Now showing items 1-20 of 2268

    • The Clinical Translation of Cardiac Xenotransplantation

      Goerlich, Corbin; Mohiuddin, Muhammad M.; Singh, Nevil (2022)
      Patients with end-stage heart failure requiring heart transplantation die simply because allografts are in short supply. Cardiac xenotransplantation from genetically-modified pig source animals has been proposed to bridge the gap between supply and demand. However, there are two predominant barriers to clinical translation and are poorly understood. The first, is a type of primary graft dysfunction, termed perioperative cardiac xenograft dysfunction (PCXD), which causes failure of the xenograft within 48 hours after transplantation. The second, post-transplantation xenograft growth, which causes a life-limiting diastolic heart failure within the first month after transplantation. We demonstrate that PCXD can be overcome with cardiac preservation techniques that minimize ischemia, either with blood-based cardioplegia induction or non-ischemic continuous preservation (NICP). In a heterotopic PCXD model, we further demonstrate that PCXD is likely a phenomenon rooted in xenograft dysfunction resulting from activation of innate immunity within the xenograft. While further studies need to be done, we demonstrate evidence that PCXD is augmented by the synergistic inflammatory effects of both cardiopulmonary bypass and cross-species transplantation. We also provide evidence that polymorphisms from TLR4 of S. scrofa (swine) compared to H. sapiens, may explain a possible target for the unique inflammatory signaling, that results in xenograft dysfunction after cardiac xenotransplantation. We also demonstrate that post-transplantation xenograft growth is multifactorial, but can be limited by growth hormone receptor knockout donors, along with genetic modifications that reduce immunogenicity of the xenograft. This growth, within 6 months after transplantation, is not a result of physiologic mismatch or cardiomyocyte hypertrophy. Therefore, recipients do not need to be treated for tachycardia and hypertension as previously thought. Lastly, we demonstrate the clinical translation of cardiac xenotransplantation by applying knowledge and expertise obtained from these studies. Expanded access (“compassionate use”) FDA authorization was based on demonstration of principle from our preclinical model. An ECMO-dependent patient without other therapeutic options was transplanted a genetically-modified cardiac xenograft, with 10-gene edits, combined with non-ischemic cardiac preservation and anti-CD40 monoclonal antibody-based immunosuppression. The patient was able to successfully wean from ECMO, participate in active rehabilitation and survived 60 days after transplantation without evidence of rejection.
    • Investigating the Role of Microglia and Extracellular Vesicles in Spinal Cord Injury-Induced Brain Dysfunction

      Khan, Niaz; Faden, A. I. (2022)
      Spinal cord injury (SCI) causes brain neurodegeneration leading to cognitive and affective changes, including memory loss and mood alterations. Using SCI models, we and others have demonstrated that progressive neurodegeneration is accompanied by neuroinflammation, including sustained microglial activation. The primary goal of this dissertation was to test the hypothesis that SCI triggers brain microglia-mediated neuroinflammation and secondary neurological dysfunction and to study the underpinning mechanisms including changes in systemic and central extracellular vesicles (EVs). First, we probed the mechanisms responsible for microglia activation and examined the effect of pharmacological depletion of microglia on posttraumatic neuropathology and cognitive/depressive-like behavior in a mouse SCI model. Microglial depletion significantly improved neuronal cell loss in key brain regions and associated cognitive/depressive-like behavioral outcomes after SCI. The transcriptomes of the spinal cord and brain were also substantially altered, supporting our hypothesis that microglia significantly contribute to changes related to inflammation, neurotransmission, and apoptosis after SCI. Second, we studied changes in circulating EVs after SCI. EVs are biological nanoparticles released from cells that contribute to intercellular communication and can become altered with disease. We found a significant increase in plasma tetraspanin CD81+ EVs after SCI at 1d post-injury. Surface CD81 was decreased on astrocytes at the injury site, suggesting that these cells may release CD81+ EVs into circulation. Total plasma EV microRNA content was also significantly modified, similar to the profile previously described in inflammatory astrocyte EVs. Notably, when injected into the cerebroventricular system, plasma EVs from SCI mice increased brain expression of several inflammatory genes, including markers of astrocyte reactivity. Finally, we examined the brain transcriptional profile and EV changes 19 months post-SCI in male and female mice. While we observed strong sex-dependent differences in the overall brain transcriptome after SCI, the homeostatic microglial phenotype was reduced in both sexes. Chronic SCI increased EV count in the brain and modified their microRNA content, which may explain the observed transcriptional changes. Plasma EV markers were also elevated late after injury, especially in males. Collectively, these experiments are the first to characterize EV dynamics after SCI and suggest that EVs may be involved in posttraumatic brain inflammation.
    • Characterization of Temperature Dependent Activity in a Model Polyextremophilic Beta-galactosidase Enzyme Through Kinetic and Structural Analysis

