Now showing items 1-20 of 1984

    • Wholly Rickettsia! Metabolic Profile of the Quintessential Bacterial Parasite of Eukaryotic Cells

      Driscoll, Timothy P.; Verhoeve, Victoria I.; Guillotte, Mark L.; Lehman, Stephanie S.; Rennoll, Sherri A.; Beier-Sexton, Magda; Rahman, M. Sayeedur; Azad, Abdu F.; Gillespie, Joseph J. (2018-06)
    • Genetic and Functional Studies of the Evolutionarily Oldest Natural Killer Receptor, NKp30

      Kinlein, Allison; Ohta, Yuko; 0000-0002-3944-2530 (2021)
      NKp30 is a Natural Cytotoxicity Receptor (NCR) expressed by Natural Killer (NK) cells and other lymphocytes. NKp30 is the only evolutionarily conserved NCR found in all jawed vertebrates and it coevolves with its ligand B7-H6. Using sharks as a model, we found NKp30 gene expression in subsets of mature and immature T cells, suggesting that T cells and NK cells share common features in these primitive vertebrates. To understand NKp30’s evolutionary origin, we examined genomic regions containing NKp30 and its homologs in vertebrates in different Classes. We observed that loci in paralogous regions containing NKp30 homologs are well conserved, suggesting the presence of NKp30 ancestors and other linked immune genes before the emergence of vertebrates 550 million years ago. Indeed, the corresponding region is also present in invertebrates. We hypothesize that this genomic region encompassed the “Primordial Immune Complex,” containing genes playing roles in immunity at the origin of vertebrates.
    • Inhibition of GPR68 Sensitizes GBM to Temozolomide Treatment via the NF-kB Pathway

      Ahmad, Jovanni; Hong, Charles C., 1967-; 0000-0001-9550-8090 (2021)
      Glioblastoma Multiforme (GBM) remains as one of the most aggressive and lethal cancer types, often resulting in poor prognosis. Currently, Temozolomide (TMZ) is the standard chemotherapy for combating GBM. However, GBM’s upregulation of O-6-Methylguanine-DNA Methyltransferase (MGMT) mitigates the alkylating effects of TMZ treatment, generating a dire new need for novel or adjuvant therapy. U138MG is a TMZ-resistant GBM cell line used for the development of new chemotherapy. Here, we investigated whether inhibition of proton sensing GPR68 would decrease MGMT expression and sensitize U138MG cells to TMZ treatment. Using various genetic, protein, and cell-based assays we determined that inhibition of GPR68 may be decreasing MGMT protein expression and sensitizing U138MG to TMZ via the Gq/NFkB pathway. Furthermore, we identified that co-administration of TMZ and OGM resulted in a synergistic decrease in cell growth compared to OGM treatment alone.
    • Actin-Like Protein 6A (ACTL6A) Suppresses p21Cip1 Expression to Maintain an Aggressive Cancer Phenotype

      Shrestha, Suruchi; Eckert, Richard (Richard L.); 0000-0002-9550-816X (2021)
      Epidermal squamous cell carcinoma (SCC) and mesothelioma are two distinct but highly aggressive forms of cancer. SCC is a common and highly invasive cancer that arises from the epidermis. The major cause of epidermal SCC is repeated exposure to ultraviolet light and other DNA damaging agents such as oxidative stress which causes mutations eventually leading to increased expression of pro-tumor genes and reduced expression of tumor suppressors. Mesothelioma is highly invasive and lethal cancer that arises from the mesothelial lining and is linked to exposure to asbestos and other toxic agents. Actin-like protein 6A (ACTL6A, BAF53A) is a member of SWI/SNF chromatin remodeling complex that has been implicated in many cancers as a driver of cancer survival and tumor formation. We show that ACTL6A functions to maintain an aggressive cancer phenotype in both SCC and mesothelioma. We further show that ACTL6A reduces expression of the p21Cip1 cyclin-dependent kinase inhibitor and tumor suppressor protein. Biochemical studies reveal that loss of ACTL6A leads to increased p21Cip1 promoter activity, and mRNA and protein expression suggesting transcriptional regulation of p21Cip1 gene. Moreover, chromatin immunoprecipitation studies show that ACTL6A interacts at the p21Cip1 promoter proximal Sp1 site and distal p53-responsive enhancer sites to suppress transcription. We further report that the increase in p21Cip1 upon ACTL6A knockdown is required to suppress the SCC and mesothelioma cancer phenotypes. This suggests that p21Cip1 is the key mediator of ACTL6A function in SCC and mesothelioma. p53 is a key tumor suppressor that interacts with the p21Cip1 promoter to increase expression; however, we show that it may not play a regulatory role in these cancers. These findings suggest that ACTL6A suppresses p21Cip1 transcription to reduce p21Cip1 function as a mechanism to maintain an aggressive cancer phenotype in SCC and mesothelioma.
    • Calcium and BK Potassium Channel Regulation of Circadian Rhythms in the Suprachiasmatic Nucleus

