Laboratory medicine

Permanent URI for this collectionhttps://digital.lib.washington.edu/handle/1773/19658

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Methods and preliminary CTE and neurodegenerative disease data in the Pacific Northwest Brain Donor Network, a military and trauma-based brain bank
(2026-04-20) Anderson, Stephanie Rose; Keene, C Dirk
The Pacific Northwest Brain Donor Network is a brain repository focused on chronic traumatic brain injury (TBI). This cohort is significant for its focus on TBI in military veterans, its use of cases with non-military TBI or no TBI as controls, and the relatively young age of brain donors. In the first 161 donors, the median age was 48 years, and there were 110 donors with TBI and 66 with a history of military service. 39 veterans, 33 non-veterans, and 38 donors with unknown military service had a history of known TBI. Brains underwent ex vivo magnetic resonance imaging followed by standardized neuropathologic evaluation. Chronic traumatic encephalopathy was identified in 45 cases (28%, 19 high stage and 26 low stage), 39 of whom had a history of TBI, and 16 of whom were veterans. Alzheimer’s disease neuropathologic change was identified in 56 cases (35%), and age-related tau astrogliopathy in 61 cases (37.9 %). Smaller numbers of donors had other neurodegenerative diseases including cerebral amyloid angiopathy (13%), Lewy body disease (9.3%), limbic-predominant age-related TDP43 encephalopathy (7.5%), primary age-related tauopathy (5%), and argyrophilic grain disease (3.1%).
Advances in liquid biopsy for preeclampsia risk prediction and tumor gene expression inference from cell-free DNA
(2026-04-20) Adil, Mohamed; Haffner, Michael; Ha, Gavin
Liquid biopsy has the potential to address critical diagnostic needs; however, substantial innovations are still required. Here we describe two unique approaches that explore epigenetic features of cell-free DNA using practical, scalable, and cost-effective assays to infer clinically relevant insights. In the first study, we leveraged a routine prenatal cfDNA screening (PDNAS) assay to predict the risk for preeclampsia early in pregnancy up to 5 months before onset of clinical symptoms with an area under the receiver operating characteristic curve (AUC) 0.85. For this we developed the PEARL (preeclampsia early assessment of risk from liquid biopsy) framework which infers nucleosome accessibility to determine placental and endothelial dysfunction in a non-invasive manner. In the second study, we expand the diagnostic spectrum of liquid biopsies by developing DRAGN-X (Deep Regulatory Autoencoder for Gene Expression), a framework that infers quantitative, tumor-specific gene expression directly from cfDNA methylation. DRAGN-X integrates novel insights into the relationship between DNA methylation and gene expression with deep learning models, achieving a high correlation (Pearson’s r = 0.91) between cfDNA-inferred expression and ground-truth tumor tissue expression. We demonstrate broad clinical utility of DRAGN-X across multiple cancer and liquid biopsy types using a scalable, cost-efficient, low-pass whole-genome cfDNA methylation assay. Together, these findings establish advances in cfDNA technologies that can enhance precision medicine and improve clinical diagnostics.
Engineered Heart Tissues for Advanced Disease Modeling
(2026-02-05) Goldstein, Alex; Sniadecki, Nathan J
Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are a powerful, in vitro tool for investigating cardiac pathologies. Although hiPSC-CMs have a relatively immature phenotype compared to primary adult cardiomyocytes, their physiological relevance can be improved by incorporating them into a biomimetic three-dimensional environment to generate engineered heart tissues (EHTs). The EHT platform matures hiPSC-CMs by providing the environmental cues present in the in vivo myocardium while allowing for assessments of cardiac function. Although heart failure is the leading cause of death in patients with Duchenne muscular dystrophy (DMD), a severe, X-linked, neuromuscular disease, the mechanisms underlying the associated dilated cardiomyopathy (DCM) are not completely understood. This is due in part to the lack of suitable animal models of DMD-associated DCM which can accurately reproduce the disease’s progression in human patients. We addressed this deficiency by developing a novel in vitro model of DMD using EHTs generated with hiPSC-CMs which had been edited with CRISPR to lack dystrophin. These dystrophic EHTs recapitulated key aspects of the DMD-associated DCM including impaired contractile function and slower kinetics. Dystrophic EHTs also showed elevated beat rate variability, reduced Ca2+ transients and delayed kinetics, and smaller cardiomyocyte size and sarcomere length. Additionally, we improved our EHT platform by addressing its throughput and lack of regional heterogeneity, two limitations of the system that exist in its current form. To increase the throughput, we designed a miniaturized EHT (mEHT) platform that is compatible with a standard 96-well culture plate. The mEHTs generated on this platform produce measurable, uniaxial, synchronous contractions and have the potential to be used for high throughput screening of cardiomyopathy phenotypes and candidate therapies. We also developed a method for multi-region suspended tissue patterning with collaborators. Using this method, we generated EHTs with localized fibrosis that showed alterations in contraction kinetics and spontaneous beat rates compared to tissues lacking a fibrotic region. Future studies may be able to use spatial heterogenous EHTs for more complex investigations of myocardial development or pathologies.
