Comparative medicine

Permanent URI for this collectionhttps://digital.lib.washington.edu/handle/1773/36358

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Short-term treatment of mice with GHK peptide enhances resilience to age-related cognitive decline
(2025-08-01) Mazzola, Jordan Margaret; Ladiges, Warren C
Age-related cognitive decline (ARCD) is a neurodegenerative process accompanied with memory loss, neuronal dysfunction, and more such as chronic inflammation and increased cellular senescence. It impacts a large percentage of the aging population, increases in prevalence with age, and is a known risk factor for more severe neurodegenerative diseases such as Alzheimer’s Disease. Currently, there are no known treatments to stop or reverse the process of ARCD which presents a clear knowledge gap and opportunity to investigate potential gerotherapeutics. Glycyl-L-histidyl-L-lysine (GHK) is a naturally occurring peptide that forms complexes with copper (II) to improve wound healing and improve several age-related skin changes. Several studies have connected treatment of GHK-Cu to improvement of several hallmarks of aging via the inhibition of TGF-β and provides reasoning for exploring GHK-Cu as a potential gerotherapeutic. To test this potential, a short-term five-day treatment of GHK-Cu in middle-aged mice was performed. C57BL/6 mice, 20 to 22-months of age, were administered an intraperitoneal (IP) injection of GHK-Cu daily for five days. Cognitive function was assessed on the fifth day of treatment via a spatial navigation learning task. Several markers in the brain were assessed via immunohistochemistry (IHC) of the hippocampus and cerebellum. These markers include TGF-β1, GFAP, and phosphorylated-SMAD2 to assess the TGF-β pathway, synaptophysin and PSD95 to assess pre- and post-synaptic function, and p21 and MCP-1 to investigate cellular senescence and inflammation respectively. Aging pathways and gene regulation of GHK-Cu was investigated by RNA sequencing of the hippocampus. Results suggest improvement of cognitive function in male mice treated with GHK-Cu across several aspects including learning and memory shown via a behavioral assay, and decreased inflammation and cellular senescence. Results also revealed strong sex-dependent phenotypes within this treatment pathway as female mice treated with GHK-Cu showed less cognitive rescue when compared to male mice. This study provides support that a short-term treatment of GHK-Cu can positively impact cognitive function in middle-aged mice in a sex-dependent manner and provides potential pathways of interest for further investigation into the sex-dependent phenotypes observed. This provides support for GHK-Cu’s potential as a gerotherapeutic and progress in alleviating the negative effects of ARCD.
Excessive Cytokine and IgE Production in Murine Hem1 Immunodeficiency Contributes to Allergic Airway Disease in a House Dust Mite Model
(2025-08-01) Tsai, Julia Yung-Ru; Iritani, Brian M
Inborn errors of immunity (IEI) are a group of genetic diseases in humans that can present as increased susceptibility to infections, autoimmunity, hyperinflammation, allergic disease, and occasionally malignancy. A recently identified gene linked to IEI, NCKAP1L, encodes for Hematopoietic Protein-1 (HEM1), an adaptor protein which is critical for normal actin polymerization. Clinical investigation and immunophenotyping of HEM1 deficient children and murine models have documented similar characteristic features of IEIs, including recurrent infections and autoimmunity. However, the cellular mechanisms behind an increased prevalence of allergic airway disease (asthma) observed in HEM1 deficient children has not been further evaluated. In this study, we evaluated the development of asthma and primary immune cell populations and cytokines driving airway and lung pathology in various Hem1 deficient mouse models. We hypothesized that Hem1 deficient T cells were driving asthma pathogenesis in part by increasing release of proinflammatory cytokines. Constitutive Hem1 deficient mice (Hem1-/-), conditional T cell specific Hem1 deficient mice (Hem1fl/flCD4Cre), conditional B cell specific Hem1 deficient mice (Hem1fl/flMb1Cre), and age-matched control mice (10-25 week old males, n=5-8/group) received saline or house dust mite via the oropharyngeal route on days 0 and 14 for sensitization and days 26, 27, and 28 for challenge. On day 29, lungs, bronchoalveolar lavage fluid (BALF), and mediastinal lymph node were collected for flow cytometric analysis to identify representations of different myeloid and lymphoid cell populations. Serum and supernatant of the BALF were analyzed for cytokine and IgE levels via multiplex immunoassay. Lungs were formalin-fixed for histopathologic evaluation using H&E and PAS staining. Our results indicate that in the face of allergic airway disease, Hem1 deficient mice had decreased total numbers of myeloid and lymphoid immune cells in the airways, lung interstitium, and lymph node. However, Hem1 deficiency resulted in increased levels of asthma-related, proinflammatory cytokines (IL-17) and IgE in the BALF, resulting in asthma-related lung pathology. These results suggest that dysregulated cytokine and IgE production may contribute to asthma in Hem1 deficient humans and mice.