      Plante, Amber; Meredith, Andrea L.; 0000-0002-1010-2348 (2021)
      Mammalian circadian rhythms are driven by a network of neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN exhibits daily (24-hour) rhythms in spontaneous action potential (AP) firing rate that encodes a time-of-day signal that coordinates the timing of circadian physiological and behavioral processes. Large-conductance Ca2+-activated K+ (BK) channels have a major role in driving the diurnal patterns of spontaneous firing in SCN neurons. BK K+ currents are larger at night, correlating with reduced neuronal excitability. The diurnal variation in BK current in the SCN is required for setting the day-night difference in firing frequency. BK currents undergo multi-level regulation by genetic and posttranslational mechanisms as well as functional coupling to Ca2+ channels. Intracellular Ca2+ (Ca2+i) is required for BK channel activation and previous studies have shown BK current is predominantly coupled to two types of Ca2+ sources in the SCN: L-type Ca2+ channels (LTCCs), and ryanodine receptors (RyRs). Circadian rhythms in Ca2+i have also been identified in SCN neurons. However, the Ca2+ channels involved in generating both AP and Ca2+i rhythms have not been clearly identified. First, to determine which Ca2+ channels are involved in AP rhythms, this study measured the impact of Ca2+ channel agonists and antagonists on the circadian parameters of spontaneous AP activity from organotypic SCN slice cultures grown on multi-electrode arrays. Next, to determine which Ca2+ channels are involved in Ca2+i rhythms, this study tested the effects of the same Ca2+ channel pharmacology on the circadian parameters of Ca2+i measured from SCN slice cultures transfected with a fluorescent Ca2+ sensor. Lastly, this investigated a potential mechanism by which LTCCs contribute to firing rate in SCN neurons by examining their ability to activate BK channels under controlled conditions. This study provides insight into the roles of specific Ca2+ sources in neural coding of the circadian time signal in the SCN.
    • Theileria infections in African cattle and buffalo: understanding genetic variation and speciation

      Palmateer, Nicholas; Carneiro da Silva, Joana; 0000-0002-4307-8049 (2021)
      East Coast fever, caused by the apicomplexan parasite Theileria parva, has an estimated annual death toll of over a million cattle in endemic sub-Saharan regions. The African Cape buffalo is the natural reservoir of T. parva and rarely exhibits clinical symptoms when infected, but transmits the parasite to cattle via a tick vector. Previous studies based on a few genetic markers showed that buffalo-derived T. parva subpopulations contain greater antigenic diversity than those from cattle. Interestingly, cattle are infected and killed by T. parva of buffalo origin, but cannot transmit those parasites, suggesting that a degree of host specificity exists. The characterization of genetic variation within and between cattle- and buffalo-derived T. parva is critical to understand the molecular mechanism(s) of host specificity. To overcome obstacles in T. parva biology that prevent the straightforward acquisition of sufficient DNA for whole genome sequencing (WGS), we adapted a DNA capture approach to select T. parva from a mix of parasite and bovine DNA obtained from T. parva-infected bovine lymphocyte cultures. To gain access to variable genomic regions that cannot be characterized through read mapping approaches, we assembled the captured reads de novo. From starting material of <1%-4% parasite DNA in a mixed sample from host and parasite, >98% of sequence reads post-capture are of parasite origin and >97% of the genome is recovered, reflecting the method’s high specificity and sensitivity. We used this whole genome DNA capture followed by sequencing to generate WGS data from 15 cattle- and 24 buffalo-derived T. parva isolates. This resulted in the generation of the first assembly of a buffalo-derived T. parva isolate. Furthermore, we determined that cattle- and buffalo-derived T. parva isolates differ in various measures at levels consistent with speciation. Finally, capture and analysis of members of the T. parva repeat (Tpr) multigene family, which encode some of the most variable antigen families in the species, enabled the study of Tpr evolution and initial inferences of its possible involvement in parasite-host interactions. These results have greatly advanced the study of the T. parva genome and improved our understanding of the evolution of this parasite population.
    • Cancer Mortality among US Solid Organ Transplant Recipients: Novel Methodologies to Estimate Cancer Burden using Linked Population-Based Registries

