Involvement of the glymphatic/meningeal lymphatic system in Alzheimer's disease: insights into proteostasis and future directions.

BACKGROUND:  Alzheimer's disease (AD) is pathologically characterized by the abnormal accumulation of Aß and tau proteins. There has long been a keen interest among researchers in understanding how Aß and tau are ultimately cleared in the brain. The discovery of this glymphatic system introduced a novel perspective on protein clearance and it gained recognition as one of the major brain clearance pathways for clearing these pathogenic proteins in AD. This finding has sparked interest in exploring the potential contribution of the glymphatic/meningeal lymphatic system in AD. Furthermore, there is a growing emphasis and discussion regarding the possibility that activating the glymphatic/meningeal lymphatic system could serve as a novel therapeutic strategy against AD. OBJECTIVES:  Given this current research trend, the primary focus of this comprehensive review is to highlight the role of the glymphatic/meningeal lymphatic system in the pathogenesis of AD. The discussion will encompass future research directions and prospects for treatment in relation to the glymphatic/meningeal lymphatic system.

A Method to Collect Cerebrospinal Fluid from Mouse Cisterna Magna to Determine Extracellular Tau Levels.

Despite being a cytoplasmic protein abundant in neurons, tau is detectable in various extracellular fluids. In addition to being passively released from dying/degenerating neurons, tau is also actively released from living neurons in a neuronal activity-dependent mechanism. In vivo, tau released from neurons first appears in brain interstitial fluid (ISF) and subsequently drains into cerebrospinal fluid (CSF) by glymphatic system. Changes in CSF tau levels alter during the course of AD pathogenesis and are considered to predict the disease-progression of AD. A method to collect CSF from various mouse models of AD will serve as a valuable tool to investigate the dynamics of physiological/pathological tau released from neurons. In this chapter, we describe and characterize a method that reliably collects a relatively large volume of CSF from anesthetized mice.

Detection of Glymphatic Outflow of Tau from Brain to Cerebrospinal Fluid in Mice.

Glymphatic system denotes a brain-wide pathway that eliminates extracellular solutes from brain. It is driven by the flow of brain interstitial fluid (ISF) and cerebrospinal fluid (CSF) via perivascular spaces. Glymphatic convective flow is driven by cerebral arterial pulsation, which is facilitated by a water channel, aquaporin-4 (AQP4) expressed in astrocytic end-foot processes. Since its discovery, the glymphatic system receives a considerable scientific attention due to its pivotal role in clearing metabolic waste as well as neurotoxic substances such as amyloid b peptide. Tau is a microtubule binding protein, however it is also physiologically released into extracellular fluids. The presence of tau in the blood stream indicates that it is eventually cleared from the brain to the periphery, however, the detailed mechanisms that eliminate extracellular tau from the central nervous system remained to be elucidated. Recently, we and others have reported that extracellular tau is eliminated from the brain to CSF by an AQP4 dependent mechanism, suggesting the involvement of the glymphatic system. In this chapter, we describe the detailed protocol of how we can assess glymphatic outflow of tau protein from brain to CSF in mice.

Late-onset Myoclonic Seizure in a 78-year-old Woman with Gaucher Disease.

We herein report a 78-year-old woman with Gaucher disease (GD) who was initially diagnosed with GD type 1, had been receiving long-term enzyme replacement therapy since 58 years old, and developed neurological manifestations in her 70s. The neurological manifestations included myoclonic seizures and progressive cognitive decline. Although it is rare for GD patients to first develop neurologic manifestations at such an advanced age, physicians engaged in long-term care for GD patients should be alert for this possibility.

Astrocytic APOE4 genotype-mediated negative impacts on synaptic architecture in human pluripotent stem cell model.

The APOE4 genotype is the strongest risk factor for the pathogenesis of sporadic Alzheimer's disease (AD), but the detailed molecular mechanism of APOE4-mediated synaptic impairment remains to be determined. In this study, we generated a human astrocyte model carrying the APOE3 or APOE4 genotype using human induced pluripotent stem cells (iPSCs) in which isogenic APOE4 iPSCs were genome edited from healthy control APOE3 iPSCs. Next, we demonstrated that the astrocytic APOE4 genotype negatively affects dendritic spine dynamics in a co-culture system with primary neurons. Transcriptome analysis revealed an increase of EDIL3, an extracellular matrix glycoprotein, in human APOE4 astrocytes, which could underlie dendritic spine reduction in neuronal cultures. Accordingly, postmortem AD brains carrying the APOE4 allele have elevated levels of EDIL3 protein deposits within amyloid plaques. Together, these results demonstrate the novel deleterious effect of human APOE4 astrocytes on synaptic architecture and may help to elucidate the mechanism of APOE4-linked AD pathogenesis.