      Laye, Victoria; DasSarma, Shiladitya (2022)
      The Antarctic haloarchaeon, Halorubrum lacusprofundi, contains a polyextremophilic family 42 β-galactosidase, which we are using as a model for polyextremophilic enzymes. Divergent amino acid residues in this 78 kDa protein were identified through comparative genomics and hypothesized to be important for cold activity. Six amino acid residues were previously mutated and five were shown by steady-state kinetic analysis to have altered temperature-dependent catalytic activity profiles via effects on Km and/or kcat compared to the wild-type enzyme. Double-mutated enzymes were constructed and tested for temperature effects from 0-50 ºC, including two new tandem residue pairs, two potential loop insertions, and pairwise combination of the single residue mutations. The observed temperature and salinity dependent kinetic effects were compared to root-mean-square fluctuations to determine structural mechanisms of polyextremophilic activity. All the mutated enzymes were found to be more catalytically active at higher and/or less active at colder temperatures compared to the wild-type, with both Km and kcat effects observed for the two tandem mutations. For pairwise combinations, a Km effect was seen when the surface-exposed F387L mutation located in a domain A TIM barrel α helix 19 Å from the active site was combined with two internal residues, N251D or V482L. When another surface-exposed residue, I476V, was paired with N251D or V482L, primarily a kcat effect was observed. The temperature dependent kinetic effects were continued to 25-50 ºC with a dominant Km effect observed in all mutated enzymes. By deleting the identified insertions/loops, we are able to determine the kinetic effects of these insertions. The domain B deletion resulted in a less active enzyme overall converging with the wild-type at higher temperatures while the domain C deletion resulted in reduced cold-activity and improved activity at higher temperatures. Domain B may confer cold-activity without changing the thermal stability while domain C confers cold activity, at least in part, at the expense of activity at higher temperatures. With molecular dynamics simulations, increased flexibility was observed with higher temperature and salinity and in the mutations, indicating that the enzyme’s function is related to its flexibility and there is a balance required for optimal polyextremophilic activity.
    • Visual Deficits in a Model of Gestational Hypothyroidism

      Tarasiewicz, Agnieszka; Medina, Alexandre E. (2022)
      Hypothyroidism prevalence among pregnant women is between 0.5 to 4% (Carney et al., 2014). Most studies look at the prolonged or severe reduction in thyroid hormone (TH) levels. We looked at how the reduced levels of TH during the third trimester of human gestation and the first weeks after birth in rodents impact the visual system. Additionally, we try to answer whether PTU treatment would affect neuronal plasticity in the visual cortex. We used the Visual Evoked Potential recordings to assess contrast sensitivity, spatial frequency acuity, and ocular dominance plasticity. In addition, we look into the expression of the photoreceptors in the retina. PTU exposure impacts the contrast sensitivity but not the spatial frequency acuity or ocular dominance plasticity. The expression level of the photoreceptor Opsin-M was also impacted. The reduced levels of the thyroid hormones during this crucial time have long-lasting consequences for the proper visual system processing.
    • Effectiveness of an Intelligent Sleep Management System in the US Military: Preliminary Results