The domestic house cricket as a versatile model for functional aging and therapeutic testing
(2025-08-01) Liao, Gerald Yu; Ladiges, Warren
Introduction: Aging impairs physiological and cognitive function across species, yet invertebrate models often lack the organ complexity to reflect vertebrate aging. We establish the house cricket (Acheta domesticus) as a scalable, biologically rich model for high-throughput assessment of age-related decline in locomotion, cognition, and behavior. Using defined life stages, we evaluate functional aging and test rapamycin, acarbose, and phenylbutyrate, individually and in combination, for their therapeutic potential.Methods: House crickets were reared under standardized conditions and fed diets containing rapamycin, acarbose, phenylbutyrate, or their combination (SLAM). Animals underwent age-targeted or longitudinal treatment and were evaluated using open field, treadmill, Y-maze, and odor-guided escape assays. Locomotor patterns were classified via k-means clustering and cognitive flexibility was assessed through tetragram analysis. Lifespan was monitored, and post-mortem-tissues were collected for histology. Behavioral and survival outcomes were analyzed using machine learning, parametric/non-parametric tests, and Kaplan-Meier estimates. Analyses were conducted in Python and GraphPad Prism. Results: Across >1,100 crickets, phenylbutyrate, rapamycin, and acarbose extended lifespan (HR’s = 0.50 to 0.61, P’s < 0.05), with strongest effects in females. SLAM conferred early but unsustained survival benefits (HR = 0.37, P < 0.0001). Exploratory strategy and entropy declined with age (adults vs. geriatrics: d = 1.04, P < 0.0001), but were unaffected by treatment (d’s < 0.34, P’s > 0.05). Olfactory discrimination diminished with age, but was rescued by acarbose, rapamycin, and phenylbutyrate (d’s = -1.82 to -1.28, P’s < 0.004), with rapamycin restoring preference in both sexes. Locomotor performance declined with age but improved with treatment. Rapamycin restored distance, speed, and running efficiency to juvenile levels (e.g., total distance: d = -1.09, P = 0.0026). Central exploration deficits were selectively reversed by rapamycin and SLAM (e.g., central time: d = -1.48, P = 0.0001). On treadmill assays, rapamycin, phenylbutyrate, and SLAM restored maximum velocity (d’s = -2.30 to -1.32, P’s < 0.0001) and running time (d’s = -2.30 to -1.32, P’s < 0.0001), whereas acarbose had no effect. Jumping distance declined only in females (d = 1.33, P = 0.0063), with partial rescue by rapamycin and acarbose in a sex-specific manner. In the escape task, aging impaired weight gain, learning, and memory (e.g., adult vs. geriatric task success: RR = 7.20, P < 0.0004). Mid-age crickets showed prolonged decision times and delayed goal-arm arrival (e.g., reward latency: d’s = -1.42 to 1.64, P’s < 0.0001 to 0.014), while geriatric performance was relatively preserved. Conclusion: House crickets demonstrate age-related decline across survival, sensory, locomotor, and cognitive domains, which are attenuated by geroprotective interventions. Rapamycin consistently rescued performance across multiple assays, while acarbose and phenylbutyrate showed selective benefits. These findings establish the house cricket as a robust and tractable invertebrate model for aging research and cross-species drug discovery.
Exploring the Neuroprotective Effects of GHK-Cu in-vitro with astrocyte C8-S and in-vivo with 5XFAD transgenic mice
(2025-08-01) He, Qianpei; Ladiges, Warren C.