Designing an Assay to Evaluate NBS Results Using Targeted Long-Read Sequencing
(2025-08-01) YE, SHENYI; Miller, Danny; Scott, Anna
NBS (NBS) is a routine screening process that identifies selected genetic, metabolic, and endocrine disorders that can affect a newborn’s health. Unfortunately, limitations in the follow-up process can create barriers to confirming screening results. Here, we demonstrate the ability of targeted long-read sequencing (T-LRS) in evaluating NBS results. Using adaptive sampling on the Oxford Nanopore platform on 8 positive control samples from Seattle Children’s Hospital, we computationally targeted more than 500 genes relevant to metabolic and non-metabolic disorders and searched for pathogenic variants using a single data source. We detected all genomic variants identified by prior genetic testing, as well as additional variants not previously identified. T-LRS demonstrates to be an efficient and cost-effective method to evaluate individuals after a positive NBS result.
Regulation of cellular structure and function through amphipathic motifs
(2025-08-01) Zhang, Lin; Bai, Jihong
Curvature-sensing mechanisms assist proteins in executing particular actions on various membrane organelles. Here, we investigate the functional specificity of curvature-sensing amphipathic motifs in Caenorhabditis elegans through the study of endophilin, an endocytic protein for synaptic vesicle recycling. We generate chimeric endophilin proteins by replacing the endophilin amphipathic motif H0 with other curvature-sensing amphipathic motifs. We find that the role of amphipathic motifs cannot simply be extrapolated from the identity of their parental proteins. For example, the amphipathic motif of the nuclear pore complex protein NUP133 functionally replaces the synaptic role of endophilin H0. Interestingly, non-functional endophilin chimeras have similar defects—producing fewer synaptic vesicles but more endosomes—and this indicates that the curvature-sensing motifs in these chimeras have a common deficiency for reforming synaptic vesicles. Finally, we convert non-functional endophilin chimeras into functional proteins by changing the cationic property of amphipathic motifs, successfully reprogramming the functional specificity of curvature-sensing motifs in vivo.
Differential Expression of IFN-Gamma Stimulated Antiviral Genes in Human Herpes Simplex Virus Infection
(2025-08-01) Zimenko, Angelina; Zhu, Jia
Herpes simplex virus type 2 (HSV-2) causes highly prevalent and recurrent ulcerative disease that affects millions of individuals worldwide. Clinical outcomes vary widely, ranging from asymptomatic, subclinical illness to severe, painful ulcerations. HSV-2 prevalence is further associated with an elevated risk of human immunodeficiency virus (HIV) acquisition, due to the recruitment of HIV-susceptible CD4+ T cells to sites of mucocutaneous infection. As HSV can establish latency in sensory ganglia and reactivate throughout an individual’s lifetime, a host's innate and adaptive immune system must continuously surveil and contain viral activity. Viral containment is primarily attributed to HSV-specific CD8+ T cells that persist at sites of prior infection, alongside the network of chemokines and cytokines that coordinate local antiviral immune responses. Within the herpes lesion microenvironment, interferon gamma (IFN-γ) plays a critical role in viral control by inducing the expression of interferon-stimulated genes (ISGs) in neighboring cells, most notably keratinocytes and fibroblasts, which produce antiviral proteins, microbicidal molecules, phagocytic receptors, and additional immune modulators that help establish an antiviral state. In this study, we investigated donor-dependent IFN–γ–induced gene expression in primary structural cells isolated from individuals with asymptomatic or severe HSV-2 disease. By assessing the transcriptional response of specific ISGs in keratinocytes and fibroblasts exposed to IFN-γ, we identified distinct patterns of gene expression based on both donor phenotype and cell type. Our findings suggest that variability in the magnitude and regulation of IFN-γ-responsive genes may underlie the heterogeneous clinical manifestations of HSV-2 infection. Understanding the mechanisms that govern effective immune control can help us identify determinants of disease severity and inform the development of targeted therapeutic strategies aimed at long-term containment and eradication of HSV-2.