      Mounts, Charles; Thomas, Mary Ann; Adornetti, Julianna P.; Wade, Christine E.; Wickwire, Emerson M.; Collen, Jacob (2022-09-12)
    • The Role of Colonization Factors CFA/I and CS21 in Enterotoxigenic E.coli (ETEC) Pathogenesis in the Human Enteroid Model

      Smith, Emily; Barry, Eileen M. (2022)
      Enterotoxigenic Escherichia coli (ETEC) is a primary causative agent of diarrhea in travelers and in young children in low-to-middle income countries (LMICs). ETEC adhere to intestinal epithelia via colonization factors (CFs) and secrete heat-stable toxin (ST) and/or heat-labile toxin (LT), causing dysregulated cellular ion transport and water secretion. ETEC isolates often harbor genes encoding more than one CF and are prime targets as vaccine antigens. Many clinical isolates express CFA/I and CS21; however, a role for CS21 alone or with CFA/I has not been defined. We hypothesize that expression of both CFs confers increased adherence and toxin delivery to the human enteroid. Clinical strains expressing CFA/I and/or CS21 were evaluated, and CF-deficient mutants were engineered. After confirming CF expression using western blot and electron microscopy, assays demonstrated CFA/I was important for CFA/I-CS21 ETEC adherence, as CFA/I-deficient mutants and strains pre-incubated with anti-CFA/I antibody had significantly reduced adherence to enteroid monolayers compared to wildtype. In contrast, CS21 was not required as CS21-deficient mutants and strains pre-incubated with anti-CS21 antibody adhered at similar levels as wildtype. These data demonstrate that targeting CFA/I in CFA/I-CS21 ETEC is sufficient for significant adherence reduction. Delivery of ST by CFA/I-CS21 ETEC was evaluated. Strain-specific levels of toxin delivery were detected but CF-dependent ST delivery was not observed, which may reflect the lack of flow and stretch in the current enteroid model. Upon investigation of host responses to ETEC, the enteroid monolayer integrity was not disrupted, as shown by the increase in transepithelial electrical resistance and the lack of inflammatory cytokines produced. Infection with ETEC strains resulted in decreased mucus (MUC2) production, but this was not CF-dependent. Further studies of strain-specific CFA/I expression revealed that it may be transcriptionally or post-transcriptionally regulated, following observation of nearly identical CFA/I operon sequences and many shared CF-specific regulators at the genomic level. Overall, these data support the role of CFA/I in CFA/I-CS21 ETEC adherence and reinforces CFA/I as a main target for vaccines. These data also highlight the human enteroid model to study ETEC pathogenesis and for evaluation of preclinical therapeutics.
    • Novel Modules in the T Cell Signaling Circuit Which Enable Synergy Between Responses to Self and Foreign Peptides

      Wolf, Gideon; Singh, Nevil (2022)
      T cell activation occurs when a T cell receptor (TCR) engages with cognate agonistic peptides in the context of Major Histocompatibility Complexes (pMHCs) on antigen presenting cells (APCs). This initiates a series of intracellular signaling events proximal to the TCR and associated CD3 complexes, mediated by unique kinases together with scaffolding and lattice molecules. The downstream cascades ultimately lead to transcriptional changes that promote the cellular program of conventional T cell activation—for example, cytoskeletal rearrangement, cytokine production, cellular proliferation, and differentiation. Understanding signaling mechanisms not only allows us to decipher the regulation of T cell activation but also to manipulate immune responses pharmacologically. TCR signaling by agonistic pMHCs is well studied, but much less is known about signaling by a parallel universe of self-peptides that also engage TCRs in vivo. The focus of this thesis is to understand how T cells perceive these signals without fully acquiring effector responses as a result. The central hypothesis of my thesis is that self-peptide ligands signal uniquely through the TCR to alter the fate and function of a T cell both with and without presence of their cognate agonist. We evaluated this hypothesis using approaches that globally increased self-peptide presentation in vivo (using FLT3L to generate more DCs), deprived T cells of self-peptide (by culturing away from APCs) or stimulated a TCR with a known self-peptide in the presence or absence of the strong agonist. The significant findings from these studies are that (i) boosting self-peptide presentation transiently increases a narrow T cell effector subset; (ii) depriving T cells of self-engagement lowers basal phosphorylation in the key signaling adapter LAT; (iii) self-peptides do not trigger cellular activation on their own, but synergize to enhance activation as measured by CD69, pERK, and several other parameters (iv) self-peptides initiate TCR signaling up to the level of pMEK and (v) self-peptides elicit a unique transcriptional profile. Together, these results not only define the unique contributions of self-peptides to T cell activation but also demonstrate a distinct wiring profile in the TCR-signaling network that limits self-peptide sensing at the ERK step.
    • The Role of Granzyme B in Tumor Migration and Metastasis