Astrocytes play a critical role in the progression of Alzheimer’s disease (AD), contributing to synaptic dysfunction, neurodegeneration, and altered neural homeostasis through reactive gliosis and phenotypic shifts. However, therapeutic strategies specifically targeting astrocyte dysfunction in AD remain underexplored. Glycyl-L-histidyl-L-lysine copper (GHK-Cu), an endogenous tripeptide with tissue-reparative properties, has recently emerged as a potential modulator of glial biology. This study investigated the efficacy of intranasally administered GHK-Cu in modulating astrocyte-driven neuroinflammation in the 5xFAD transgenic mouse model of AD and examined underlying mechanisms using an astrocyte cell line stimulated with lipopolysaccharide (LPS). Results revealed a significant sex-dependent reduction in hippocampal astrocytic activation, as indicated by decreased GFAP expression and reduced MCP-1 levels in GHK-Cu–treated female 5xFAD mice compared to untreated controls. Correspondingly, in vitro experiments using murine C8-S astrocytes demonstrated that GHK-Cu treatment, when applied either before or after lipopolysaccharide (LPS) exposure, mitigated astrocyte reactivity. These findings suggest that GHK-Cu modulates key features of astrocytic pathology in AD, including reactive transformation and stress-associated signaling. The data support further exploration of GHK-Cu as a therapeutic candidate targeting astrocyte dysfunction in neurodegenerative disease.
Investigation of cellular and molecular targets in the brain of mice given intranasal GHK peptide to treat age-related cognitive decline
(2025-08-01) Rosenfeld, Manuela; Ladiges, Warren
There are currently 1.2 billion people worldwide over the age of 60 and this number is projected to double by 2050. With the world population over 60 reaching 22% within the next 50 years, there is an increasing need to understand the mechanisms and processes of aging. An area of major interest is brain aging given that approximately 2 out of 3 Americans experience some level of cognitive impairment by age 70. Neuropathology associated with this cognitive impairment can lead to more severe neuropathology associated with Alzheimer’s disease and other dementias providing a need to develop interventions to treat and potentially prevent or reverse age related cognitive decline. One particular therapeutic of interest is a naturally occurring peptide called glycyl-L-histidyl-L-lysine (GHK) as recent studies suggest that GHK in its Cu bound form has the potential to age related cognitive decline. In order to further investigate the potential of GHK-Cu as a therapeutic for age related cognitive decline, mouse hippocampal tissue from 20-month-old C57BL/6 mice treated with 15mg/kg of intranasal GHK-Cu daily for 8 weeks were used for immunohistochemistry and RNA sequencing to discover cellular and molecular targets of this peptide. Results suggested that mice treated with intranasal GHK-Cu had lower expression of potentially harmful astrocytes. RNA sequencing results revealed that mice treated with GHK-Cu had upregulation or downregulation of various gene pathways in a way that supports healthy brain aging. Sex differences were also observed with certain pathways being affected differently in males and females. These findings provide rationale for further investigation of GHK-Cu as a therapeutic for age related cognitive decline in preclinical and clinical studies.
Dysfunctional Activation and Apoptotic Fragility in Hem1-Deficient B Cells
(2025-01-23) Anderson, Andreas; Iritani, Brian M
Loss-of-function mutations in NCKAP1L and its associated protein, HEM1, cause severeprimary immunodeficiency (PID) in children associated with mature B lymphopenia and impaired humoral immune response to foreign antigens, but also with B cell hyperactivation and production of autoantibodies. However, the mechanism by which loss of HEM1 causes this phenotype in B cells is poorly understood. In this study, we examined increased activation and metabolic activity in Hem1-deficient murine B cells from a transgenic conditional knockout mouse model. By fixing the B cell receptor repertoire using MD4 (IgHEL), we were able to confirm higher activation in Hem1-deficient B cells independent of BCR specificity. We demonstrate that this hyperactivated phenotype is directly associated with vulnerability to apoptosis, where risk of cell death rises with increasing strength of BCR activation. We observed increased mitochondrial mass and ROS in Hem1-deficient cells, which may both increase signal strength and promote cytotoxicity leading to apoptosis. Our results are supported by RNASeq validated by RT-qPCR, which showed upregulation of genes associated with growth, metabolism, proliferation, and DNA synthesis following disruption of Hem1.
Evaluation of Doxorubicin vs. Aclarubicin Exposure in C57BL/6J Mice as a Chronic Model of Anthracycline Induced Cardiotoxicity
(2024-09-09) Tu, Darleen Song; Iritani, Brian M.; Lawlor, Elizabeth R.