Development of Third-Generation Gold Nanoparticles (CRISPR-AuNP) for Enhanced CRISPR Delivery to Hematopoietic Stem and Progenitor Cells
(2025-08-01) Gottimukkala, Karthikeya Srinivasa Varma; Adair, Jennifer E
Efficient CRISPR ribonucleoprotein (RNP) delivery into hematopoietic stem and progenitor cells (HSPC, CD34+) enables stable genome editing with potential for lifelong therapeutic benefits from a single intervention. However, current methods like electroporation require specialized equipment and procedures, whereas viral vectors are costly and require living cells to assemble, limiting access in research and clinical translation. Synthetic nanoparticles offer a promising alternative. We previously reported a gold-based CRISPR-AuNP nanoformulation that delivered gene editing in HSPCs, favoring Cas12a over Cas9. Here, we demonstrate that Cas9 loads poorly into this formulation. By elucidating Cas9's physicochemical interactions with AuNP surfaces, we optimized the nanoformulation by pre-forming RNP complexes in a 2nd generation CRISPR-AuNP. This formulation demonstrated increased active Cas9 and Cas12a loading in tube but failed to deliver gene editing in primary cells in vitro. Further analysis of the CRISPR-AuNP physiochemistry with HPSC endosomes guided further optimization. Preformed RNP polyplexes were formed with thiolated poly(ethyleneimine)-poly(ethylene glycol) (PEI-PEG-SH) polymers at a 2 N/P ratio and conjugated to AuNP. This 3rd generation Cas9 CRISPR-AuNP achieved 13.23 Â± 0.12% indels at the β-2-microglobulin (B2M) gene in HSPCs at a 100 pmol dose, with no observed loss in cell viability and cell entry within 6 hours. The platform's modularity extended to two additional CRISPR systems: Cas12a (15.07 Â± 1.9% indels) and MG29-1 (13.39 Â± 1.5% indels), highlighting its versatility. Most importantly, this nanoformulation can be assembled in a few hours at benchtop for <$70 per 1 million HSPC treated. We also demonstrate early data that this formulation can be modified with surface antibodies to facilitate active cellular targeting, with a future goal of testing these formulations in vivo for possible clinical translation. Very preliminary in vivo studies using untargeted 3rd generation Cas9 CRISPR-AuNP targeting the murine B2m gene in wild-type C57Bl6 mice showed detectable gene editing in multiple tissues with no elevated inflammatory responses or increased liver enzymes. These advancements position gold-polymeric nanoparticle hybrids as a simple, low-cost non-viral delivery system for CRISPR into HSPCs. The ability to rapidly assemble these particles with different CRISPR systems and polymers will greatly increase research access for multiple cell types.