      Ellis, Tibbs; Cao, Xuefang (2022)
      The granule exocytosis pathway has classically been described as our most potent response to pathogens, cancers, as well as autoreactive disorders. This is executed by the action of perforin and a family of granule-associated serine proteases known as granzymes (Gzm). Many studies have shown that these proteases exert their effects by killing affected cells, creating an obvious correlation with cytotoxic T cells (CTLs) and natural killer cells (NK) greatly expressing GzmB. Studies have found that the tumor cells ability to create a suppressive environment is dependent on Gzm expression by regulatory T lymphocytes (Treg). Research has shown GzmB can target and cleave extracellular molecules such as fibronectin, vitronectin and laminin. Bird et al published data showing that GzmB aids in the transmigration of CTLs, however the ability of GzmB to aid in the migration of non-T cells has not been investigated. We hypothesize that GzmB secretion may cause extracellular matrix (ECM) remodeling in the tumor microenvironment, aiding in the outgrowth of the tumor cells via promoting higher rates of invasion and/or metastasis. First, we designed experiments to test in-vivo and in-vitro conditions that generate GzmB production in multiple cell types. We were able to induce GzmB production by Tregs both in-vitro and in-vivo. We also identified multiple mechanisms that allow for CD8 T cells to produce GzmB. We next investigated mechanisms that would allow for GzmB to be found in the extracellular environment. We found that CD8 T cells have the ability to secrete GzmB without the need of an immunological synapse. In fact, GzmB secretion is not dependent on exogenous IL-2. GzmB improves the migration and invasive potential of tumor cells. In Treg-specific GzmBko (Foxp3creGzmBfl/fl) mice, GzmB+ Tregs increase the rate of tumor metastasis in the lungs of the mice. In addition, GzmB+ CD8 T cells can potentiate tumor metastasis, in contrast to GzmB-deficient CD8 T cells. These findings shed light on the protumorigenic potential of secretory GzmB, which could serve as a unique biomarker for predicting metastasis.
    • Flight Relevant Hypobaria Worsens Neuropathologic and Behavioral Outcomes in Rats Following the Combination of Under-Vehicle Blast plus Controlled Cortical Impact

      Fiskum, Gary; Proctor, Julie; Miller, Catriona H.; Piskoun, Boris; Castillo, Lidia; Rangghran, Parisa; Hrdlick, Amanda; Leiste, Ulrich; Fourney, William (2022-09-09)
    • Using PacBio SMRT platform to assess the genetic variation of Plasmodium falciparum Circumsporozoite Protein (PfCSP) in Kalifabougou, Mali