Multifaceted Roles of Aquaporins in the Pathogenesis of Alzheimer's Disease.

The central nervous system is highly dependent on water, and disturbances in water homeostasis can have a significant impact on its normal functions. The regulation of water balance is, at least in part, carried out via specialized water channels called aquaporins. In the central nervous system, two major aquaporins (AQPs), AQP1 and AQP4, and their potential involvements have been long implicated in the pathophysiology of many brain disorders such as brain edema and Neuromyelitis optica. In addition to these diseases, there is growing attention to the involvement of AQPs in the removal of waste products in Alzheimer's disease (AD). This indicates that targeting fluid homeostasis is a novel and attractive approach for AD. This review article aims to summarize recent knowledge on the pathological implications of AQPs in AD, discussing unsolved questions and future prospects.

An adolescent and young adult (AYA) cancer education initiative aimed at healthcare students in Japan.

BACKGROUND: The focus on cancer in adolescents and young adults (AYA) has increased in recent years. We participated in an event called AYA week 2021 as part of a research project of the Ministry of Health, Labour and Welfare (MHLW) and conducted a fact-finding survey to collect information for raising public awareness of AYA-generation cancers and to improve support for AYA-generation cancer patients. METHODS: A 25-item questionnaire survey was conducted through requests sent to the deans of medical schools and presidents of related universities across Japan and by advertising on social networking sites and friendship networks. Furthermore, the effects of a lecture given by three cancer survivors on their cancer experience were examined. RESULTS: A total of 1288 healthcare students participated. The most common age group was between 20 and 24 years, with the majority being medical students (83%). The AYA cancer-educated population had more knowledge about AYA-generation cancers than the overall group. At present, very few people are familiar with AYA-generation cancers (30.5%), which highlights the importance of school education. There were 163 participants who attended the lecture given by the cancer survivors, of whom 108 completed the questionnaire. The results showed high participant satisfaction related to the lecture given by cancer survivors, suggesting that such lectures could help educate and raise awareness about AYA-generation cancers. CONCLUSIONS: The knowledge survey and lecture given by cancer survivors had educational effects. Many healthcare students responded positively, suggesting the efficacy of these types of initiatives.

Glymphatic system clears extracellular tau and protects from tau aggregation and neurodegeneration.

Accumulation of tau has been implicated in various neurodegenerative diseases termed tauopathies. Tau is a microtubule-associated protein but is also actively released into the extracellular fluids including brain interstitial fluid and cerebrospinal fluid (CSF). However, it remains elusive whether clearance of extracellular tau impacts tau-associated neurodegeneration. Here, we show that aquaporin-4 (AQP4), a major driver of the glymphatic clearance system, facilitates the elimination of extracellular tau from the brain to CSF and subsequently to deep cervical lymph nodes. Strikingly, deletion of AQP4 not only elevated tau in CSF but also markedly exacerbated phosphorylated tau deposition and the associated neurodegeneration in the brains of transgenic mice expressing P301S mutant tau. The current study identified the clearance pathway of extracellular tau in the central nervous system, suggesting that glymphatic clearance of extracellular tau is a novel regulatory mechanism whose impairment contributes to tau aggregation and neurodegeneration.

Lemur tail kinase 1 (LMTK1) regulates the endosomal localization of ß-secretase BACE1.

Lemur tail kinase 1 (LMTK1), previously called apoptosis-associated tyrosine kinase (AATYK), is an endosomal Ser/Thr kinase. We recently reported that LMTK1 regulates axon outgrowth, dendrite arborization and spine formation via Rab11-mediated vesicle transport. Rab11, a small GTPase regulating recycling endosome trafficking, is shown to be associated with late-onset Alzheimer's disease (LOAD). In fact, genome-wide association studies identified many proteins regulating vesicle transport as risk factors for LOAD. Furthermore, LMTK1 has been reported to be a risk factor for frontotemporal dementia. Then, we hypothesized that LMTK1 contributes to AD development through vesicle transport and examined the effect of LMTK1 on the cellular localization of AD-related proteins, amyloid precursor protein (APP) and ß-site APP cleaving enzyme 1 (BACE1). The ß-cleavage of APP by BACE1 is the initial and rate-limiting step in Aß generation. We found that LMTK1 accumulated BACE1, but not APP, to the perinuclear endosomal compartment, whereas the kinase-negative(kn) mutant of LMTK1A did not. The ß-C-terminal fragment was prone to increase under overexpression of LMTK1A kn. Moreover, the expression level of LMTK1A was reduced in AD brains. These results suggest the possibility that LMTK1 is involved in AD development through the regulation of the proper endosomal localization of BACE1.