Doxorubicin is an anthracycline chemotherapeutic limited by dose-dependent cardiotoxic effects, while Aclarubicin is a promising alternative anthracycline shown to be less cardiotoxic. Future clinical trials involving Aclarubicin will need to be tested in a relapse setting where patients have already received Doxorubicin. The study’s objective was to evaluate the cardiac effects of Aclarubicin following administration of Doxorubicin and develop a standardized mouse model for assessing cardiotoxicity of oncologic adjuvants after prior Doxorubicin exposure. C57BL/6J mice all received an initial once weekly dose of 5 mg/kg intraperitoneal injections for 4 weeks and rested for an additional 4 weeks. Mice were then administered intraperitoneal injections of one of the following agents once-a-week for 4 weeks: Doxorubicin (5 mg/kg), Aclarubicin (5 mg/kg) or equivalent volumes of saline. Mice were humanely sacrificed after an additional 12-week rest period. Mice that received 40 mg/kg cumulative Doxorubicin had the lowest survival rate and the most weight loss. There were no detectable echocardiographic measurement differences between groups. Overall cardiac changes for all three mice groups were mild via histologic examination by periostin immunohistochemistry, trichrome, and hematoxylin and eosin staining. The results indicate Aclarubicin does not further potentiate Doxorubicin cardiotoxicity.
Immunogenicity and Protection of the mXCL1-PyCSP Fusion Protein Prime-and-Trap DNA Vaccine in a Murine Malaria Immunization/Challenge Model
(2024-09-09) Boey, Kenneth; Murphy, Sean C
Malaria is a life-threatening parasitic disease caused by Plasmodium spp. and is transmitted byfemale Anopheles spp. mosquitoes. Annually, there are nearly 250 million cases worldwide causing over 600,000 deaths, primarily in children under 5 years of age in sub-Saharan Africa. Currently, no highly efficacious (>85–90%) vaccine exists, hence the development of such a vaccine against human malaria infection is of paramount importance. The chemokine ligand XCL1, also known as lymphotactin, binds to its chemokine receptor XCR1. Recent studies have shown that XCL1-antigen fusion proteins efficiently induce CD8+ T cell responses by preferentially delivering antigens to cross-presenting dendritic cells expressing XCR1. In this study, we evaluated the immunogenicity of a fusion protein of the murine XCL1 chemokine and the Plasmodium yoelii circumsporozoite protein (mXCL1-PyCSP) in our “Prime-and-Trap” vaccine in a murine model of malaria. We hypothesized that this fusion protein vaccine would increase immunogenicity and protection outcomes compared to the standard unfused PyCSP vaccine. In summary, we showed that the fusion of PyCSP with the mXCL1 chemokine enhanced cell-mediated immune responses and significantly increased immunogenicity in male and female BALB/cJ mice, while not hampering protection outcomes in female mice. Fusion with mXCL1 may also reduce the need for cluster priming and improve vaccine scheduling. Overall, the data obtained in the present study could contribute to the overall goal of improving the efficacy of pre-erythrocytic malaria vaccines.
CFTR Mutation: Evaluation of Uptake Kinetics of Ferret Proximal Tubule Cells and Impact of CFTR Modulators on Renal Excretory Profile
(2024-09-09) Peagler, Kristen; Kelly, Edward J
Pre-clinical testing is an important step in drug development. Generally, in vivo, and in vitro methods are used during pre- clinical stages to test for toxicities and efficacy. In vivo models provide a medium for evaluating pharmacokinetics, pharmacodynamics and margin of safety; however, even with this testing, many drugs may perform well in pre-clinical trials but are discontinued during early phases of clinical trials. This gap is large as many of the drugs developed fall into this category and what may have once looked promising in animal studies and 2D studies do not make it to the patient’s bedside. Microphysiological systems may hold the answer to closing this gap. Microphysiological systems offer unique environments that if validated, can serve to reduce the number of animals used in toxicity studies. These models may be able to identify certain drug properties that are not amenable to be used as therapies earlier in the pre-clinical process. While work has been done to create a ferret kidney on a chip, rats and canines are the most used animal species in the drug development phase. Thus, more work should be done to create a model of rat and canine kidney on a chip utilizing proximal tubule cells that are able to effectively identify species specific toxicities as a result of treatment with drug compounds. We hypothesize that MPS systems will allow for rat proximal tubule cells to maintain normal morphology and will effectively model species -specific nephrotoxicity when exposed to an in vivo relevant toxicant by exhibiting increased kidney injury molecule 1 levels. Of note, antibiotics such as aminoglycosides, polymyxin B and chemotherapeutics are known to carry the risk of nephrotoxicity. Not only is this nephrotoxicity important in seemingly healthy patients, but also patients with diseases such as Cystic Fibrosis who commonly require treatment of complicated infections with high doses of these antibiotics. While there are many models of cystic fibrosis, the ferret model closely resembles a majority of the pathology that human patients face. With alterations in the CFTR gene and increased cubilin shedding in the urine, we hypothesize that kidneys collected from ferrets with a CFTR mutation will have decreased cubilin/megalin complexes at the apical surface of ferret proximal tubule epithelial cells, while the presence of urinary cubilin will be increased at the initial stage of treatment in human CF patients. Renal uptake of proteins and aminoglycosides will be altered due to this phenotype. While some data has been collected in regard to rat proximal tubule cells in 2D culture and MPS as well as evidence of differential uptake of the protein albumin and gentamicin, an amino glycoside, more data is needed to develop a conclusion in both arenas.