      Noone, Anne-Michelle; Dorgan, Joanne; Engels, Eric A.; 0000-0001-6997-4004 (2021)
      Background: The solid organ transplant population has an elevated risk of cancer compared with the general population. Excess risk is largely due to immunosuppression. As this population grows, understanding long-term health risks such as cancer is critical. Population-based estimates of cancer mortality are needed since they measure the downstream outcome following a cancer diagnosis. Furthermore, quantifying deaths attributable to cancer can inform priorities to reduce the cancer burden. Methods: Linked transplant and cancer registry data were used to identify incident cancers and deaths among solid organ transplant recipients in the United States (1987-2014). Population-attributable fractions (PAFs) of deaths due to cancer and corresponding cancer-attributable mortality rates were estimated. Cancer-attributable mortality rates computed using the PAF were compared to cancer-specific mortality rates computing using cause of death (COD). The life-years lost (LYL) to cancer were estimated using two methods: an approach using matching to construct a cancer-free cohort and an approach using Cox proportional hazards regression models. Results: Among 221,962 transplant recipients, 15,012 developed cancer. Thirteen percent of deaths (PAF=13.2%) were attributable to cancer, corresponding to a cancer-attributable mortality rate of 516 per 100,000 person-years. Lung cancer was the largest contributor to mortality (PAF=3.1%), followed by non-Hodgkin lymphoma (NHL, PAF=1.9%), colorectal cancer (PAF=0.7%), and kidney cancer (PAF=0.5%). Overall, the cancer-specific mortality rate lower, 368 per 100,000 person-years. Within 10 years post-transplant, the mean LYL was 0.16 years per transplant recipient and 2.7 years per cancer. Cancer accounted for 1.9% of the total LYL expected in this population. Lung cancer was the largest contributor, accounting for 24% of all LYL, and NHL had the next highest contribution (15%). Conclusions: Cancer is a substantial cause of mortality among solid organ transplant recipients resulting in excess deaths and a shortened lifespan. Lung cancer and NHL are major contributors to the cancer burden including LYL to cancer, highlighting opportunities to reduce cancer mortality through prevention and screening.
    • The epidemiology of patient to patient transmission of MRSA among critical care patients