Overexpressing low-density lipoprotein receptor reduces tau-associated neurodegeneration in relation to apoE-linked mechanisms.

APOE is the strongest genetic risk factor for late-onset Alzheimer's disease. ApoE exacerbates tau-associated neurodegeneration by driving microglial activation. However, how apoE regulates microglial activation and whether targeting apoE is therapeutically beneficial in tauopathy is unclear. Here, we show that overexpressing an apoE metabolic receptor, LDLR (low-density lipoprotein receptor), in P301S tauopathy mice markedly reduces brain apoE and ameliorates tau pathology and neurodegeneration. LDLR overexpression (OX) in microglia cell-autonomously downregulates microglial Apoe expression and is associated with suppressed microglial activation as in apoE-deficient microglia. ApoE deficiency and LDLR OX strongly drive microglial immunometabolism toward enhanced catabolism over anabolism, whereas LDLR-overexpressing microglia also uniquely upregulate specific ion channels and neurotransmitter receptors upon activation. ApoE-deficient and LDLR-overexpressing mice harbor enlarged pools of oligodendrocyte progenitor cells (OPCs) and show greater preservation of myelin integrity under neurodegenerative conditions. They also show less reactive astrocyte activation in the setting of tauopathy.

Amlexanox enhances the antitumor effect of anti-PD-1 antibody.

Cancer immunotherapy, especially treatment with monoclonal antibodies (mAbs) that block programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) signaling, has attracted attention as a new therapeutic option for cancer. However, only a limited number of patients have responded to this treatment approach. In this study, we searched for compounds that enhance the efficacy of anti-PD-1 mAb using mixed lymphocyte reaction (MLR), which is a mixed culture system of the two key cells (dendritic and T cells) involved in tumor immunity. We found that amlexanox enhanced production of interferon (IFN)-γ, an indicator of T cell activation, by anti-PD-1 mAb. Amlexanox also induced PD-L1 expression in dendritic cells in MLR, whereas it did not stimulate interleukin-2 production by Jurkat T cells. These results suggest that amlexanox acts on dendritic cells, not T cells, in MLR. Furthermore, it enhanced the antitumor effect of the anti-PD-1 mAb in vivo in a mouse tumor-bearing model. The combination of amlexanox and anti-PD-1 mAb increased the expression of Ifng encoding IFN-γ, IFN-γ-related genes, Cd274 encoding PD-L1, and cytotoxic T cell-related genes in tumors. In conclusion, amlexanox stimulates the antitumor effect of anti-PD-1 mAb by acting on dendritic cells, which in turn activates cytotoxic T cells in tumors.

Behavioral and electrophysiological evidence for a neuroprotective role of aquaporin-4 in the 5xFAD transgenic mice model.

Aquaporin-4 (AQP4) has been suggested to be involved in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD), which may be due to the modulation of neuroinflammation or the impairment of interstitial fluid bulk flow system in the central nervous system. Here, we show an age-dependent impairment of several behavioral outcomes in 5xFAD AQP4 null mice. Twenty-four-hour video recordings and computational analyses of their movement revealed that the nighttime motion of AQP4-deficient 5xFAD mice was progressively reduced between 20 and 36 weeks of age, with a sharp deterioration occurring between 30 and 32 weeks. This reduction in nighttime motion was accompanied by motor dysfunction and epileptiform neuronal activities, demonstrated by increased abnormal spikes by electroencephalography. In addition, all AQP4-deficient 5xFAD mice exhibited convulsions at least once during the period of the analysis. Interestingly, despite such obvious phenotypes, parenchymal amyloid ß (Aß) deposition, reactive astrocytosis, and activated microgliosis surrounding amyloid plaques were unchanged in the AQP4-deficient 5xFAD mice relative to 5xFAD mice. Taken together, our data indicate that AQP4 deficiency greatly accelerates an age-dependent deterioration of neuronal function in 5xFAD mice associated with epileptiform neuronal activity without significantly altering Aß deposition or neuroinflammation in this mouse model. We therefore propose that there exists another pathophysiological phase in AD which follows amyloid plaque deposition and neuroinflammation and is sensitive to AQP4 deficiency.