A Diet High in Saturated Fat and Sucrose Increases Susceptibility to Aspects of Alzheimer’s Disease in Aging Mice
(2024-04-26) Johnson, Chloe; Ladiges, Warren C
Alzheimer’s disease (AD) is a progressive, degenerative disorder of the brain and is the most common form of dementia in older adults. Prominent behavioral manifestations of AD include memory impairments and decline in other cognitive and non-cognitive domains. Clinical data suggest that the pathophysiological processes of AD begin more than a decade prior to the diagnosis of dementia. Therefore, in addition to increasing age, other factors could impose significant risks for developing AD. Diets rich in saturated fats and sugar (HFS) have been implicated in increasing risks for age-related diseases including AD but studies linking AD to HFS diets have been limited and at times controversial. A model of AAV vector (Aβ42 and pTau) mediated early stage AD in aging C57BL/6 mice was used to determine the impact of a HFS diet on the development of AD-associated cognitive impairment and neuropathology. Mice were started on the HFS diet and administered AAV-AD vectors intravenously at 20 months of age and followed for three months when they were tested for cognitive and non-cognitive behavior, white blood cell counts, and physical performance. Mice were then euthanized, and tissues collected for immunohistochemistry. The HFS diet generally increased cognitive impairment, anxiety, and incoordination and decreased strength in mice with AAV-AD in a sex-dependent manner. Neuronal Aβ42 showed increased density in the presence of AAV-AD and in association with decreased density of microglia and astrocytes. In conclusion, observations from this project suggest that behavioral and performance phenotypes may be associated with AD neuropathology but in a diet and sex dependent manner. The HFS diet is suspected of increasing neuronal susceptibility to AAV-AD in aging C57BL/6 mice, especially females, suggesting that a metabolically stressful diet could potentially increase the risk for neurodegeneration and development of cognitive decline and behavioral dysfunction.
A simple spatial navigation paradigm in aging mice connects heterogeneous behavioral phenotypes with neuropathology and Alzheimer’s disease
(2024-04-26) Park, Joo Young; Ladiges, Warren C
Aging of the brain affects everyone, but some develop signs and symptoms, such as a decline in cognitive function, more quickly than others. Spatial navigation confusion and memory deficits with increasing age may be one of the first indicators of more serious cognitive impairment associated with Alzheimer’s disease (AD). This project was designed to evaluate a spatial navigation learning task coined Boxmaze to predict resistance to AD progression. C57BL/6 female and male mice, 20 months of age, were tested for cognition using the Boxmaze, and then intravenously administered AAV-Aβ42 and AAV-pTau, or an AAV sham vector and followed for two months. Mice were then tested in the Y-maze and retested in the Boxmaze, followed by euthanasia and collection of brain tissues for formalin-fixation and immunohistochemistry using antibodies specific for neuropathology associated aging and AD. A spatial learning index, reflecting individual learning rates, was developed to measure distinctions in cognitive decline. Behavioral data showed that mice had differing susceptibilities to further cognitive decline with correlation of pre-AAV learning index and post-AAV learning index. Cognitive impairment phenotypes were not associated with neuropathology or other aging markers in the brain. Data from this project suggest that the Boxmaze spatial learning index could potentially predict early signs of learning impairment and screen for indicators of resistance or susceptibility to AD.
