Siris syndrome (CSS), a congenital disorder characterized by coarse craniofacial features and intellectual disability. Approximately 50% of individuals with CSS harbor variants in one of the mutually exclusive BAF subunits, ARID1A/ARID1B. While Arid1a deletion in mouse neural crest causes severe craniofacial phenotypes, little is known about the role of ARID1A in CNCC specification. Using CSS-patient-derived ARID1A[thorn]/ induced pluripotent stem cells to model CNCC specification, we discovered that ARID1A- haploinsufficiency impairs epithelial-to-mesenchymal transition (EMT), a process necessary for CNCC delamination and migration from the neural tube. Furthermore, wild-type ARID1A-BAF regulates enhancers associated with EMT genes. ARID1A-BAF binding at these enhancers is impaired in heterozygotes while binding at promoters is unaffected. At the sequence level, these EMT enhancers contain binding motifs for ZIC2, and ZIC2 binding at these sites is ARID1A-dependent. When excluded from EMT enhancers, ZIC2 relocates to neuronal enhancers, triggering aberrant neuronal gene activation. In mice, deletion of Zic2 impairs NCC delamination, while ZIC2 over- expression in chick embryos at post-migratory neural crest stages elicits ectopic delamination from the neural tube. These findings reveal an essential ARID1A-ZIC2 axis essential for EMT and CNCC delamination
A brief chronicle of research on human pluripotent stem cells.
No full textToday, human pluripotent stem cell technologies find widespread application across biomedical research, as models for early human development, as platforms for functional human genomics, as tools for the study of disease, drug screening and toxicology, and as a renewable source of cellular therapeutics for a range of intractable diseases. The foundations of this human pluripotent stem cell revolution rest on advances in a wide range of disciplines, including cancer biology, assisted reproduction, cell culture and organoid technology, somatic cell nuclear transfer, primate embryology, single-cell biology, and gene editing. This review surveys the slow emergence of the study of human pluripotency and the exponential growth of the field during the past several decades
Hypertonic Saline Induces Host Protective Immune Responses against
Get PDFHypertonic saline (HTS) resuscitation can enhance immune responses against various pathogens, however, the effect of HTS on brucellosis is yet to be defined. In this study, we found that HTS inhibited Brucella infection in mice by augmenting Th1 immunity. HTS treatment enhanced the serum cytokines production and the expression of nitric oxide synthase (NOS2) and nuclear factor kappa B (NF-ĸB) p50 and p65, crucial anti-Brucella effectors in splenocytes. In addition, HTS treatment also inhibited the phosphorylation of MAPK signaling, accompanied by the down-regulation of the autophagy marker LC3B-II. Due to directing an appropriate immune response, HTS treatment substantially decreased bacterial burden in spleen and liver tissues. In summary, corroborating previous studies showing the antimicrobial effects of HTS, our findings indicate that HTS treatment triggers a protective immune response against Brucella infection. Additionally, these results provide promising evidence of the immunomodulatory role of HTS in controlling bacterial infections
Investigating Novel Mutations and Developmental Timing of Congenital Diaphragmatic Hernia
No full textCongenital Diaphragmatic Hernia (CDH) is a mammalian-specific muscle critical for respiration and division of the thoracic and abdominal body cavities. CDH affects 1:3000 live births with a mortality rate of 30%, characterized by muscle weakness/partial loss of diaphragm muscle accompanied by lung hypoplasia. Development of CDH is primarily driven by genetic mutations, however, the total number of CDH critical genes remains a question in the field. This research presents a method to screen CDH patient mutations in mice, demonstrated by investigation of two novel gene mutations—Wt1 and Kalrn. To introduce mutations into fertilized mouse zygotes (E0.5 embryos) CRISPR/Cas9 technology was used. Edited E0.5 are transplanted into donor mice to allow for embryonic development past the stage of diaphragm patterning. Edited embryos were then analyzed for structural defects using X-Ray Micro-Computed Tomography to assess diaphragmatic integrity and lung development. The experimental results showed CDH-like phenotypes in mutant mouse embryos and further understanding of the roles of Wt1 and Kalrn mutations in CDH. We also were able to have a better understanding when herniations arose within Cdc42bpb from embryonic days E13.5 -E16.5 and next steps to take when targeting the specific embryonic day with Cnot1. The project\u27s impact lies in advancing our understanding of CDH pathogenesis, potentially paving the way for targeted therapies aimed at correcting genetic abnormalities implicated in this life-threatening condition
Pathophysiology and Etiology of a Novel Mouse Model for Vasculitis