Seeding Activity-Based Detection Uncovers the Different Release Mechanisms of Seed-Competent Tau Versus Inert Tau via Lysosomal Exocytosis.

The pathological aggregation of tau characterizes a set of neurodegenerative diseases collectively referred to as tauopathies. Recent studies using cellular and animal models have suggested that tau pathology progresses by trans-cellular propagation. The process of propagation is mediated by certain species of extracellular tau, which are taken up by recipient cells and serve as a seed for tau aggregation. Tau propagation is currently one of the most active areas of research in dementia. Previous efforts to identify the specific tau molecules involved in propagation have suggested that multiple forms of tau with different molecular weights derived from recombinant tau or brain lysates exert seeding activity. Nonetheless, the molecular characteristics of the "extracellular" seed-competent tau as well as its release mechanisms remain to be elucidated. Given that tau is physiologically released into the extracellular space, it is critical to distinguish seed-competent tau from normal monomeric tau. Utilizing biosensor cells expressing P301S mutant tau fused to CFP/YFP, here we discriminated between seed-competent tau and inert monomer tau released from HEK293 cells. By analyzing the size-exclusion fractions of the media, we found that seed-competent tau was enriched in high molecular weight fractions of >2,000 kDa, while the majority of soluble tau in the media positively detected by ELISA was in low molecular weight fractions. We also found that lysosomal stress not only increased Ca2+-dependent release of seed-competent tau but also altered its molecular size. Inhibiting lysosomal exocytosis specifically decreased release of seed-competent tau without influencing total tau. These data underscore the differential response of seed-competent tau and inert tau to lysosomal stress and indicates the presence of distinct release mechanisms via lysosomes.

Differential effects of diet- and genetically-induced brain insulin resistance on amyloid pathology in a mouse model of Alzheimer's disease.

BACKGROUND: Based on epidemiological and experimental studies, type 2 diabetes mellitus (T2DM), especially insulin resistance that comprises the core mechanism of T2DM, has been recognized as a significant risk factor for Alzheimer's disease (AD). Studies in humans and diabetic AD model mice have indicated a correlation between insulin resistance and increased amyloid deposition in the brain. Paradoxically, mice with targeted disruption of genes involved in the insulin signaling pathway showed protective effects against the AD-related pathology. These conflicting observations raise an issue as to the relationship between dysregulation of insulin signaling and AD pathophysiology. METHODS: To study the causal relations and molecular mechanisms underlying insulin resistance-induced exacerbation of amyloid pathology, we investigated the chronological changes in the development of insulin resistance and amyloid pathology in two independent insulin-resistant AD mouse models, i.e., long-term high-fat diet (HFD) feeding and genetic disruption of Irs2, in combination with dietary interventions. In addition to biochemical and histopathological analyses, we examined the in vivo dynamics of brain amyloid-ß (Aß) and insulin by microdialysis technique. RESULTS: HFD-fed diabetic AD model mice displayed a reduced brain response to peripheral insulin stimulation and a decreased brain to plasma ratio of insulin during the hyperinsulinemic clamp. Diet-induced defective insulin action in the brain was accompanied by a decreased clearance of the extracellular Aß in vivo and an exacerbation of brain amyloid pathology. These noxious effects of the HFD both on insulin sensitivity and on Aß deposition in brains were reversibly attenuated by dietary interventions. Importantly, HFD feeding accelerated Aß deposition also in the brains of IRS-2-deficient AD mice. CONCLUSIONS: Our results suggested a causal and reversible association of brain Aß metabolism and amyloid pathology by diet-dependent, but not genetically-induced, insulin-resistance. These observations raise the possibility that the causal factors of insulin resistance, e.g., metabolic stress or inflammation induced by HFD feeding, but not impaired insulin signaling per se, might be directly involved in the acceleration of amyloid pathology in the brain.

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes.

