Alzheimer's disease (AD) is an incurable disease characterized by a slow, progressive decline in cognitive functions as well as the presence of amyloid-beta (Aβ) plaques and neurofibrillary tangles. Interestingly, two thirds of AD patients are women who have a faster disease progression. Despite this clinical profile, sex differences in AD pathophysiology are largely ignored at the basic and clinical levels. Current therapies provide only mild to moderate improvement in patient symptoms. There is, therefore, an urgent need to expand our understanding of the underlying pathophysiology of AD, and to obtain alternative hypotheses and therapeutics. A recent and promising development involves the sigma-1 receptor (Sig1R), a protein regulated by steroid hormones, which has been implicated in AD. Most interestingly, Sig1R agonists have been shown to ameliorate cognitive deficits in an AD mouse model. Here, we investigated the role of Sig1Rs in an Aβ25-35-infusion mouse model of AD, using behavioural paradigms. Previous studies employing this model have demonstrated Aβ-induced impairments in learning and memory in young male rodents, while no work has been done on females. We examined cognitive function following Aβ25-35 infusion in wild-type and knock-out Sig1R adult male and female mice using the Morris water maze, spontaneous alternation in the Y-maze, and forced alternation in the Y-maze tasks. Overall, the data unexpectedly shows that genotype, Aβ25-35-treatment, and sex had no effect on cognitive functions. These results suggest that additional efforts are required to obtain a working Aβ25-35-infusion model in our Sig1R mice and behavioural tasks. Future experiments will hopefully shed some light on the link between Sig1Rs and AD, which could lead to the development of therapeutics and disease prevention.

The lung was recently identified to consist of a complex microenvironment made up of microorganisms that interact with one another and the host cells via direct and indirect interactions. As a result, understanding the dynamic of the microbiome in chronic respiratory diseases has become the focus of pulmonary researches. In cystic fibrosis (CF), chronic infections are a comorbidity associated with the genetic disorder. Recently, it was noted that the interactions of the fungus, Aspergillus fumigatus, and the bacterium, Pseudomonas aeruginosa together contribute to more severe disease outcomes in CF patients. In vitro co-cultures show that P. aeruginosa and A. fumigatus can affect one another’s growth and pathogenicity, but very few studies have attempted to model interactions of these microorganisms in vivo. Based on clinical and basic research, we developed a co-exposure model in which we could compare non-allergic and allergic animals co-exposed to Pseudomonas aeruginosa and Aspergillus fumigatus. While both groups had significant neutrophilia and production of acute phase response cytokines and chemokines, the allergic co-exposed group had a greater mortality with 34.8% of the animals expiring by 24h in comparison to 12.5% for the non-allergic co-exposed animals and 100% survival in the controls. A contributing factor to the more severe disease outcomes in the allergic co-exposed group is the increase in eosinophilic inflammation and IL-17A production, which only occurs when both microorganisms are viable. In addition, it was found that viable P. aeruginosa but not A. fumigatus causes interstitial inflammation, significant neutrophilia, and even death during co-exposures. The decline in health of animals co-exposed to the fungus and bacteria could be attributed not only to the host’s inflammatory response, but also to the spatial and temporal co-localization in the lung. To address this, we performed in vitro studies finding an aggregation of the microorganisms that could also be identified in vivo. This current research emphasizes the need for in vivo studies on polymicrobial interactions. / ND Agricultural Experiment Station; National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R155AI137886

