abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Mannose-binding lectin (MBL) functions as pattern recognition molecule to mediate first-line host defense against invading pathogens. Although MBL is well-known for its anti-bacterial action, its role towards virus infection is less comprehensively understood. In 2009, the pandemic H1N1 2009 (pdmH1N1) influenza A virus caused more than 18,000 deaths worldwide and is still circulating in human community as a seasonal strain. In this study, the role of MBL in pdmH1N1 infection was investigated. Using in vitro microtiter capture assay, MBL was found to bind to pdmH1N1 virus via its carbohydrate recognition domain. Under transmission electron microscope (TEM), MBL was clearly visible on the surface of pdmH1N1 virus. By infecting C57B6/J wild-type (WT) and MBL knockout (KO) mice with a sub-lethal dose of pdmH1N1 virus, WT mice displayed greater weight loss and more severe lung damage than MBL KO mice. Using flow cytometry-based profiling analysis of the lung homogenates isolated from infected mice, a variety of proinflammatory cytokines and chemokines were found to be significantly up-regulated. These results indicate that the presence of MBL can cause excess proinflammatory cytokine production and result in a more severe pdmH1N1 infection. To provide physiologically relevant insight into the immunomodulating role of MBL, the investigation was further extended to the use of human cell line model. Infection of A549 cells, which is a human lung epithelial cell line, with MBL-bound pdmH1N1 virus elevated the production of MCP1, RANTES and IL-8 significantly more than unbound pdmH1N1 infection. The increased production of chemokines also enhanced recruitment of monocytes as demonstrated by transwell migration assay. Interestingly, MBL did not affect viral entry or replication kinetics. TEM and confocal imaging revealed the presence of MBL-bound pdmH1N1 inside infected A549 cells, suggesting that the endocytosed MBL may interact with intracellular components to promote the release of cytokines and chemokines. To this end, expressions of Toll-like receptors were examined (TLR3, TLR7, TLR8 and TLR9) and found that TLR3 expression was dramatically enhanced upon pdmH1N1 infection. Interestingly, in MBL-bound pdmH1N1 infection, TLR3 mRNA and protein expression was significantly higher than unbound pdmH1N1 infection in A549 cells. In addition, the NF-κB signaling was further activated in the presence of MBL-bound pdmH1N1. A novel physical interaction between MBL and TLR3 was also delineated as evidenced by MBL’s capability to bind to TLR3 in vitro; and their colocalization in the endosomes of the infected A549 cells. In summary, MBL can bind to pdmH1N1 virus but fails to inhibit its infection in human lung epithelial cell line. Upon pdmH1N1 infection, MBL is internalized with the virus into the cell, where it may associate with TLR3 to further amplify the NF-κB signaling and augment the cytokine production in the human lung epithelial cells. The present findings advocate the adverse immunomodulating role of MBL during pdmH1N1 infection. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

The mechanisms responsible for the enhanced inflammatory response in type 2 diabetes (T2DM) and its contribution to the severe ischemia/reperfusion (I/R) injury observed in the T2DM heart are unclear. I/R is associated with an acute inflammatory response recognized by reactive oxidant production, complement activation, and leukocyte-endothelial cell adhesion, among others. Complement activation plays an important role in the inflammatory response and is involved in the manifestation of I/R injury in the non-diabetic heart, and is a potent chemoattractant for circulating neutrophils (PMNs). The purpose of this dissertation research was to test the hypothesis that the complement system, predominantly the lectin pathway, is a significant contributor to the excessive response of the Zucker Diabetic Fatty (ZDF), a rat model of T2DM, to myocardial I/R injury. Following 30min of coronary artery occlusion and 120min of reperfusion we measured C3 deposition, PMN accumulation, PMN CD11b expression, and ICAM-1 expression. We found significantly more C3 deposition, PMN accumulation, ICAM-1 and PMN CD11b expression in diabetic samples compared to non-diabetic samples. To elucidate a role for complement system activation, we treated animals with FUT-175, a broad complement inhibitor. In vivo, FUT-175 treatment significantly decreased complement deposition (66%), PMN accumulation (59%), and infarct size (55%) compared to untreated animals in both non-diabetic Sprague-Dawley and diabetic ZDF rats. To specifically examine the role of the lectin pathway, we selectively inhibited rat MBL-A prior to myocardial I/R in ZDF rats. Anti-MBL treatment significantly decreased infarct size, C3 deposition and PMN accumulation in the ZDF post-ischemic left ventricle (LV). Genomic analysis revealed that gene expression of the pro-inflammatory cytokines IL-6 and IL-1α was enhanced in the ZDF heart following reperfusion, and quantitative RT-PCR results confirmed IL-6 upregulation. We found significantly increased complement C5a receptor (CD88) expression on diabetic neutrophils prior to ischemia, suggesting that diabetic PMNs are "primed" to respond to complement activation. Taken together, these results provide evidence that 1) the ZDF rat is a good model for chronic inflammation in the setting of T2DM, 2) lectin pathway activation plays a significant role in the inflammatory response to I/R injury in the ZDF heart, and 3) anti-complement therapy may be particularly cardio-protective in T2DM.