A Drug Cocktail of Rapamycin, Acarbose, and Phenylbutyrate Enhances Resilience to an Early-Stage Model of Alzheimer’s Disease
(2024-02-12) Wezeman, Jackson; Ladiges, Warren
The process of aging is defined by the breakdown of critical maintenance pathways leading to an accumulation of damage and its associated phenotypes. Aging affects many systems and is considered the greatest risk factor for a number of diseases. Therefore, interventions aimed at establishing resilience to aging should delay or prevent the onset of age-related diseases. Recent studies have shown a three-drug cocktail consisting of rapamycin, acarbose, and phenylbutyrate delayed the onset of physical, cognitive, and biological aging phenotypes in old mice. To test the ability of this drug cocktail to impact Alzheimer’s disease (AD), an Adeno-Associated-Viral vector model of AD was created. Mice were fed the cocktail 2 months prior to injection and allowed 3 months for phenotypic development. Cognitive phenotypes were evaluated through a navigation learning task. To quantify neuropathology, immunohistochemistry was performed for AD proteins and pathways of aging. Age related lesions in the brain were evaluated through a geropathology grading platform. Results suggested the drug cocktail was able to increase resilience to cognitive impairment, inflammation, age-related lesions, and AD protein aggregation while enhancing autophagy and synaptic integrity, preferentially in female cohorts. In conclusion, female mice were more susceptible to the development of early stage AD neuropathology and learning impairment, and more responsive to treatment with the drug cocktail in comparison to male mice. Translationally, a model of AD where females are more susceptible would have greater value as women have a greater burden and incidence of disease compared to men. These findings validate past results and provide the rationale for further investigations into enhancing resilience to early-stage AD by enhancing resilience to aging.
Increased Anxiety in Aging Mice is Associated with Cognitive Decline and Decreased Wheel-running
(2023-09-27) Li, Kunyuan; Ladiges, Warren C
This research project explores the relationship between anxiety and aging in mice, focusing on various physiological and behavioral parameters. The study used open field tests, cognitive function assessments, grip strength and rotation tests, and voluntary wheel running observations to examine anxiety levels and their association with memory and motor function in two strains (C57BL/6 and CB6F1) of male mice at increasing ages. Results indicated that older mice, particularly the CB6F1 strain, showed more anxiety as measured by the open field test. Higher levels of anxiety were associated with decreased cognitive performance as evidenced by impaired long-term memory, and reduced running distance. No significant association was found with grip strength or rotational performance. This study contributes to an understanding of anxiety and its consequences in aging and provides insights into potential therapeutic interventions for aging.
The Actin Regulatory Protein Hem1 Regulates T cell Homeostasis and Cytokine Production in a T Cell Specific Manner
(2023-08-14) Christodoulou, Alexandra; Iritani, Brian
Hematopoietic Protein-1 (Hem1) is a hematopoietic cell specific component of the actin regulatory WAVE complex, which is activated downstream of multiple immune receptors and a mediator of f-actin polymerization. Mutations in the NCKAP1L gene encoding HEM1 have recently been found to result in severe Primary Immunodeficiency Disease (PID) in children. We utilized mouse models of constitutive and conditional deletion of Nckap1l to study the role Hem1 plays in T cell development, activation, and function. Hem1 deficient T cells exhibit ed a shift towards decreased naÃ¯ve and increased memory T cells, and increased frequency of regulatory T cells. Loss of Hem1 specifically in T cells resulted in hallmarks of CD4 T cell exhaustion, including CD4 T cell lymphopenia, decreased activation and proliferation, increased expression of the inhibitory receptors PD-1 and Tim3, and increased IL-10 production. In vitro TCR stimulation of CD4 T cells resulted in significantly decreased expression of IL-2, and significantly increased production of the Th2 cytokines IL-4, IL-5, IL-13; increased Th17 cytokines IL-17, IL-22; and increased Treg cytokine IL-10. This correlated with decreased F-actin capping and increased expression of CD107a indicative of increased granule membrane fusion, suggesting increased release of cytokines secondary to cortical actin disruption. These results collectively suggest that Hem-1 and the WAVE Regulatory Complex (WRC) are critical for maintaining CD4 T cell homeostasis and regulated cytokine production following T cell activation.