Characterization of Somatic Mutations in the Normal Colon Using Duplex Sequencing to Evaluate Colorectal Cancer Risk
(2025-08-01) Blokker, Alexis; Risques, Rosana
The accumulation of somatic mutations in normal tissues has been established over the last decade. However, we lack an understanding of how these mutations might predispose individuals to cancer. In this study we expand on previous findings to demonstrate that duplex sequencing enables the detection of variants at extremely variant allele fractions (VAFs) in normal colon from patients without cancer or polyps, with polyps, and those with colorectal cancer (CRC). By applying advanced computational techniques, we characterize the potential pathogenicity of these mutations, developing a framework for assessing positive selection in the normal colon. Our data indicates that individuals with CRC have a higher frequency of mutations in key CRC driver genes in their normal colon, specifically in APC, FBXW7, and PIK3CA, than patients without cancer. Furthermore, patients with cancer exhibit a higher frequency of pathogenic large clones (pathogenic variants with >1 duplex read) in KRAS and TP53 in normal colon, indicating an increased prevalence of positive selection and clonal expansions driven by mutations in those genes. This selection is not random, with mutations clustering in colorectal cancer gene hotspots. These results reveal that the clonal landscape of CRC driver genes differs between the normal colon of individuals with cancer and without cancer, providing insight into how the somatic genome may predict a patient's risk for developing cancer.
Non-viral Gold Nanoparticle Mediated Delivery of CRISPR-Cas9 Ribonucleoprotein and Long DNA Transgenes into Primary Blood Cells
(2025-08-01) Cunningham, Rachel; Adair, Jennifer E
Gene editing using CRISPR systems has gained traction for its potential to treat various diseases. However, current gene editing therapies suffer from lack of affordable, scalable and synthetic delivery, especially for complex cargo such as the CRISPR ribonucleoprotein (RNP) complex and DNA templates required for insertion of specific genomic sequences. Development of an effective non-viral approach to RNP delivery could overcome these limitations and would be transformative for clinical translation and research. We previously reported a simple, synthetic gold-based nanoparticle which can deliver various CRISPR systems as RNP into primary hematopoietic stem and progenitor cells without the need for complex protein engineering (CRISPR-AuNP). Here, we describe a gold-based nanoparticle to simultaneously deliver CRISPR systems and large DNA templates which can encode transgenes (HDT-CRISPR-AuNP). HDT-CRISPR-AuNP can carry templates as long as ~2.1kb, with potential for larger cargo. We evaluated HDT-CRISPR-AuNP for gene editing at two loci of interest with different transgenes. These nanoparticles were able to successfully engineer primary human T cells and hematopoietic stem and progenitor cells with insertion of an antigen-specific T-cell receptor (TCR) transgene. This proof-of-concept immune engineering study shows transgene integration and expression from various hematopoietic lineages and suggests a potential for efficacy in vivo.
Determining if the Developmental Origin of Cardiac Fibroblasts Determines the Fate of Activation in Adult-Onset Hypertension
(2025-08-01) Gifford, Amy Leigh; Davis, Jennifer
Heart disease remains a leading cause of morbidity and mortality worldwide, responsible for 1 in5 deaths, with cardiac fibrosis playing a critical role in heart failure progression [13, 18, 17]. Cardiac fibrosis, characterized by excessive extracellular matrix (ECM) deposition and tissue stiffening, impairs cardiac function, reducing contractility and increasing the risk of arrhythmias [13, 17]. Resident cardiac fibroblasts, originating from epicardial and endothelial progenitors, are essential regulators of fibrosis [7, 9, 10]. Periostin expression is canonically known as a marker of the activated fibroblast state in disease, but it is also upregulated during postnatal day 1 through postnatal day 11 [6] in development. We hypothesize that a neonatal Periostin- expressing fibroblast population is poised to activate in adulthood when challenged with a profibrotic stimulus. Previous work in the Davis Lab has shown that fibroblasts retain intrinsic memory, where when exposed to injury they exhibit a heightened reactivity upon re-injury [1, 4, 10, 12]. However, it appears that there is not a new population that activates when challenged with a second fibrotic injury after a period of recovery (Figure 4B). To test this, we used genetic lineage tracing via a tamoxifen-inducible Cre model to label Periostin+ fibroblasts in neonatal mice during the first 9 days in the postnatal period, before challenging them with AngII/PE in adulthood to determine if they reactivate upon injury. Histological analysis of whole hearts confirmed treatment dependent ECM remodeling following injury. This thesis demonstrates that Postn-lineage derived fibroblasts are poised to activate and expand when responding to injury or stress. Currently, the postnatal role of Postn-lineage fibroblasts remains poorly understood however, this is important to understand fibrotic regression and how it depends on both ECM remodeling and the deactivation of Postn-lineage myofibroblasts during postnatal development. In the Davis Lab, this information can be utilized to understand the role this population of fibroblasts plays in wound healing for other models such as dilated cardiomyopathy and myocardial infarction. These insights may inform therapeutic strategies targeting Postn-lineage fibroblasts to reverse fibrosis and improve overall heart function.