The potential of lignin and mannanoligosaccharides (MOS), as alternatives to antibiotic growth promoters was evaluated in broilers. Dietary treatments included: (1) negative control (CTL-, antibiotic free); (2) positive control (CTL+, 11 mg/kg virginiamycin); (3) MOS (diet 1 + Bio-Mos: 0.2% to 21 d and 0.1% thereafter); (4) LL (diet 1 + 1.25% Alcell lignin); (5) HL (diet 1 + 2.5% Alcell lignin). Bodyweight and feed conversion were not different when broilers were fed the CTL+, MOS, LL or HL diet. Birds fed MOS or LL had increased jejunum villi height (P < 0.05) and greater goblet cell number per villus (P < 0.05) when compared to those fed the CTL+ diet. MOS and LL increased (P < 0.05) the cecal populations of Lactobacilli and Bifidobacteria when compared to CTL+ fed birds. However, Lactobacilli and Bifidobacteria loads were lowest (P < 0.05) in birds fed the CTL+ or HL diet. Litter E. coli load was reduced (P < 0.05) when birds were fed MOS than when fed the CTL+ diet, but comparable to LL or HL fed birds. In birds challenged with pathogenic strains of E. coli (O2 and O88 serotypes) and fed the MOS or HL diet, the cecal population of total E. coli was lower (P < 0.05) than those fed the CTL+ diet; LL fed birds tended to have lower E. coli load than CTL+ fed birds. In summary, birds fed the MOS or LL diet had comparative advantage over CTL+ fed birds as evidenced by increased cecal populations of Lactobacilli and Bifdobacteria, increased villi height and greater goblet cell number in the jejunum, lower E. coli load in the litter, and lower cecal population of E. coli after an in vivo challenge with pathogenic strains of E. coli. Therefore, MOS and lignin could be regarded as natural alternatives to antibiotic growth promoters in poultry production. / Key words. Antibiotics, mannanoligosaccharides, lignin, gut health, broilers.

Background.  Sepsis is a syndrome associated with high mortality rates, substantial morbidity and high costs of care. The incidents of sepsis is reported to be high and controversy exists whether gender affect severity or outcome. Little is known about factors determining suscepti­bility for developing the syndrome and severity of the syndrome once developed. Early detection and adequate antibiotic administration are the mainstay of treatment and means to identify patients with particular high risk of adverse outcome are desirable. There are data to suggest that the course of sepsis and outcome from the syndrome may be influenced by inherited differences in the immunological response among humans Aims: Paper I: Assess incidence and outcome for ICU-treated sepsis patients in this region; Paper II: Assess if there are gender differences related to characteristics, aspects of treatment or out­come in sepsis in this region. Paper III: Assess the association of baseline levels of leptin and adiponectin and future sepsis event, and association of these adipokines in the cute phase and sepsis severity and outcome. Paper IV: Assess association of baseline levels of mannose-binding lectin (MBL) and future sepsis event, and MBL levels in the acute phase in relation to sepsis severity and outcome. Results. Paper I:  Overall ICU mortality rate was 25%, while the ICU mortality for patients with septic shock was 58% in this retrospective single university hospital cohort analysis. Cardio­vascular disease and diabetes were the most prevalent comorbidities among patients who died during hospital stay.  Paper II:  No gender-related differences in mortality or length of stay was found in this prospective single center observational study. Differences in aspects of treatment were related to differences in site of infection. Men had more often infections in skin and skin-structures, whereas women more often had abdominal infections. Early organ dysfunction asses­sed as SOFA score at admission was a stronger predictor for hospital mortality for women than for men. The discrepancy was related to the SOFA coagulation-sub score.  Paper III: In this nes­ted case-referent study hyperleptinemia at baseline predicted a first-ever sepsis event, even after adjustment for BMI and other cardiovascular risk factors. Hyperleptinemia in the acute sepsis phase was associated with reduced risk of in-hospital death in men, but associated with increased risk of in-hospital   death in women.  Paper IV: In the same matched cohort as in Paper III high baseline levels of MBL predicted a first ever sepsis event. High MBL levels in the acute phase or an increase from baseline to the acute phase associate with increased in-hospital death in women but not in men. Low MBL levels was not identified as a risk for acute sepsis or in-hospital death. Conclusions. Mortality from severe sepsis is high, equally affecting men and women. There are differences in patient characteristics and inflammatory markers, which associate with in-hospital mortality differentially in men and women. Aspects of gender should be mandatory, and genetic analysis are desired in future sepsis research.