Purpose of Review: To emphasize key gaps in knowledge impacting efforts to control single infection and co-infections with Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG), the most common bacterial sexually transmitted infections (STIs) worldwide. Recent Findings: Clinical and epidemiological studies describe gaps in understanding about female rectal CT infection, screening effectiveness, pelvic inflammatory disease, and influence of the microbiome. For NG, gaps in knowledge include factors increasing incidence in men who have sex with men, correlations between treatment and antibiotic resistance, the role of pharyngeal infection, and microbiome influence. CT/NG co-infections are poorly understood, and adequate models to explore pathophysiological consequences of co-infection urgently needed. The sole existing CT/NG co-infection mouse model showed that CT/NG interactions in vivo modulate host response and NG load/shedding—encouraging further consideration of this model and potential alternatives. Summary: We stress key challenges in controlling these important STIs. Appropriate, quality-assured animal models are essential to improve understanding of the pathogenic interplay in CT/NG co-infections.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13466号 / 論医博第2253号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 村川 泰裕, 教授 中島 貴子, 教授 藤田 恭之 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Yes / Backgorund Prior data suggest pre-diagnostic aspirin use impacts breast tumour biology and patient outcome. Here, we employed faithful surgical resection models of HER2+ and triple-negative breast cancer (TNBC), to study outcome and response mechanisms across breast cancer subtypes. Method NOD/SCID mice were implanted with HER2+ MDA-MB-231/LN/2-4/H2N, trastuzumab-resistant HER2+ HCC1954 or a TNBC patient-derived xenograft (PDX). A daily low-dose aspirin regimen commenced until primary tumours reached ~250 mm3 and subsequently resected. MDA-MB-231/LN/2-4/H2N mice were monitored for metastasis utilising imaging. To interrogate the survival benefit of pre-treatment aspirin, 3 weeks post-resection, HCC1954/TNBC animals received standard-of-care (SOC) chemotherapy for 6 weeks. Primary tumour response to aspirin was interrogated using immunohistochemistry. Results Aspirin delayed time to metastasis in MDA-MB-231/LN/2-4/H2N xenografts and decreased growth of HER2+/TNBC primary tumours. Lymphangiogenic factors and lymph vessels number were decreased in HER2+ tumours. However, no survival benefit was seen in aspirin pre-treated animals (HCC1954/TNBC) that further received adjuvant SOC, compared with animals treated with SOC alone. In an effort to study mechanisms responsible for the observed reduction in lymphangiogenesis in HER2+ BC we utilised an in vitro co-culture system of HCC1954 tumour cells and mesenchymal stromal cells (MSC). Aspirin abrogated the secretion of VEGF-C in MSCs and also decreased the lymph/angiogenic potential of the MSCs and HCC1954 by tubule formation assay. Furthermore, aspirin decreased the secretion of uPA in HCC1954 cells potentially diminishing its metastatic capability. Conclusion Our data employing clinically relevant models demonstrate that aspirin alters breast tumour biology. However, aspirin may not represent a robust chemo-preventative agent in the HER2+ or TNBC setting. / Funding is acknowledged from the Irish Cancer Society Collaborative Cancer Research Centre under BREAST- PREDICT grant CCRC13GAL (www.breas tpred ict.com). I.S.M, E.M and A.T.B, are members of the EurOPDX Consortium, and receive funding from the European Union's Horizon 2020 research and innovation programme, grant agreement no. #731105 (EurOPDX Research Infrastructure, www.europ dx.eu).

Over 1.1 billion people smoke worldwide despite the association of smoking with numerous diseases including chronic obstructive pulmonary disease (COPD). The decline in lung function observed in COPD patients is thought to be related to smoke-induced inflammation. COPD patients are also at increased risk of acquiring lung bacterial infections that are associated with exacerbations, characterized by worsened disease symptoms and inflammation. The focus of this thesis is on how cigarette smoke impacts bacterial-host interactions and bacterial community interactions to promote infection and disease. In chapter 3.1, we sought to understand how cigarette smoke primed the lungs towards an amplified inflammatory response to bacterial infection reflective of COPD exacerbations that accelerate disease progression. We present a novel finding that exacerbated neutrophilia elicited by nontypeable Haemophilus influenzae (NTHi) lung challenge in smoke-exposed mice occurred dependent on IL-1α. Smokers and patients with COPD are additionally at increased risk of acquiring bacterial infection that may be related to impaired containment of nasally colonizing pathogens. In chapter 3.2, we found that cigarette smoke predisposed mice to invasive pneumococcal disease (IPD) following nasal pneumococcal colonization associated with attenuated nasal inflammatory responses. To our knowledge, this is the first study to describe the progression from asymptomatic nasal pneumococcal colonization to the development of IPD in the context of cigarette smoking. It has been suggested that smokers have higher rates of pathogen colonization as a consequence of cigarette smoke-induced nasal microbiome dysbiosis. The last study in chapter 3.3 advanced knowledge in the field by testing this hypothesis. We observed that cigarette smoke alone did not alter the mouse nasal microbiome and concluded that microbiome dysbiosis observed in smokers likely occur as a consequence of nasal pathogen colonization. Overall, work presented in this thesis advanced our understanding of how cigarette smoking alters bacterial-host interactions to promote infection and disease. / Thesis / Doctor of Philosophy (PhD) / Over 1.1 billion people smoke worldwide and can develop chronic obstructive pulmonary disease (COPD), a serious inflammatory disease compromising lung function. Additionally, smokers and COPD patients have higher rates of bacterial infection. The goal of this thesis is to understand how smoking impacts our ability to combat infection. Lung infection in COPD patients causes exacerbation, with worsened disease symptoms. Using mouse models, we learned how smoking causes increased lung inflammation following bacterial infection, contributing to damage reflective of COPD exacerbations, and identified a potential intervention. We elucidated smokers may have increased infections due to impaired immune responses in the nose, a major pathogen entry point. It is thought smoking reduced beneficial bacteria that counter pathogen acquisition in the nose. We confirmed smoking did not impact these bacteria, directing research focus towards other ways smokers acquire pathogens. Overall, this thesis advanced knowledge and will help efforts to control disease in smokers.