Method validation for dried blood spot Plasmodium 18S rRNA RT-PCR on Roche cobas automated molecular diagnostic platform
(2025-08-01) MDENYO, JAPHETH KENETH ALULA; Murphy, Sean C; Shears, Melanie J
Malaria, caused by Plasmodium spp., is a major health burden worldwide. Plasmodium falciparum is responsible for the most severe cases and most human mortality in sub-Saharan Africa. Conventional diagnostic approaches like microscopy and rapid diagnostic tests (RDTs) provide point-of-care screening but lack sufficient sensitivity for low-density infections and surveillance in endemic regions. Molecular diagnostic platforms, particularly nucleic acid tests (NATs), which target highly abundant Plasmodium 18S ribosomal RNA (rRNA), have emerged as sensitive and specific platforms for malaria detection and quantification. The FDA approved Roche cobasÂ® 6800 system, which is a fully automated, high-throughput molecular platform assay for the qualitative detection of Plasmodium spp. from whole blood samples by targeting the 18S rRNA.This study aimed (1) to adapt the cobasÂ® malaria assay for off-label use with non-standard sample types, including 50 μL of whole blood and dried blood spot (DBS) samples, and (2) to generate a standard curve for absolute quantification of the parasites. To achieve this, asexual-stage P. falciparum cultures were established and diluted to generate a full panel of validation samples across clinically relevant densities (1 x 10⁸–1 x 10Â¹ parasites/mL). Fifty- microliter blood samples were spotted onto DBS cards. Together, whole blood liquid and DBS samples were processed then tested using the cobasÂ® 6800 system. Plasmodium Armored RNA calibrators were used to generate standard curves for quantification. The study evaluated performance metrics parameters, that included pilot feasibility, standard curve assessment, linearity, limit of detection (LoD), precision, accuracy, carryover, DBS RNA stability under different storage conditions, calibrators-sample matrix matched assessment and extraction buffer elution assessment. The modified FDA-approved Roche cobasÂ® 6800 malaria assay demonstrated successful adaptation for detecting and quantifying P. falciparum 18S rRNA in 50 ÂµL whole blood samples. However, performance was reduced for DBS samples. Both whole blood and DBS sample formats achieved ≥95% detection at the 100 parasites/mL threshold. A matrix mismatch using liquid calibrators to quantify DBS samples resulted in systematic underestimation bias of +0.79 log₁₀ parasites/mL due to poor RNA recovery from DBS samples. The use of DBS-specific calibrators corrected the loss with a reduced bias of -0.07 log₁₀ parasites/mL, which enabled for the reliable quantification of P. falciparum concentration in the DBS samples. Low (100 parasites/mL) density DBS samples stored at room temperature (22–25 Â°C) and -80Â°C consistently maintained a 100% positivity hit rate across all the two timepoints (weeks 1 and 6). All MID (2000 parasites/mL) parasite density DBS samples exhibited 100% positivity across both time points and temperature storage conditions. The stability of DBS samples indicated reliable qualitative detection of P. falciparum RNA for DBS samples stored at 22-25Â°C or -80Â°C conditions, which supports their use in decentralized sample collection. Overall, these results suggest that the modified FDA- approved Roche cobasÂ® 6800 malaria assay performs well with 50 μL liquid whole blood samples but improvements are needed to achieve optimal results for 50 μL DBS samples.