      Adediran, Timileyin; Harris, Anthony D.; Thom, Kerri (2021)
      Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen that leads to an increase in morbidity and mortality. To decrease the spread of MRSA, there is a need to elucidate factors that lead to patient-to-patient transmission in critical care settings. Objectives: Aim 1: To understand if patient-to-patient transmission via HCP mediator differs between high-risk activities by determining the odds of MRSA contamination of the patient from the gown and gloves of the health care personnel (HCP). Aim 2: To determine if isolates found on the gown and gloves of HCP are similar to patient isolates after performing an HCP-patient interaction, using comparative genomic techniques. Methods: Aim 1: This was an observational study of MRSA-positive patients and the HCP who cared for them. We conducted a simulation study of patient-to-patient transmission of MRSA from a HCP vector to a manikin (proxy for the subsequent patient). Using a generalized linear mixed model, we determined the odds of manikin contamination after performing HCP-patient interactions. Aim 2: We selected 95 patient MRSA isolates and their co-isolated HCP gown or glove MRSA isolates using a stratified sampling method. Comparative genomics analyses such as phylogenetic analysis, spa-typing, multi-locus sequence typing (MLST), large-scale blast score ratio (LSBSR), and single nucleotide variant (SNV) analysis were used to achieve this aim’s objective. Results: Aim 1: We observed 103 HCP-patient interactions with 65 MRSA-positive patients and found that subsequent transmission of MRSA from HCP gown and gloves to the manikin proxy occurred 10.7% of the time. There was no association between high-contact patient care activities and MRSA contamination of the manikin following patient care activity (p-value=0.1). Aim 2: Using multiple typing methods, we found that the majority of our isolates were genetically similar. The phylogenetic analysis revealed that 85.2% of paired isolates were similar, and the spa-typing and the LSBSR found that more than 75% of our paired isolates were concordant. However, SNV and MLST identified more than 40% of our paired isolates as discordant. Conclusion: The studies conducted demonstrated patient-to-patient transmission of MRSA via HCP vector, indicating the importance of contact precautions and infection control practices.
    • Prescription Opioids and Traumatic Brain Injury in Older Adults

      Herrera, Anthony; Albrecht, Jennifer S.; 0000-0001-5345-1241 (2021)
      Older adults using opioids in the US increased annually from 1999 to 2011. This is a significant public health concern because prescription opioid use increases fall-risk, the most common cause of traumatic brain injury (TBI) among older adults and a leading cause of disability and mortality. No studies have investigated the association between prescription opioid use and TBI. This dissertation characterized prescription opioid use in Medicare beneficiaries aged ≥65 years and examined the relationship between opioid use and incident TBI. Using Medicare administrative claims data from 2010 – 2015, I assessed how older adult prescription opioid use has changed over time, as well as changes in opioid prescriber specialties and pre-opioid diagnoses/ procedures. I also used these data to estimate risk for TBI associated with prescription opioid use. Next, I used R Adams Cowley Shock Trauma Center Registry (STR) data from 2015 – 2019 to explore the relationship between prescription opioid use and TBI injury mechanisms and severity. I found that the percent of older adults using prescription opioids decreased from 35.4% to 32.9% (p<0.001). Primary care physicians prescribed the most opioids, but their share of prescriptions dropped from 59.0% to 52.8% (p<0.001). Back pain remained the most common diagnosis among older adult opioid users, unchanged over time (24.1% to 25.3%, p=0.594). Opioid use increased TBI risk (odd ratio: 1.34, 95% confidence interval: 1.28 - 1.40) among older adults, regardless of opioid dosage and duration differences. Compared to non-users in the STR data, older adult opioid users who sustained TBI were 85% more likely to be injured in a fall compared to a motor vehicle incident (OR 1.85, 95% CI 1.20 – 2.86). Opioid use was associated with a 39% increase in sustaining more severe TBIs. (OR 1.39, 95% CI 1.09 – 1.79). This dissertation found opioid prescriptions decreasing among older adults. This parallels reductions in primary care opioid prescribing as well as changes in opioid-related diagnoses and procedures. These are the first studies to provide evidence that prescription opioids raise TBI risk and severity in older adults. Future studies could refine the association between opioid and TBI using data with definite dosing details
    • The Impact of Sex and Cognition on Recovery and Mortality Post Hip Fracture