      Ouedraogo, Nadiatou; Laurens, Matthew B. (2022)
      Objectives: In this study, we assessed the genetic variation in CSP sequences from Kalifabougou, Mali, by evaluating the diversity of haplotypes in the N- and C- Terminal region, the repeat regions, and the T- and B-cell epitopes. Methods: Sequences used for the study were generated from 59 sequences collected during a malaria transmission season conducted in Kalifabougou, Mali. Generated using the PacBio SMRT platform. Results: Results showed that the central repeat and C-terminal Th2R/Th3R epitope regions were highly polymorphic in this study, whereas the N-terminal non-repeat region was less variable. Furthermore findings highlighted CSP polymorphism and suggested that the repeat and B-cell epitope of our samples is similar to those of 7G8 PfCSP, whereas the C-terminal is similar to that of the 3D7 PfCSP strain. Finally, findings the CSP sequences from the Kalifabougou site are more similar to those from Navrongo than to those from Cape Coast.
    • Membrane-Anchored Serine Proteases in Ovarian Cancer Dissemination and Metastasis

      Pawar, Nisha; Antalis, Toni M. (2022)
      Ovarian cancer (OvCa) is the most fatal gynecological malignancy due to delayed clinical presentation, passive metastasis, frequent tumor recurrence, and lack of effective targeted therapies. OvCa tumors predominantly shed as single cells or spheroids and disseminate throughout the peritoneal cavity. Spheroids are considered critical metastatic units that contribute to disease recurrence and chemoresistance. Despite recent advances in treatment strategies, there has been little improvement in patient survival. There is an urgent need to develop a better understanding of molecular mechanisms that are fundamental for OvCa dissemination, in order to provide alternative therapeutic options for long-term remission. Matriptase, a membrane-anchored serine protease (MASP), and its substrate protease-activated receptor-2 (PAR-2) both exhibit elevated expression in OvCa compared to normal ovary tissues which worsens patient survival, suggesting a critical role for activation of this pathway in OvCa progression. Overactive matriptase, induced by an imbalanced ratio with its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) has been implicated in disruption of barrier integrity and epithelial carcinogenesis, although the mechanism is not known. This study, demonstrates a functional role for an imbalanced matriptase:HAI-1 ratio in OvCa progression, and identifies a matriptase/PAR-2/PI3K/Akt/MMP-9/E-cadherin signaling axis that disrupts cell-cell interactions and promotes formation of pro-metastatic loose spheroids in vitro, in in vivo orthotopic xenograft models, and in patient-derived tumor cells. This matriptase/PAR-2 pro-metastatic signaling is in direct contrast to another MASP family member, testisin, suggesting a novel mechanism of biased agonism that orchestrates OvCa dissemination. Since over-activity of MASPs is associated with advanced OvCa, we have developed a MASP-activated pro-drug based on a re-engineered anthrax toxin (PAS:LF) that is selectively activated by zymogen-activating proteases on the tumor cell surface to inhibit tumor cell-survival pathways. This therapeutic strategy is radically different from anti-proliferative mechanisms of standard chemotherapeutic agents. We have demonstrated anti-tumor efficacy in a range of OvCa cells and spheroids in vitro, in various in vivo orthotopic xenograft models of OvCa dissemination, in patient ascites-derived tumor cells and in patient-derived xenograft models in vivo, with no off-target adverse effects. Clinical translation of these preclinical findings could establish PAS:LF as a promising treatment strategy to improve patient outcomes.
    • Nitric Oxide’s Role in Mechanically-Stimulated Sclerostin Degradation

      Buck, Heather; Torre, Olivia; Gould, Nicole; Ward, Christopher, Ph.D.; Stains, Joseph P. (2022-09)
    • A Specialized Metabolic Pathway Partitions Citrate in Hydroxyapatite to Impact Mineralization of Bones and Teeth

      Dirckx, Naomi; Zhang, Qian; Chu, Emily Y.; Tower, Robert J.; Li, Zhu; Guo, Shenghao; Yuan, Shichen; Khare, Pratik A.; Zhang, Cissy; Verardo, Angela; et al. (2022-09)
    • Evaluating Long-QT Type 8 Mutations in Induced Pluripotent Stem Cell Derived Cardiomyocytes