BCG vaccination induces TCR-dependent effector functions among Vδ1/3 T cells that are associated with protection against tuberculosis
(2025-05-12) Maerz, Megan; Seshadri, Chetan
Intravenous vaccination with Mycobacterium bovis strain bacillus Calmette-Guérin (IV-BCG) protects macaques against Mycobacterium tuberculosis (Mtb) challenge and intradermal BCG protects human infants from disseminated tuberculosis. γδ T cells expressing a Vδ1+ or Vδ3+ T cell receptor (TCR) are enriched at mucosal surfaces and recognize mycobacterial antigens, but their role in protection against Mtb is largely unknown. We used multimodal single-cell RNA sequencing, mass cytometry, and flow cytometry to profile γδ T cells from human infants and macaques after protective BCG vaccination. A subset of Vδ1/3 T cells in BCG-vaccinated human infants shows evidence of clonal expansion and differentiation into pro-inflammatory and cytotoxic effector cells that respond to Mtb. In macaques, IV-BCG induced pro-inflammatory and cytotoxic responses to Mtb among Vδ1/3 T cells that were enriched in the airway compared to the blood. Notably, these responses were dependent on signaling through the TCR, and clonal expansion was most prominent among cytotoxic Vδ1/3 T cells. Finally, the total frequency of Vδ1/3 T cells in the lung and frequency of cytokine-expressing Vδ1/3 T cells in the airway were associated with protection against Mtb challenge. Thus, Vδ1/3 T cells are activated by BCG in a TCR-dependent manner and accumulate in the lung, where they upregulate cytotoxic and pro-inflammatory functions that may contribute to protective immunity against Mtb.
The Complexities of Adapting and Validating a Novel Assay for Adherence Monitoring of Oral HIV Pre-Exposure Prophylactics
(2024-10-16) Sheraden, Rachel; Greninger, Alex
Oral pre-exposure prophylaxis (PrEP) is effective in preventing HIV, but its efficacy depends on adherence. The gold standard for adherence monitoring, liquid chromatography-mass spectrometry, is accurate but costly and complex. Therefore, a simpler method is needed. The REverSe TRanscrIptase Chain Termination (RESTRICT) assay measures oral PrEP drug levels in blood by detecting tenofovir-diphosphate, an active PrEP metabolite. RESTRICT has a turnaround time of under 24 hours, is easy to prepare, and uses standard clinical lab equipment. The dose-dependent normalized fluorescence result can be plotted as a standard curve for semi-quantitative adherence measurement. The assay must be validated as a Laboratory Developed Test, assessing sensitivity, specificity, accuracy, precision, range, linearity, and interference. The validation showed RESTRICT as a promising alternative to mass spectrometry, potentially improving PrEP uptake and adherence.
Modeling sex as a personalized biological variable using the Drosophila metabolome
(2024-10-16) Coig, Rene; Promislow, Daniel
The research community now routinely considers “Sex as a Biological Variable” (SABV) in medical research and healthcare, because differences between genders/sexes can significantly impact disease prevalence, progression, and responses to treatment. Personalized medicine considers genetics and SABV, but a truly precise approach requires an understanding of how genetics and sex interact (GxS) to influence an individual’s internal biochemistry. Currently, empirical research is lacking that assesses the degree to which genetic diversity contributes to variability in SABV. Animal models with robust genetic toolkits, such as Drosophila melanogaster, can be used to measure the prevalence of GxS interactions in a population, interactions that are difficult to detect in humans. This dissertation presents the results from two complementary projects measuring GxS interactions in the metabolome of Drosophila–a model for natural genetic variation and a model for monogenic, Mendelian genetic variation in sex characteristics (VSC). This work conceptualizes sex differences in metabolite levels as genotype-specific biological effects. The analyses presented here reveal that the effect of sex on the metabolome is far from binary or dichotomous, but rather falls along a continuum, with genotype playing a larger role than sex alone on most metabolite levels. I show that genetic variation in external, measurable dimorphic traits are associated with genetic variation in sex differences at a molecular level, which effectively renders biological sex a variable as unique and diverse as any individual genome. These studies underscore the importance of considering genetic context in research that incorporates SABV, and the potential pitfalls of analyzing sex as a fixed effect in statistical models.