      Mutchie, Heather; Gruber-Baldini, Ann L.; 0000-0002-6964-5283 (2021)
      Introduction The overall objective of this work was to estimate misclassification of cognitive impairment (CI) by sex among hip fracture patients. The effects of sex and identified CI on hip fracture recovery outcomes, including trajectories of cognitive and physical function and mortality (all-cause and cognition-specific), were subsequently examined. Methods This study used secondary data from a cohort of hip fracture patients recruited from 8 Baltimore-area hospitals between 2006-2011 (N=339), with frequency matched enrollment of females (n=171) and males (n=168), tested within 22 days of hip fracture admission and at 2, 6, and 12 months. Death data were derived from the National Death Index 2006-2018 (n=260; females=113, females=147). Analyses differentiated source of CI identification (SCI, n=330) between clinical diagnosis/documentation and direct testing [Modified Mini Mental State Examination (3MS)] of cognition at baseline by patient sex. Analyses identified cognitive and functional recovery using group-based trajectory modeling (GBTM) and joint trajectory models; and estimated time to death for all-cause and cognition-related cause of death (CR-COD) by SCI and sex. Results Males had increased odds of CI identified by both hospital record and 3MS after adjusting for age, education, and comorbidities. There were 2-4 distinct groups of recovery for cognitive and functional recovery. In joint models of recovery, high levels of cognitive function were only seen in high physical functioning groups. Significantly more men died than women (147 vs 113, p<.0001) and died sooner from all-cause mortality (41 vs 54 months, p=0.001) but not CR-COD. Males and those with SCI Both were independently at increased risk for all-cause mortality but there was not a significant interaction. Those with SCI Both were 14 times more likely to die of CR-COD (p<.0001). Conclusions There is clinical underdiagnosis of active CI in males. Additional CI ascertainment can identify a sub-population of patients at excess risk for mortality and CR-COD. Cognitive testing after hip fracture should be instituted as standard practice to avoid sex bias in identification of CI. Cognition changes little over 12 months post fracture. Pre-fracture functional status informs pattern of physical function recovery. Sex and CI increase mortality risk, and to some extent cause-specific mortality.
    • μ-Crystallin: A Novel Protein Regulator of Mammalian Metabolism

      Kinney, Christian; Bloch, Robert J.; 0000-0002-9799-9097 (2021)
      Thyroid hormones control many aspects of physiology such as metabolism and thermogenesis. Because thyroid hormones control such crucial bodily functions, their levels in the body are tightly regulated. Hypo- and hyperthyroidism can result when the level of thyroid hormone is too low or too high, respectively, with potentially serious pathophysiological consequences. µ-Crystallin is an NADPH-regulated thyroid hormone binding protein. The protein is minimally expressed in the skeletal muscle of most people; however, some individuals express relatively high baseline levels of µ-crystallin. We generated a transgenic mouse, the Crym tg mouse, that expresses high levels of µ-crystallin in its skeletal muscle, in order to explore the consequences of expressing high levels, comparable to those seen in some humans. The Crym tg mouse expresses mouse µ-crystallin at levels 2.6-147.5-fold higher than control mice in their skeletal muscle. Consequently, intramuscular triiodothyronine (T3) levels are elevated ~190-fold in the tibialis anterior, while serum thyroxine levels are decreased by 1.2-fold. Crym tg mice have a decreased respiratory exchange ratio that corresponds to a 13.7% increase in fat utilization as an energy source. Female Crym tg mice gained weight faster on high fat or high simple carbohydrate diets. Gene ontology enrichment analysis of transcriptomic and proteomic data revealed alterations to the expression of genes associated with metabolism and fiber type, while the fiber sizes of Crym tg soleus muscle are significantly smaller than those of controls. Taken together, these results suggest that µ-crystallin may play a role in regulating metabolism, perhaps through the control of thyroid hormone in muscle. Thus, humans who naturally express higher levels of µ-crystallin in their skeletal muscle may have an altered metabolism, comparable to the Crym tg mice.
    • Multi-omic analysis of hearing difficulty risk loci and gene regulatory networks in the mammalian cochlea