Role of macrophage-derived versican in the innate immune response to LPS
(2022-07-14) Mosca, Leandra F; Frevert, Charles
Versican, a chondroitin sulfate proteoglycan (CSPG), is an integral component of the extracellular matrix (ECM) necessary for embryonic development, but thereafter is expressed in very low levels in healthy adult lungs. As a key component of the extracellular matrix (ECM), versican modulates the innate immune response through interactions with other extracellular matrix molecules, regulating cell migration and influencing the accumulation and release of cytokines and growth factors. An essential feature of versican is that its impact is highly contextual. Among other factors, the influence of versican depends on the cellular source, specific agonist, and phase of the inflammatory response. Although previous work from our group shows versican has both pro and anti-inflammatory effects, the influence on pulmonary function, inflammation, and injury is not well understood. Here, we investigated the role of macrophage-derived versican in a mouse model of LPS-induced lung injury. We demonstrate that bone marrow-derived macrophages (BMDMs) and mice deficient in macrophage-derived versican show an altered acute immune response to Toll-Like Receptor-4(TLR4) activation compared to wild-type (WT) controls. The significance of these findings is furthered by histologic evidence that a lack of macrophage-derived versican contributes to increased leukocyte recruitment at 48 hours post-challenge. This study demonstrates that versican is an immunomodulatory molecule and controls the innate immune system.
A cocktail of rapamycin, acarbose and phenylbutyrate prevents age-related cognitive decline in mice by altering aging pathways
(2022-07-14) Jiang, Zhou; Ladiges, Warren C
Aging is a primary risk factor for cognitive dysfunction and exacerbates multiple biological processes in the brain, including but not limited to nutrient sensing dysregulation, insulin sensing dysfunction and histone deacetylation. Therefore, pharmaceutical intervention of aging that targets several distinct but overlapping pathways provides a basis for testing combinations of drugs in a cocktail. A recent study showed that middle-aged mice treated with a drug cocktail of anti-aging drugs rapamycin, acarbose, and phenylbutyrate for three months had increased resilience to age related cognitive decline. This finding provided the rationale to investigate the comprehensive transcriptomic and molecular changes within the brain of mice that received this cocktail treatment or control substance. Transcriptome profiles were generated through RNA sequencing and pathway analysis was performed by gene set enrichment analysis to evaluate the overall TNA message effect of the drug cocktail. Molecular endpoints representing aging pathways were measured through immunohistochemistry to further validate the attenuation of brain aging in the hippocampus of mice that received the cocktail treatment, each individual drug or controls. Results indicated that biological processes that aggravate with increasing age were suppressed and autophagy, which decreases with aging, was enhanced in the brains of mice given the drug cocktail. The molecular endpoint assessments indicated that the treatment with the drug cocktail was overall more effective than any of the individual drugs for relieving cognitive impairment by targeting multiple aging pathways.
Fibroblast p38-MAPK signaling modulates cardiac function and phenotype in mouse models of inherited dilated and hypertrophic cardiomyopathy
(2021-08-26) Zabrecky, Kristin; Davis, Jennifer
Inherited cardiomyopathies are common causes of heart failure in the general population that present with two main clinical phenotypes: dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). Little is known regarding the pathogenesis and subsequent clinical impact of early extracellular matrix (ECM) remodeling in these diseases. By genetically modulating the ECM via fibroblast p38-MAPK, the mechanical basis of ECM-cardiomyocyte feedback and consequence on whole heart remodeling in DCM and HCM was examined via non- invasive and invasive physiology metrics. Both structural and functional phenotype was demonstrated to be susceptible to matrix modifications at the whole heart and cardiomyocyte level. This study demonstrates that fibroblast state and downstream matrix remodeling play an active role in the pathogenesis of cardiomyopathies and offers insights into early diagnostic tools and targeted therapeutic approaches.
Murine norovirus inhibits B cell development in bone marrow, but does not impair antibody production in Stat1-/- mice
(2021-08-26) Eldridge, Daniel E; Hsu, Charlie C.
Murine norovirus (MNV) is used as a model system to study human noroviruses, can infect macrophages/monocytes, neutrophils, dendritic, intestinal epithelial, T and B cells, and is highly prevalent in laboratory mice. We previously showed that MNV infection significantly reduces bone marrow B cell populations in a Stat1-dependent manner. We demonstrate here that while MNV-infected Stat1-/- mice have significant bone marrow B cell losses, expansion of splenic B cells capable of mounting an antibody response to novel antigens remains intact. We also investigated increased granulopoiesis as a mechanism of B cell loss, and we show that administration of anti-G-CSF antibody inhibits the pronounced bone marrow granulopoiesis induced by MNV infection of Stat1-/- mice, but this inhibition does not rescue bone marrow B cell losses. Therefore, these results show that MNV-infected Stat1-/- mice can still exhibit a robust humoral immune response in spite of decreased bone marrow B cells, and suggests that further investigation will be needed to identify other indirect factors or mechanisms that are responsible for the bone marrow B cell losses seen after MNV infection. Additionally, this work adds to the knowledge about the potential physiologic effects on mice with Stat1-related disruptions in research mouse colonies that may be endemically infected with MNV.