Detrimental immune and inflammatory responses contribute to the pathogenesis of various conditions, including Crohn’s disease, Lupus, and sepsis.1,2,3 Unfortunately, novel treatments for detrimental immune and inflammatory responses have been met with little success. Mesenchymal stromal cells (MSCs) represent a promising cellular therapy to treat immune and inflammatory disorders due to their ability to suppress the immune system. However, despite their promise, clinical trials that have employed MSC cellular therapies have produced varying and sometimes conflicting results. These discrepancies have been partially attributed to the cellular heterogeneity within MSC populations. To address these discrepancies, I performed transcriptomic and proteomic analysis of MSCs with varying immunomodulatory capacity to identify robust immunomodulatory biomarkers and gain better mechanistic insights into MSC immunomodulatory function. In this study, MSCs with differing immunomodulatory function were identified and the effect of in vitro passaging and proinflammatory induction on immunomodulatory ability was characterized. To characterize MSC immunomodulatory control mechanisms, RNA sequencing and proteomic analyses were performed on MSCs with different immunomodulatory capabilities. These analyses enabled the identification of potential immunomodulatory biomarkers and regulatory mechanisms. Finally, to test the therapeutic efficacy of immunomodulatory MSC subpopulations, I developed a humanize mouse model for sepsis. Overall, this work contributes to our understanding of MSC immunomodulation and to the development of a robust MSC cellular therapeutics.

Whether a patient presents with deep vein thrombosis (DVT) or pulmonary embolism (PE) varies based on clinical factors. Patients with factor V Leiden (FVL) typically present with DVT while cancer patients present with PE. The biological mechanisms that determine DVT stability in the progression of DVT to PE are not known. Thus, little is known about the mechanism of thrombus stability, the factors involved, or the effect of anticoagulants on embolization and PE burden. In order to answer these questions, we first need to (i) develop a mouse model to evaluate DVT stability and its relationship with PE burden when treated with anticoagulants, (ii) determine if anticoagulants, by inhibiting thrombin, require FXIII to decrease thrombus stability, (iii) determine the effects of attenuating fibrinolysis, using epsilon aminocaproic acid (ε-ACA or EACA), supplemental FXIII and α2-AP, on clot stability and (iv) utilize our model to explain the FVL paradox. For our thrombus stability model, the femoral vein of C57BL/6, FXIII deficient (FXIII-/-), FVL heterozygous, or FVL homozygous female mice was subjected to ferric chloride (FeCl3) injury to initiate a non-occlusive thrombus. Treatment with saline, dalteparin, dabigatran, EACA or FXIII was administered 12 minutes after thrombus formation. Intravital videomicroscopy recorded the thrombus sizes and embolic events leaving the thrombus for 2 hours. Lungs were harvested, sectioned and stained for the presence of PE. Total and large embolic events were highest after dabigatran treatment compared to saline or dalteparin in wild-type (WT) mice. Variations in amounts of embolic events were not attributed to variations in thrombus size since thrombus size was similar between the groups. The number of emboli per lung slice was higher in dabigatran-treated mice. Large embolic events correlated positively with the number of emboli per lung slice independent of treatment. Dabigatran treatment in FXIII-/- mice did not alter embolization patterns suggesting that FXIII is required for dabigatran to decrease thrombus stability. EACA increases thrombus size significantly and therefore would not be a feasible alternative to IVC filters, as it will increase DVT size. FXIII marginally increased thrombus size. Treatment with FXIII decreases total and large embolic events in saline-, dalteparin- or dabigatran-treated mice, similar to EACA-treated mice. The number of emboli per lung slice was reduced after treatment with FXIII and EACA compared to non-treated mice. PE burden was not significantly different between FXIII anticoagulated mice or EACA-treated mice. The large embolic events correlate positively with PE burden. FVL heterozygous and homozygous mice had significantly reduced embolization and thrombus size grew significantly over time, this contrasted with WT mice, where thrombus size remained similar to the initial injury. PE burden was significantly reduced in the FVL mice compared to WT. Collectively, these data shows that we have successfully developed a mouse model of acute venous thrombus stability that can quantify emboli and PE burden. Consistent with clinical data, dabigatran, a DTI, was shown to acutely decrease thrombus stability and increase PE burden compared to LMWH or saline; an effect that was FXIII-dependent. Also, attenuating fibrinolysis with EACA, but not FXIII, increases thrombus size; but both increase DVT stability and decrease PE burden. Supplementing α2-AP did not alter thrombus stability. This suggests that administration of FXIII may be a better treatment option for DVT patients who are bleeding than EACA, since EACA may increase DVT size. Lastly, our model can explain the FVL paradox. Those with FVL have stable thrombus formation leading to an increased incidence of symptomatic DVT and a decreased risk of PE. / Thesis / Doctor of Philosophy (PhD)