      Kalra, Gurmannat; Ament, Seth A.; 0000-0002-4176-6489 (2021)
      The sensory cells of the mammalian cochlea do not have the capacity to regenerate. Studies have identified some genes and pathways that are critical for hair cell formation; however, the conditions necessary to regenerate fully functional hair cells remain unknown, as the genetic and genomic architecture of hearing loss and hair cell regeneration is only partially defined due to different experimental conditions and models used in each study. This dissertation improves our understanding of these conditions by taking a systems biology approach, combining diverse multi-omic data into genome-scale models for predicting target genes in the mammalian cochlea and in vitro systems. Using a meta-analysis of summary statistics from hearing-related traits, I identified 31 genome-wide significant risk loci for self-reported hearing difficulty. I then investigated the regulatory and cell specific expression for these loci and found that risk-associated genes were most strongly enriched for expression in cochlear epithelial cells, as well as for genes related to sensory perception and known Mendelian deafness genes, supporting their relevance to auditory function. My epigenomic and statistical fine-mapping most strongly supported 50 putative risk genes. To derive target genes from a model of hair cell regeneration, I characterized cochlear organoids derived from murine progenitor cells through bioinformatic analysis of single-cell RNA sequencing and bulk RNA sequencing data. For comparison, I integrated data from six previous studies of cochlear and utricular cell types in vivo and report an improved list of marker genes for each inner ear cell type. I found that cells in organoids mimic nearly all subtypes of supporting cells and hair cells in the cochlea and that the resulting hair cells reach a mature identity. I reconstructed a gene regulatory model from these data to gain insight into the transcription factors driving the trans-differentiation of progenitors to hair cells. My model identified known regulators of hair cell development and predicts novel regulatory factors. I validated these networks across transcriptional datasets, demonstrating dynamic changes in the expression of these transcription factors. Overall, I report new risk genes for hearing difficulty and new transcription factors that play a role in hair cell regeneration.
    • Identifying the Molecular Mechanisms of Thymine DNA Glycosylase (TDG) Substrate Specificity

      Dow, Blaine Jacob; Drohat, Alexander Clark (2021)
      Thymine DNA glycosylase (TDG) helps maintain genomic integrity by removing thymine from G·T mispairs arising via deamination of 5-methylcytosine (mC). TDG employs strict regulation for both the opposing guanine, as well as the base downstream of the target thymine, in order to limit removal of thymine from canonical A·T pairs, as erroneous removal of thymine from A·T pairs is mutagenic and cytotoxic. TDG also excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), oxidation products of mC generated by ten-eleven translocation (TET) enzymes during active DNA demethylation. Remarkably, using single-turnover kinetics reactions to determine the maximum rate of substrate removal, k¬max, we find that TDG activity for fC and caC shows little dependence on the opposing base or the downstream base, revealing a major difference in specificity for excision of fC and caC relative to T. Using a novel 19F NMR approach to determine the flipping equilibrium for thymine into the TDG active site, we establish that specificity during thymine excision manifests largely by modulating the stability for thymine flipping in the active site. Structure-function analysis employing a variety of opposing bases reveals that both the thermodynamic stability (ΔH) of A·T pairs, as well as direct contacts between TDG and the opposing base, contribute to opposing base specificity. The differences in specificity observed for thymine versus fC or caC are likely explained by interactions between these substrates and the TDG active site. Structural information obtained from x-ray crystallography, combined with TDG mutational studies, identified several TDG active site residues that form stabilizing interactions with fC and caC, helping to both stabilize base flipping into the active site, as well as enhance the chemical steps of base excision. Conversely, two conserved residues in TDG, A145 and H151, limit stability of thymine in the active site, and destabilize thymine as a leaving group. As a result, additional contacts between both the opposing guanine, as well as the guanine downstream of the target thymine, appear necessary to orient thymine in a manner which produces stable, productive flipping into the active site.
    • Identification of core genes involved in Streptococcus pneumoniae host-pathogen interactions under diverse infections, and their potential as therapeutic targets