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an established method that measures relatively small molecules including amino acids or neurotransmitters, it has been recently used to also assess dynamics of larger molecules in ISF using probes with high molecular weight cut off membranes. Upon using such probes, microdialysis has to be run in a push-pull mode to avoid pressure accumulated inside of the probes. This article provides step-by-step protocols including stereotaxic surgery and how to set up microdialysis lines to collect proteins from ISF. During microdialysis, drugs can be administered either systemically or by direct infusion into ISF. Reverse microdialysis is a technique to directly infuse compounds into ISF. Inclusion of drugs in the microdialysis perfusion buffer allows them to diffuse into ISF through the probes while simultaneously collecting ISF. By measuring tau protein as an example, the author shows how its levels are altered upon stimulating neuronal activity by reverse microdialysis of picrotoxin. Advantages and limitations of microdialysis are described along with the extended application by combining other in vivo methods.

Characterization of oil-producing yeast Lipomyces starkeyi on glycerol carbon source based on metabolomics and 13C-labeling.

Lipomyces starkeyi is an oil-producing yeast that can produce triacylglycerol (TAG) from glycerol as a carbon source. The TAG was mainly produced after nitrogen depletion alongside reduced cell proliferation. To obtain clues for enhancing the TAG production, cell metabolism during the TAG-producing phase was characterized by metabolomics with 13C labeling. The turnover analysis showed that the time constants of intermediates from glycerol to pyruvate (Pyr) were large, whereas those of tricarboxylic acid (TCA) cycle intermediates were much smaller than that of Pyr. Surprisingly, the time constants of intermediates in gluconeogenesis and the pentose phosphate (PP) pathway were large, suggesting that a large amount of the uptaken glycerol was metabolized via the PP pathway. To synthesize fatty acids that make up TAG from acetyl-CoA (AcCoA), 14 molecules of nicotinamide adenine dinucleotide phosphate (NADPH) per C16 fatty acid molecule are required. Because the oxidative PP pathway generates NADPH, this pathway would contribute to supply NADPH for fatty acid synthesis. To confirm that the oxidative PP pathway can supply the NADPH required for TAG production, flux analysis was conducted based on the measured specific rates and mass balances. Flux analysis revealed that the NADPH necessary for TAG production was supplied by metabolizing 48.2% of the uptaken glycerol through gluconeogenesis and the PP pathway. This result was consistent with the result of the 13C-labeling experiment. Furthermore, comparison of the actual flux distribution with the ideal flux distribution for TAG production suggested that it is necessary to flow more dihydroxyacetonephosphate (DHAP) through gluconeogenesis to improve TAG yield.

Extracellular α-synuclein levels are regulated by neuronal activity.

BACKGROUND: α-Synuclein is a presynaptic protein abundant in the cytoplasmic compartment of neurons, whereas its presence in the extracellular space has also been observed under physiological conditions. Extracellular α-synuclein has pathological significance, exhibiting cellular toxicity and impairment of synaptic transmission. Notably, misfolded α-synuclein drives the cell-to-cell propagation of pathology via the extracellular space. However, the primary mechanism that regulates the extracellular levels of α-synuclein remains to be determined. METHODS: Using several mechanistically distinct methods to modulate neuronal/synaptic activities in primary neuronal culture and in vivo microdialysis, we examined the involvement of neuronal/synaptic activities on α-synuclein release. RESULTS: We demonstrate here that physiological release of endogenous α-synuclein highly depends on intrinsic neuronal activities. Elevating neuronal activity rapidly increased, while blocking activity decreased, α-synuclein release. In vivo microdialysis experiments in freely moving mice revealed that ~ 70% of extracellular α-synuclein arises from neuronal activity-dependent pathway. Selective modulation of glutamatergic neurotransmission altered extracellular α-synuclein levels, implicating this specific neuronal network in the mechanism of activity-dependent release of α-synuclein. While neuronal activity tightly regulated α-synuclein release, elevated synaptic vesicle exocytosis per se was capable to elicit α-synuclein release. We also found that extracellular α-synuclein exists as high molecular weight species. CONCLUSIONS: The present study uncovers a novel regulatory pathway associated with α-synuclein release, whose dysregulation might affect various pathological actions of extracellular α-synuclein including its trans-synaptic propagation.

Extracellular Tau and Its Potential Role in the Propagation of Tau Pathology.