Investigating sex differences in mouse models of malaria vaccination using acute, reversible hormone manipulation
(2024-09-09) Sen, Nilasha; Shears, Melanie J
In many species, including mice and humans, biological sex impacts immunity. In general, females mount greater innate and adaptive immune responses, leading to sex differential vaccine outcomes. Prior research with mouse malaria models has shown female mice are better protected after vaccination and challenge than males. Prior studies further showed that castration significantly improved protection in males, suggesting that testosterone in intact males may be immunosuppressive. Here, we investigated the extent to which testosterone suppresses the formation of productive immune responses at vaccination compared to challenge in the context of malaria vaccination. We used a well-known GnRH antagonist, acyline, to suppress testosterone in BALB/cJ males. After dose optimization studies, mice were vaccinated with a two-dose vaccine and then challenged with wild type P. yoelii mouse malaria parasite. Acyline was injected subcutaneously on days 4 and 5 at a dose of 300 μg before either vaccination or challenge. Protection was measured using our standard endpoint assay, quantitative Plasmodium 18S rRNA RT-PCR. Male mice that had testosterone suppressed at challenge were better protected than those who had testosterone suppressed at vaccination. Adding exogenous testosterone abolished the protective effect of acyline. Taken together, this suggests that testosterone specific immunosuppression decreases protection to a greater extent during challenge compared to vaccination. Additionally, we also correlated suppressed peripheral testosterone levels with decreased androgen receptor expression in the liver as a surrogate measure for the liver hormone environment. Finally, the protective effect of suppressed testosterone during challenge was validated in another mouse and parasite model, C57BL/6 and P. berghei. Insight into the role of testosterone in vaccine response could help to engineer a better malaria vaccine, and further our understanding of sex differential outcomes in immunity.
Optimization of Multiparametric Flow Cytometry Assays: A Comprehensive Assessment of Immunophenotypic Characteristics of Monocytic and Blastic Plasmacytoid Dendritic Cell Neoplasms
(2024-04-26) Heard, Jung; Cherian, Sindhu
This study aimed to design and optimize multiparametric flow cytometry (MFC) assays forin-depth characterization of monocytic cells and plasmacytoid dendritic cells (pDCs) in the context of hematological neoplasms, such as monocytic leukemia and blastic plasmacytoid dendritic cell neoplasm (BPDCN). Building on previous research, we integrated novel markers, including LILRB1, LILRB4, and CD300e for the monocytic assay panel and CD303 and CD304 for the pDC assay panel, to provide a comprehensive characterization of antigenic expression associated with maturation and differentiation of these populations. We established unique expression patterns of novel markers and highlighted distinctive immunophenotypic features of monocytic cells and pDCs in their normal and neoplastic state, demonstrating robust performance and reliability of the optimized assays.
Decoding T Cell Responses to Herpes Simplex Virus Infection in Skin Using Highly Multiplexed Tissue Imaging
(2024-04-26) Yeckel, Amanda Karin Stole; Zhu, Jia
Herpes Simplex Virus (HSV) is a prevalent global health concern, with two main subtypes, HSV-1 and HSV-2, infecting billions of people. While HSV-1 typically causes cold sores and HSV-2 is associated with genital herpes, both can manifest in various forms. Infections often result in painful lesions, but many remain asymptomatic. Immunocompromised individuals face severe complications, including increased susceptibility to HIV infection. HSV establishes latency in neurons, periodically reactivating to cause viral shedding. Most reactivations are subclinical, contributing to the high global prevalence of HSV. Despite ongoing research, there is no cure or vaccine available. T cells play a crucial role in controlling HSV infections, with tissue-resident memory cells (TRMs) influencing reactivation frequency. Chronic infections can lead to T cell exhaustion, characterized by diminished effector functions and memory T cell development, leading to an inadequate immune response. Our study examined T cell responses in HSV-infected skin, exploring activation, exhaustion, regulation, and survival during lesion healing. Using advanced imaging and machine learning techniques, we observed a shift from activated T cells to TRMs post-healing. Additionally, regulatory T cell density was elevated in patients with higher shedding rates. Currently, there is no vaccine available to prevent HSV infection and transmission. However, a deeper understanding of T cell dynamics during lesion healing has the potential to directly guide vaccine development efforts aimed at leveraging T cell responses for effective prevention.
The Relationship Between Neurosteroid Structures and Inhibition of Pyroptotic Lysis
(2024-02-12) Huston, Hanna Clare; Fink, Susan L.