      D'Mello, Adonis; 0000-0001-6995-3617; Tettelin, Hervé (2021)
      Streptococcus pneumoniae (the pneumococcus) is a human-specific opportunistic pathogen that asymptomatically colonizes the nasopharynx. It is the leading cause of otitis media, communityacquired pneumonia, bacteremia and meningitis, as it can spread from the nasopharynx. It has been estimated that ~500,000 children under the age of 5 die annually due to pneumococcal infection and pneumococcal bacteremia and meningitidis has over 60% mortality rates in the elderly. It has also been established that influenza A co-infections enhance the progression from asymptomatic colonization to invasive disease, with clinically worsened outcomes. Utilization of antibiotics and pneumococcal conjugate vaccines has resulted in the rise of antibiotic resistance and emergence of non-vaccine serotypes, requiring identification of novel therapeutic targets. We hypothesized that there exist pneumococcal conserved genes, that are involved in these diverse forms of colonization, infection, and influenza co-infection. We sought to identify such genes using a combination of in silico, in vivo and ex vivo approaches. Through pangenomics and reverse vaccinology (in silico), and multi-species transcriptomics on a mouse model of pneumococcal colonization and invasive disease (in vivo), and a primary human lung epithelial model of pneumococcal and influenza co-infection (ex vivo), we observed mechanisms underlying diverse host-pathogen interactions and identified novel potential avenues for therapy from both the host and bacterial perspectives.
    • MyD88 Co-stimulation in CD8+ T Cells Improves Tumor Immunotherapy in Allogeneic and Syngeneic Models

      Ciavattone, Nicholas; Cao, Xuefang; Davila, Eduardo, Ph.D.; 0000-0001-9265-8202 (2021)
      Improving the efficacy of T cell therapies for solid tumors and leukemias could improve clinical outcomes. In leukemia, allogeneic hematopoietic cell transplantation and donor lymphocyte infusions can be potentially curative, however, tumor evasion of the graft-versus-leukemia effects still limit their efficacy. In the solid tumor microenvironment, immune suppressive myeloid cells inhibit T cell activation which can be detrimental to anti-tumor T cell responses. Toll-like Receptor/MyD88 signaling in cytotoxic T cells can provide a strong co-stimulation signal to improve T cell activation, function, and efficacy to counteract evasion mechanisms in hematopoietic and solid tumors. Unfortunately, TLR agonists lack specificity to T cells and may induce hyper inflammation or induce pro-tumor effects. Rather than provide TLR agonist therapy in leukemia and solid tumors, there exists potential to modify or engineer T cells to provide a direct MyD88 co-stimulus. Our research group developed a CD8α:MyD88 T cell co-receptor that mimics TLR co-stimulation in conjunction with T cell receptor activation. Our goal was to first determine whether this engineered T cell co-receptor could enhance graft-versus-leukemia responses in allogeneic hematopoietic cell transplantation and donor lymphocyte infusion therapies. In a suppressive solid tumor, we asked if MyD88 co-receptor could improve T cell activation and function in a suppressive tumor microenvironment. Using multiple experimental transplant models, we found that MyD88 co-stimulation in donor CD8+ T cells could improve the graft versus tumor response with some non-lethal increases in graft-versus-host disease. Looking further, we found that the CD8α:MyD88 co-receptor increased donor cytotoxic T cell proliferation, survival, and function in vivo. Donor CD8α:MyD88 T cells were able to directly kill tumor better than transduced controls. In the second part of this project we found that cytotoxic T cells receiving a synthetic MyD88 co-stimulation maintained strong basal activation in the presence of myeloid suppression. In the suppressive B16-GMCSF melanoma model, CD8α:MyD88 T cell were able to control tumor growth and reduce populations of suppressive myeloid cells in the tumor. These data show that augmented MyD88 co-stimulation in cytotoxic T cells could benefit patients undergoing both autologous and allogeneic T cell therapies.
    • Epidemiology of Plasmodium falciparum infection and clinical malaria among infants in Malawi