      Vieira, Daiana C. O.; Owoyemi, Josiah O.; DiSilvestre, Deborah; Dick, Ivy E (2022-09)
      Long QT syndrome type 8 (LQT8) is a severe disorder caused by a point mutation in CaV1.2 L-type Ca2+ channels, resulting in a prolonged QT interval, arrhythmia, and sudden cardiac death. The canonical and most common mutation, G406R, occurs most often within the mutually exclusive exon 8a, found in 20% of cardiac CaV1.2 variants. Yet, even heterozygous expression (10% of cardiac channels) of G406R is hugely deleterious. Despite this limited expression, the patients exhibit severe symptoms, which are often fatal in early childhood. Conversely, some recently discovered CaV1.2 mutations, occur in constitutive exons, resulting in an overall higher expression (50%). We therefore compared the biophysical effects of LQT8 mutations and evaluated the impact of each mutation on the action potential profile of induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs). Patch clamp recordings showed the mutations differentially altered Ca2+ dependent inactivation (CDI), and voltage dependent inactivation (VDI). In addition, action potential recordings demonstrated significant prolongation in iPSC-CMs harboring each mutation, consistent with LQT8. Moreover, optical mapping demonstrated readily inducible arrhythmic behavior in each mutant iPSC-CM line, displayed as a spiral pattern in the propagation of the action potential across the coverslip. Thus, LQT8 iPSC-CMs recapitulate not only the expected AP prolongation of LQT8, but the arrhythmogenic episodes seen in patients, making them an ideal model system to study this disease.
    • Determining the role of the unique N-terminal 1b domain in the gating of heterotetrameric hERG1a/hERG1b potassium channels

      Crawford, Taylor R.; Trudeau, Matthew C. (2022-09-08)
      Two isoforms of the hERG (human Ether-á-go-go-Related Gene) potassium channel, hERG1a and hERG1b, associate in the heart to form the rapid delayed rectifier current (IKr), which is primarily responsible for repolarizing cardiac action potentials. Inherited mutations in both hERG1a and hERG1b cause Long QT Syndrome type 2 (LQT2), marked by prolonged cardiac repolarization and sudden death arrhythmia. The assembly of hERG1a subunits with hERG1b subunits increases the number of hERG1b subunits at the plasma membrane, but the mechanism by which hERG1a enhances hERG1b is not completely understood. Recently, it was reported that when expressed in trans, the hERG1a N-terminal region markedly increased hERG1b currents and increased biotinlabelled hERG1b protein at the plasma membrane surface. A direct structural interaction between the hERG1a N-terminal residues 216-220 and the hERG1b N-terminal region was characterized and proposed to be necessary for the upregulation of hERG1b (Johnson et al., JBC 2022). However, hERG1b channels with a deletion of the unique N-terminal 1b domain did not have a measurable increase in current density or in biotinylated protein when co-expressed with hERG1a N-terminal region pieces. This indicates that the 1b domain is required for the increase in hERG1b by hERG1a. Interestingly, the 1b domain-deleted hERG1b channels displayed a large leftward shift in the voltage dependence of activation when co-expressed with the full hERG1a N-terminal region. This is unexpected, as the 1b domain was not previously considered as a determinant of hERG gating. Here, we will investigate the mechanism of this large leftward shift and determine the role of the 1b domain in the gating properties of hERG1a/hERG1b heterotetrameric channels.
    • Dissecting the role of CaV1.2 dysfunction in the pathogenesis of autism spectrum disorder