Pyroptosis is a form of programmed cell death with inflammatory biologicalconsequences. In moderation, pyroptosis helps to regulate and maintain a healthy immune response in the presence of invading pathogens. However, excessive, or inappropriate pyroptosis causes harmful consequences including inflammation and damage to tissues and organs. There are several steps within the pyroptotic cascade that result in lysis and release of cellular components implicated in harm to healthy cells and tissues. Previously identified small molecule inhibitors, like the amino acid glycine, have been shown to inhibit pyroptosis-mediated cell lysis, in vitro. Glycine is an ion channel modulator and examination of other ion channel modulators led to the discovery that pregnenolone sulfate also inhibits cell lysis in cells undergoing pyroptosis. Pregnenolone sulfate is a more potent inhibitor than glycine, but the mechanism of action for the protection is unknown. To begin to fill the gap in knowledge of how pregnenolone sulfate, and other small molecules, may be interacting with pyroptotic cells to prevent membrane lysis, we tested a rationally selected library of neurosteroids with similar structures to pregnenolone sulfate for inhibitive activity. We found some neurosteroids retain the same level of activity in comparison to pregnenolone sulfate, but others exhibit a partial or total loss in activity. No single aspect of the structure was responsible for inhibitive activity; different parts of the molecule contribute to inhibition of pyroptosis. Together, the results of this thesis lay the foundation for a deeper knowledge of what mechanisms may provide protection and how we can design future inhibitors with these mechanisms in mind.
A simple rapid flow cytometry assay to assess malaria vaccine responses in mice
(2023-09-27) Shankar Raman, Shruthi; Murphy, Sean Dr.; Shears, Melanie Dr.
Malaria is a global threat affecting nearly half of the world's population. Efforts to control the disease include vector control, accurate testing, and post-infection treatment. Despite substantial progress in these control measures, they do not provide comprehensive protection against malaria. This leads to the need for a more effective control measure, and a malaria vaccine would indeed be helpful as a public health tool. Thus, in the Murphy Laboratory, we are on the hunt for a more effective second-generation pre-erythrocytic malaria vaccine that induces a strong CD8 T cell response and offers long term protection against subsequent malarial infections. In this thesis, we discuss in detail, a surrogate marker assay: the upregulation of CD11a / downregulation of CD8a on CD8 T cells post antigenic experience. This assay serves as a valuable tool to measure vaccine responses in mice, enabling us to evaluate the efficacy of novel vaccines developed in our laboratory. By examining the specific changes in the expression of CD11a and CD8a on CD8 T cells following exposure to antigens, we gain insights into the immune response stimulated by the vaccines.
Highly Saturated Transposon Sequencing identifies genes impacting Staphylococcus aureus pathogenesis in macrophages
(2023-08-14) Lo, HsinYu; Salipante, Stephen J.
Staphylococcus aureus is a facultative intracellular pathogen in many host cell types,facilitating its persistence in chronic infections. The genes contributing to intracellular pathogenesis have not yet been fully enumerated. Here, we cataloged genes influencing S. aureus invasion and survival within human macrophages using two laboratory strains (ATCC2913 and JE2). We developed an in vitro transposition method to produce saturated transposon mutant libraries in S. aureus, and performed Tn-Seq to identify candidate genes with significantly altered abundance following macrophage invasion. While some significant genes were strain-specific, 107 were identified in common across both S. aureus strains, with most (n=105) being required for optimal macrophage infection. We used CRISPR interference (CRISPRi) to functionally validate phenotypic contributions for a select subset of genes. Of the 20 genes passing validation, 7 had a previously identified role in S. aureus virulence, and 13 were newly implicated. Validated genes frequently evidenced strain-specific effects, yielding opposing phenotypes when knocked down in the alternative strain. Genomic analysis of de novo mutations occurring in groups (n=237) of clonally-related S. aureus isolates from the airways of chronically infected individuals with cystic fibrosis (CF) revealed significantly greater rates of in vivo selection in candidate genes than factors not associated with macrophage invasion. This study implicates a core set of genes necessary to support macrophage invasion by S. aureus, highlights strain-specific differences in phenotypic effects of effector genes, and provides evidence for selection of candidate genes identified by Tn-Seq analyses during chronic airway infection in CF patients in vivo.

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