      Andronescu, Liana; Laufer, Miriam K.; 0000-0003-3342-7820 (2021)
      Background: Few malaria interventions are designed to target infants under six months. The burden of malaria in infancy and its long-term health impact needs to be better characterized to inform surveillance and treatment guidelines for this age group. Objectives: The aims of this study were to (a) assess the effect of intermittent preventive treatment during pregnancy (IPTp) regimens on the risk of malaria in infancy, (b) characterize P. falciparum infection and clinical malaria in the first six months and the risk of subsequent malaria, and (c) assess effects of P. falciparum infection and clinical malaria exposure in first six months on weight and hemoglobin concentration after six months. Methods: Longitudinal cohort data collected from infants in southern Malawi between 2016 and 2019 was analyzed using Cox proportional hazards models, Poisson generalized estimating equations with a log link function, and linear mixed effects models. Results: Maternal IPTp regimen had no effect on infant incidence of clinical malaria (IRR=1.03; 95%CI: 0.58–1.86) or incidence of P. falciparum infection (IRR=1.18; 95%CI: 0.92–1.55) before two years. Maternal IPTp was not significantly associated with infant’s time to first infection (HR=1.05; 95%CI: 0.8–1.39) or clinical malaria (HR=0.92; 95%CI: 0.58–1.48). Exposure to any malaria before six months was associated with higher incidence of any malaria (IRR=1.27; 95%CI: 1.06–1.52) and clinical malaria (IRR=1.76; 95%CI: 1.42–2.19) between 6 and 24 months. Clinical malaria exposure before first six months was also associated with higher incidence of any malaria (IRR=1.64; 95%CI: 1.38–1.94) and clinical malaria (IRR=1.85, 95%CI:1.48–2.32) between 6 and 24 months. Exposure to asymptomatic P. falciparum infection before six months was associated with lower weight-for-age z-scores during follow-up (p=0.02) and exposure to clinical malaria before six months was associated with lower hemoglobin concentrations during follow-up (p=0.02). Conclusion: Prevention of malaria during pregnancy does not reduce infant risk of malaria and malaria infection before six months is associated with higher malaria incidence, lower weight-for-age Z-scores, and lower hemoglobin concentrations in early childhood. Early malaria infection may be an indication of high exposure risk and a marker for downstream health outcomes.
    • Characterizing the Influence of Resident Microbiota and Mosquito Factors on Plasmodium Infections of Anopheles Mosquitoes

      Bogale, Haikel; Serre, David; 0000-0002-1907-788X (2021)
      Pathogens transmitted by mosquitoes are responsible for illnesses that cause nearly 700 million cases every year. A significant proportion of this morbidity is due to malaria, a disease caused by Plasmodium parasites and spread through the bites of infected Anopheles mosquitoes. While we have seen a reduction in malaria mortality rates thanks to the development of antimalarial therapeutics and entomological controls, there is a potential of malaria resurgence associated with the emergence and spread of antimalarial and insecticide resistance, highlighting the need for additional malaria control strategies. Malaria transmission occurs only if one Plasmodium parasite develops through key stages in the mosquito, including passing through the midgut, which harbors a microbial community that can influence Plasmodium transmission. Despite the opportunities they present for novel interventions, the development of Plasmodium sporozoites and the factors that shape the microbiota in mosquitoes are incompletely understood. Here, I sought to provide new insights into the microbial variations of wild-caught mosquitoes and the transcriptional regulatory programs of Plasmodium sporozoites. To this end, I simultaneously characterized the bacterial composition of 665 individual field-caught Anopheles mosquitoes in addition to their species, insecticide resistance genotype, blood-meal status, and infection status. My analyses revealed that mosquito collection site is the main driver of the microbial diversity, while other factors showed marginal or non-significant contribution. I also generated scRNA-seq data from 36,958 sporozoites of three Plasmodium species and collected from multiple anatomical sites of the mosquito and developmental stages of the parasite in an effort to better understand parasite developmental processes critical for malaria transmission. I identified transcriptional variations among salivary gland sporozoites of different Plasmodium species, patterns of gene regulation accompanying the journey of Plasmodium berghei sporozoites, and novel candidates potentially critical for mechanisms involved in sporozoite maturation. In addition, my analyses highlighted novel extensive transcriptional heterogeneity among sporozoites isolated from the same anatomical site, indicating asynchronous sporozoite development in the mosquito, that is regulated by intrinsic and environmental factors. Altogether, my findings improve our understanding of factors that influence malaria parasite transmission and lay the groundwork for identifying key transmission components, to inform development of novel intervention strategies.
    • Addressing Children's Exposure to Violence and the Role of Health Care

      Dubowitz, Howard (American Medical Association, 2021-05-12)