      Herold, Kevin G.; Bamgboye, Moradeke A.; Vieira, Daiana C. O.; DiSilvestre, Deborah; Hussey, John; Owoyemi, Josiah O.; Meredith, Andrea L.; Dick, Ivy E (2022-09-07)
      Autism Spectrum Disorder (ASD) is a neurological disorder characterized by repetitive, restricted behaviors, interests and activities along with a range of challenges in communication skills and social interaction. Pathway and genetic analyses have indicated a strong connection between ASD and the dysregulation of Ca2+ signaling. In particular, genetic mutations within the L-type Ca2+ channel (LTCC) have been shown to be causative of ASD. In fact, mutations within the CaV1.2 LTCC have been identified as one of the few monogenic causes of ASD, resulting in a multisystem disorder known as Timothy Syndrome (TS). TS patients harbor a single de novo point mutation within CaV1.2, which causes severe cardiac arrhythmias, long-QT syndrome and ASD. TS is one of the most penetrant genetic forms of ASD, making it an ideal model system with which to gain traction on understanding the role of Ca2+ dysfunction in ASD. Interestingly, we identified marked differences in channel gating due to distinct TS mutations. Such differences may underlie the differential phenotypes of select mutations, potentially explaining why some pathogenic mutations do not lead to ASD. To probe the effects of these mutations on neuronal function, we utilized human induced pluripotent stem cell (iPSC)-derived neurons. Patch clamp recordings of these cells yield distinct LTCC currents, enabling evaluation of the mutations in the context of a human neuron. Moreover, we utilized iPSC-derived 3D neuronal organoids to gain a deeper understanding of the effect of CaV1.2 dysfunction at a network level. Overall, as Ca2+ disruption may be a recurrent feature of ASD, studying rare mutations such as TS, may provide insight into the pathogenesis ASD beyond TS.
    • The Effects of HIV Exposure and Maternal Antibodies to Hepatitis B Virus on the Immune Response of Nigerian Infants to Hepatitis B Vaccine.

      Bada, Florence; Stafford, Kristen Alyce; Campbell, James Daniel (2022)
      Introduction: Chronic hepatitis B virus (HBV) infection leads to considerable morbidity and early mortality. Development of chronic HBV infection can be prevented by a three to four dose schedule of Hepatitis B vaccines in immunocompetent infants. However, HIV exposed uninfected infants (HEU) are thought to exhibit an attenuated immune response to some vaccines. In addition, a significant proportion of pregnant women in Nigeria have antibodies to HBV, specifically Hepatitis B surface antibodies (HBsAb). Maternal antibodies inhibit infant immune responses in some instances. Objective: To estimate the effect of antenatal and perinatal exposure to maternal HIV, and the effects of maternal HBsAb on the immune response of Nigerian infants to hepatitis B vaccine. Methods: Using a retrospective cohort design, we determined the relationship between infant HIV-exposure status, and infant exposure to detectable concentrations of maternal HBsAb, and infant immune response to Hepatitis B vaccine separately using general linear models adjusted for potential confounders. Subsequently, we used Fisher’s Exact tests to compare the proportion of infants with HBsAb concentrations above 10mIU/mL: for HEU as compared to HIV unexposed uninfected infants (HUU), and for infants exposed to detectable concentrations of maternal HBsAb as compared to infants unexposed to detectable concentrations of maternal HBsAb at 24 and 52 weeks of age separately. Results: We found HIV exposure to be associated with infant immune response to hepatitis B vaccine at birth, Weeks 4 and 52 (P<0.0001, P=0.05 and P<0.0001) and also to be associated with the proportion of infants with HBsAb concentrations ≥ 10mIU/mL at Week 52 (P=0.04). Exposure to detectable concentrations of maternal HBsAb was also associated with infant immune response at Weeks 24 and 52 respectively. Conclusion: Though antenatal and perinatal exposure to HIV, and to detectable concentrations of maternal HBsAb were found to be associated with infant immune response to hepatitis B vaccine, these exposures did not appear to attenuate immune response. In addition, differences observed in the proportion of infants with HBsAb concentrations ≥ 10mIU/mL were small and do not appear to be clinically relevant.
    • Hippocampal Contributions to Stress: What the Hippocampus Tells the HPA Axis and What Could Go Wrong?