Hannover, Universiẗat, Diss., 1998.

Acanthamoeba mannose-binding protein (AcMBP) is a virulence factor of the free-living amoeba, Acanthamoeba castellanii. It is crucial for the development of Acanthamoeba keratitis (AK), a corneal infection that often causes blindness. AK is associated with contact lens use and contaminated water sources. Therapeutic unresponsiveness is attributed to similarities in the biological processes that Acanthamoeba shares with humans and its ability to form drug-resistant cysts. I aimed to characterise AcMBP as a basis for developing future drugs against Acanthamoeba. To start with, I carried out morphological studies on the two well-known life stages of Acanthamoeba and characterised a third stage: the protocysts. Mature cysts and protocysts could not interconvert directly, but always excysted to trophozoites. This is important because Acanthamoeba can potentially be trapped as protocysts, which are likely to be more susceptible to drugs. I also studied Acanthamoeba adhesion towards various surfaces and cytopathic activities towards cells (including human corneal epithelial cells). Whilst AcMBP was important for adhesion, it is not the only receptor involved. To gain structure/function information, I expressed the extracellular portion of AcMBP and three truncated fragments. AcMBP is a Ca2+-dependent lectin (~100 kDa) that binds to mannose. Ca2+ is essential for lectin activity and stability. The extracellular fragment is monomeric, indicating that trimerisation, shown previously, depends on the membrane-spanning and/or intracellular regions. Bioinformatics revealed that lectin activity is almost certainly located in a DUF 4114 domain (~10 kDa, DUF: domain of unknown function). N-terminal fragments, including the DUF4114 domain did not bind to mannose-Sepharose, suggesting that part of the cysteine-rich domain is also important. AcMBP bound to a variety of mammalian glycans so may have more than one lectin activity. Although attempts to crystallise AcMBP were unsuccessful, future structural analysis will be useful for defining the domains and determining how it binds to mannose.

En 1971, Le Dr. Américain Judah FOLKMAN a publié une hypothèse : la croissance tumorale dépend de l’angiogenèse. Le défit des recherches actuelles est de trouver un moyen pour affamer la tumeur en arrêtant son angiogenèse. L’angiogenèse est un processus physiologique complexe qui fait intervenir de nombreux récepteurs, parmi lesquels se trouve le récepteur du mannose-6-phosphate / insulin-like growth factor II (RM6P/IGFII). Nous présentons, au cours de cette thèse, la synthèse d’analogues du mannose-6-phosphate (M6P) selon deux méthodes différentes, et l’évaluation de leur activité angiogénique par la méthode de la "CAM" et de leur activité anticancéreuse sur un modèle de tumeur induite chez la souris. Afin de localiser le récepteur mis en jeux, des dérivés fluorescent du M6P ont été synthétisés et testés également selon le modèle de la "CAM". D’autre part, il a été montré que le RM6P/IGFII peut lier deux molécules de M6P ou une molécule d’oligosaccharide diphosphorylé par monomère, pour cela des molécules bidentées ont été synthétisées en appliquant la "Chimie Verte" dans le but d’évaluer leur activité angiogénique. / In 1971, the American Dr. Judah FOLKMAN published the hypothesis : tumor growth depends on angiogenesis. The challenge of current research is to find a way to starve tumors by arresting the angiogenesis. Angiogenesis is a complex physiological process that involves many receptors, among which is the receptor for mannose-6-phosphate / insulin-like growth factor II (RM6P/IGFII). We present during this thesis, the synthesis of analogues of mannose-6-phosphate (M6P) using two different methods, and assessment of their angiogenic activity by the method of "CAM" and their anticancer activity in a model of induced tumor in mice. In order to locate the receptor, fluorescent derivatives of M6P were also synthesized and tested using the model of "CAM". On the other hand, it was shown that the RM6P/IGFII can bind two molecules of M6P or diphosphoryled oligosaccharide molecule per monomer, so bidentate molecules have been synthesized using "Green Chemistry" for evaluation of angiogenic activity.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22633号 / 理博第4622号 / 新制||理||1664(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 森 和俊, 教授 平野 丈夫, 教授 川口 真也 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

It is well known that too much sugar uptake causes many health problems, including diabetes and obesity (Lustig et al. in Nature 482:27-29, 2012). However, a team of researchers led by Dr. Wanjun Chen of the National Institute of Dental and Craniofacial Research (NIDCR), National Institutes of Health (NIH), USA, have recently shown that d-mannose, a naturally occurring C-2 epimer of glucose is likely beneficial to human health. Their studies have revealed that supraphysiological levels of d-mannose that are safely achievable via drinking-water supplementation can be preventive and therapeutic to experimental autoimmune diabetes and asthmatic lung inflammation (Zhang et al. in Nat Med 23:1036-1045, 2017).