No full textVasculitis is a heterogeneous group of disorders defined by blood vessel inflammation and tissue damage (1). Preliminary data suggest that Collaborative Cross Recombinant Inbred Cross (CCRIX) mice developed a rare form of small-vessel vasculitis known as Granulomatosis with Polyangiitis (GPA). The mice presented with kidney failure leading to early death. This prognosis mirrors clinical observations, where up to 90% of patients with untreated GPA patients die of respiratory or kidney failure within 2 years. GPA patients initially show a range of respiratory symptoms, fever, and malaise followed shortly by severe damage to the ears, eyes, throat, lungs, or kidney. This diverse range of symptoms necessitates extensive characterization of clinical and subclinical features for an accurate differential diagnosis (2). The lack of models for GPA slows the discovery of biomarkers and targeted therapeutics that would improve diagnosis and treatment. CCRIX-GPA mice provide an important opportunity to do this through a better understanding of GPA genetics and disease mechanisms. Here we used a range of clinical and genomic assays to define the disorder. GPA Pathophysiology was characterized via glomerular filtration rate analysis, urinalysis, gross necropsy, and histopathological analysis of collected tissue samples. GPA etiology was investigated via RNA-sequencing analysis. This process will lead to a complete and accurate diagnosis of the vasculitis and allow us to detect the early stages of disease in live mice. These data will be critical for follow up studies that test disease mechanisms or treatments for GPA
Using machine learning to classify subtypes of rhabdomyosarcoma
No full textRhabdomyosarcoma (RMS) is a soft-tissue pediatric cancer, accounting for 5% of all solid tumor cases. The prognosis and treatment outcomes vary significantly depending on the subtype of RMS and its mutational profile – however, disparities exist in accessing genetic screening and expert pathology services, often influenced by geographical and socioeconomic factors. Our objective is to leverage neural network models to categorize RMS samples solely based on histopathology images obtained from biopsies. 386 whole-slide images (WSI) of RMS biopsies from various institutions in the United States were consolidated to test binary classification models and explore model behavior on the images. The results show that a native model – prior developed in the Chuang Lab – exhibits high accuracy in classification by relying on characteristic histological morphologies of RMS subtypes
The immunopathological landscape of human pre-TCRα deficiency: From rare to common variants.
Get PDFWe describe humans with rare biallelic loss-of-function PTCRA variants impairing pre–a T cell receptor (pre-TCRa) expression. Low circulating naive ab T cell counts at birth persisted over time, with normal memory ab and high gd T cell counts. Their TCRa repertoire was biased, which suggests that noncanonical thymic differentiation pathways can rescue ab T cell development. Only a minority of these individuals were sick, with infection, lymphoproliferation, and/or autoimmunity. We also report that 1 in 4000 individuals from the Middle East and South Asia are homozygous for a common hypomorphic PTCRA variant. They had normal circulating naive ab T cell counts but high gd T cell counts. Although residual pre-TCRa expression drove the differentiation of more ab T cells, autoimmune conditions were more frequent in these patients compared with the general population
New directions for Alzheimer\u27s disease research from the Jackson Laboratory Center for Alzheimer\u27s and Dementia Research 2022 workshop.
Get PDFINTRODUCTION: In September 2022, The Jackson Laboratory Center for Alzheimer\u27s and Dementia Research (JAX CADR) hosted a workshop with leading researchers in the Alzheimer\u27s disease and related dementias (ADRD) field.
METHODS: During the workshop, the participants brainstormed new directions to overcome current barriers to providing patients with effective ADRD therapeutics. The participants outlined specific areas of focus. Following the workshop, each group used standard literature search methods to provide background for each topic.
RESULTS: The team of invited experts identified four key areas that can be collectively addressed to make a significant impact in the field: (1) Prioritize the diversification of disease targets, (2) enhance factors promoting resilience, (3) de-risk clinical pipeline, and (4) centralize data management.
DISCUSSION: In this report, we review these four objectives and propose innovations to expedite ADRD therapeutic pipelines
Consensus and controversy in the management of paediatric and adult patients with ovarian immature teratoma: the Malignant Germ Cell International Consortium perspective.
No full textOvarian immature teratoma (IT) is a rare neoplasm comprising ∼3% of ovarian cancers, occurring primarily in young females. Management presents several challenges, including those with elevated serum alpha-fetoprotein, potential confusion regarding pathology interpretation, and paucity of data to support decision-making. MaGIC (https://magicconsortium.com/) is an interdisciplinary international consortium of GCT experts from multiple subspecialties, with members receiving frequent queries regarding IT patient management. With evidence from published literature where available, we summarise consensus management of such patients. Given lack of published data, controversy in certain areas remains. The most obvious variance in practice is between paediatric and adult teams, despite very similar outcomes. Paediatric teams typically employ a surgery-only approach, whereas in adult practice, all patients, except those with stage IA, grade 1 (low-grade) tumours, still generally receive adjuvant chemotherapy. Given the rarity of ovarian IT and lack of published data, discussion with GCT experts and/or national advisory panels is recommended