The pathological aggregation of tau protein is a hallmark of a set of neurodegenerative diseases collectively referred to as tauopathies. Tau aggregates independently in each neuron, but this aggregation can also occur in a non-cell autonomous manner in which aggregated tau is transmitted from one cell to another. Such trans-cellular propagation is initiated by the uptake of extracellular tau, which then seeds soluble tau in the recipient cells to spread the tau pathology. Accumulating evidence has demonstrated that tau is not only present in the cytoplasm of neurons but also actively released into the extracellular space. This finding has led to the idea that extracellular tau could be a novel therapeutic target to halt the propagation of tau pathology. From this perspective, the present review article focuses on recent advances in understanding the mechanisms regulating the levels of extracellular tau and discusses the role of such mechanisms in the propagation of tau pathology.

ApoE4 markedly exacerbates tau-mediated neurodegeneration in a mouse model of tauopathy.

APOE4 is the strongest genetic risk factor for late-onset Alzheimer disease. ApoE4 increases brain amyloid-ß pathology relative to other ApoE isoforms. However, whether APOE independently influences tau pathology, the other major proteinopathy of Alzheimer disease and other tauopathies, or tau-mediated neurodegeneration, is not clear. By generating P301S tau transgenic mice on either a human ApoE knock-in (KI) or ApoE knockout (KO) background, here we show that P301S/E4 mice have significantly higher tau levels in the brain and a greater extent of somatodendritic tau redistribution by three months of age compared with P301S/E2, P301S/E3, and P301S/EKO mice. By nine months of age, P301S mice with different ApoE genotypes display distinct phosphorylated tau protein (p-tau) staining patterns. P301S/E4 mice develop markedly more brain atrophy and neuroinflammation than P301S/E2 and P301S/E3 mice, whereas P301S/EKO mice are largely protected from these changes. In vitro, E4-expressing microglia exhibit higher innate immune reactivity after lipopolysaccharide treatment. Co-culturing P301S tau-expressing neurons with E4-expressing mixed glia results in a significantly higher level of tumour-necrosis factor-α (TNF-α) secretion and markedly reduced neuronal viability compared with neuron/E2 and neuron/E3 co-cultures. Neurons co-cultured with EKO glia showed the greatest viability with the lowest level of secreted TNF-α. Treatment of P301S neurons with recombinant ApoE (E2, E3, E4) also leads to some neuronal damage and death compared with the absence of ApoE, with ApoE4 exacerbating the effect. In individuals with a sporadic primary tauopathy, the presence of an ε4 allele is associated with more severe regional neurodegeneration. In individuals who are positive for amyloid-ß pathology with symptomatic Alzheimer disease who usually have tau pathology, ε4-carriers demonstrate greater rates of disease progression. Our results demonstrate that ApoE affects tau pathogenesis, neuroinflammation, and tau-mediated neurodegeneration independently of amyloid-ß pathology. ApoE4 exerts a 'toxic' gain of function whereas the absence of ApoE is protective.

[Causative Bacterial Strains and Sensitivityto Antimicrobial Agents in Female Acute Uncomplicated Cystitis].

Recently, wide spreading of fluoloquinolone resistant Escherichia coli is a serious problem inthe treatment of urinary tract infection. To investigate the causative bacterial strains of female acute uncomplicated cystitis (AUC) in the community and their sensitivity to antimicrobial agents, we retrospectively reviewed the medical records of 215 female AUC patients treated at our clinics from April 2014 to June 2015. Two hundred and nineteen strains were isolated as the causative bacteria from the patients'urine samples, including E. coli of 179 strains (82%) followed by Klebsiella pneumoniae (5.5%). One hundred and forty five strains (81%) of the isolated E. coli were sensitive to levofloxacin, whereas 32 strains (17.9%) were levofloxacin-resistant. To fosfomycin, the isolated E. coli showed the highest sensitivity (93.9%) among all antimicrobial agents tested. In univariate analysis, factors associated with levofloxacinresistant E. coli included two or more episodes of cystitis within the past year and levofloxacin use at the latest episode of cystitis. Inmultivariate analysis, two or more episodes of cystitis withinthe past year were found to be associated with levofloxacinresistan ce (p＝0.004). To prevent the increasing prevalence of infections caused by antibiotic-resistant bacteria, it is important to confirm the sensitivity of the causative agents for optimal antimicrobial therapy. The community-based surveillance data should be collected and considered when selecting empirical antimicrobial agents.