Resilience to acute sleep deprivation is associated with attenuation of hippocampal mediated learning impairment
(2020-08-14) Lee, Amanda Y; Ladiges, Warren C
Sleep deprivation is a universal issue that affects individuals in differing ways. While some individuals experience a deficit in daily performance, others experience resiliency as they can maintain high levels of physical and mental functionality. Although we know that a loss of sleep causes cognitive dysfunction in areas such as learning and memory, we do not understand which neural mechanisms contribute to the attenuation of learning impairment. Recently, our laboratory produced a cognitive assay known as the Box Maze that can assess learning impairment in sleep-deprived mice. Based on data accumulated from the box maze, we hypothesized that a grading platform could separate fast and slow learners in mice that have or have not been exposed to sleep deprivation. This grade could then be further explored with biomarkers, providing insight to the attenuation of learning impairment in the hippocampi of sleep deprived, fast learning mice. This study utilized an existing database of box maze escape times across 16-18 month old, male, C57BL/6 mice that were or were not sleep deprived with all other conditions standardized. After data mining, a total of 40 mice fit the criteria for the study. The grading platform utilizes a logarithmic trend line of the box maze trials to ultimately separate fast and slow learners. The separation was based on the R2 value which represented the learning curve of each individual mouse. The results showed that sleep deprived mice had more slow learners than fast whereas control mice showed the opposite. The hippocampus of these mice then underwent immunohistochemistry to explore biomarker levels that would be insightful of the attenuated learning impairment in sleep deprived mice that were graded as fast learners. The results showed that fast learners in the sleep deprived groups, expressed similar levels of biomarkers as that of the control fast learning groups. The data provides evidence that sleep-deprived mice that performed well in a cognitive assay show less hippocampal mediated learning impairment. These findings provide the rationale for clinical investigations into neurobiological resilience with increasing age.
Conditional Disruption of Hem-1 Results in Impaired B Cell Development and Aberrant Antibody Production
(2020-08-14) Avalos, Alan Vinicio; Iritani, Brian M
Hematopoietic protein-1 is a hematopoietic cell specific member of the actin-regulatory WAVE complex, which acts downstream of multiple immune receptors to stimulate F-actin polymerization. Loss-of-function mutations in the gene encoding Hem-1, NCKAP1L, have recently been found to result in Primary Immunodeficiency Disease in humans, characterized by recurrent bacterial and viral respiratory infections, asthma, skin infections, bacteremia, atopy, and autoimmunity. However, the cellular and molecular mechanisms of how loss of Hem-1 results in PID are not known. In this study, we generated constitutive and B cell specific Nckap1l knockout mice using the Cre-LoxP system to dissect the importance of Hem-1 in B cell development and functions. We found that mice with B cell specific disruption of Hem-1 lack mature recirculating B cells in the bone marrow and lymph nodes, along with reduced peripheral B cell populations such as B1, follicular and marginal zone (MZ) B cells due to the decreased capability to efficiently migrate to different lymphoid tissues. Immunization with the T-independent antigens NP-ficoll and heat-killed S. pneumoniae (HKSP), failed to elicit antibody production due to a lack of the innate-like B1 and MZ B cell populations. Immunization with HKSP failed to produce antibody titers sufficient to protect Hem-1 deficient mice from a challenge with a lethal dose of the bacteria Streptococcus pneumoniae. In contrast to the poor antibody response to T-independent antigens, Hem-1 deficient mice produced pronounced IgM and IgG2c antibody titers following immunization with the T-dependent antigen NP-KLH. Hem-1 deficient B cells demonstrated hyperreactive states as shown by: decreased surface IgM expression, increased and sustained calcium influx following IgM stimulation, increased expression of activation markers and higher expression of phosphorylated molecules downstream of the BCR before and after IgM stimulation. These changes in Hem-1 deficient B cells are permissive to the formation of the unique T-bet+CD11c+ B cell population that form autoantibodies. These collective findings suggest that Hem-1 is essential for normal development of B cells, B cell migration and homing and regulating BCR signaling thus providing a mouse model that may provide further insight into the mechanism of autoimmunity in children with mutations in NCKAP1L.

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