      Cole, Anthony; Thompson, Scott M. (2022)
      The links between stress and psychiatric illnesses are numerous and bidirectional. Stress is highly correlated to psychiatric illness, increasing the risk of illness and often precipitating its onset. The primary class of neuroendocrine stress hormones, glucocorticoids, is often dysregulated in patients with major depression. Understanding the neurobiological changes underlying these findings is crucial for the development of improved therapeutic strategies and better patient outcomes. Prior work suggests that the hippocampus is stress-sensitive, substantially altered in major depression, and plays an important regulatory role in HPA axis function. Our understanding of the processes regulating the stress response is incomplete, particularly in regards to the various brain regions responsible for its central regulation. In this document I strive to clarify our understanding of a specific component of stress regulation, namely the hippocampal contribution, which could prove important to understanding the various links between stress and psychiatric illness. To do this, I targeted the ventral hippocampus with injections containing virus that expressed the light-sensitive ion channel channelrhodopsin (ChR-EYFP) in glutamatergic neurons. I determined the extent of glutamatergic hippocampal terminal expression in brain regions thought to regulate the paraventricular nucleus of the hypothalamus (PVN), and then stimulated those terminals while recording from corticotropin-releasing factor-positive (CRF+) neurons in the PVN in a whole-cell voltage clamp recording configuration. Using parasaggital brain sections, I was able to induce optically-evoked inhibitory post-synaptic currents in CRF+ neurons of the PVN, demonstrating the first direct functional evidence of hippocampal inhibition of the PVN. I used a similar injection scheme to record GAD+ neurons in the BNST. Finally, I utilized in vivo optical stimulation of hippocampal terminals within the BNST during acute restraint stress and observed a significant decrease in circulating levels of corticosterone. My data indicates that the hippocampus sends dense projections to the BNST and optical excitation of these hippocampal terminals produce inhibitory currents in CRF+ neurons of the PVN. Our data suggests a disynaptic hippocampus-BNST-PVN circuit of regulation by which the hippocampus inhibits the CRF+ PVN cells. The in vivo results indicate that the hippocampal inhibition of these neurons is capable of functionally inhibiting the HPA axis.
    • Evaluation of circadian behaviour in the KCNMA1-N999S channelopathy mouse model

      Dinsdale, Ria L.; Roache, Cooper E.; Meredith, Andrea L. (2022-09)
      KCNMA1 encodes the large conductance calcium- and voltage-activated potassium (BK) channel which regulates suprachiasmatic nucleus neural excitability and circadian behavioural rhythms. Some gain-of-function (GOF) mutations disrupt circadian behaviour and clock function, evidenced by an increase in behavioural active period and amplified response to phase-shifting stimuli, with no detectable effect on motor coordination. The KCNMA1-N999S channelopathy patient variant, has been characterized as GOF, and heterozygous mice harbouring BKN999S (KCNMA1N999S/WT) are a validated model of paroxysmal dyskinesia. Whether KCNMA1N999S/WT mice have a disruption of circadian behaviour due to the GOF nature of the mutation has not been tested. Circadian behaviour in wild-type (WT) and KCNMA1N999S/WT littermates was measured by wheel running activity in constant darkness, assessing circadian period (τ) and the circadian amplitude of activity rhythms, as measured by the amplitude of a χ² periodogram and the relative power of the circadian component from the Fourier transform (FFT rPSD). We predict that if the circadian rhythm is disrupted, there would be a reduction in χ² amplitude and FFT rPSD, with a change in τ. Instead, KCNMA1N999S/WT mice display a reduction in χ² amplitude (-17 %) and FFT rPSD (-35 %), but without a τ difference compared to WT. The number of wheel rotations during the active phase decreased in KCNMA1N999S/WT mice (14667 ± 1432, N=14) compared to WT (25094 ± 1848, N=12; P=0.0002, Welch’s t test), suggesting KCNMA1N999S/WT mice have reduced locomotor function. However, when exposed to a phase-shifting zeitgeber, there is a 42-minute greater response to a pulse of light at CT16. KCNMA1N999S/WT mice also re-entrain to a phase-advance of the light:dark cycle (+6L) in 2.5 fewer days than WT. Thus, although no overt difference in circadian rhythm was identified, diminished clock function was detected as an enhanced sensitivity to phase-shifting stimuli, comparable to other GOF models.