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2 이학박사학위논문 Celdeath and Pro-inflammatory Cytokine ReleaseofRatMicroglialCelsby Naegleriafowleri 아주대학교 대학원 의학과 박창은

3 Celdeath and Pro-inflammatory Cytokine ReleaseofRatMicroglialCelsby Naegleriafowleri by Chang-Eun Park A Dissertation Submitted to The Graduate School of Ajou University in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY(SCIENCE) Supervised by Ho-Joon Shin, Ph.D. Department of Medical Sciences The Graduate School, Ajou University August, 2008

4 박창은의이학박사학위논문을인준함. 심사위원장 박 선 인 심사위원 안 명 희 인 심사위원 신 호 준 인 심사위원 김 경 민 인 심사위원 임 경 일 인 아주대학교 대학원 2008 년 6 월 23 일

5 ACKNOW LEDGEMENT 대학원의수학과정동안많은것을깨닫고느꼈습니다. 우선사람은혼자살아가는것이아니라는점을간절히느꼈습니다. 앞으로는이점을깊이간직하고베풀어가면서살아야겠다는마음을다시잡아봅니다. 학위동안에세세하게여러모로힘이되어주시고챙겨주신신호준교수님, 논문지도에열성을보여주신, 박선교수님, 김경민교수님, 안명희교수님, 임경일교수님께모두머리숙여깊은감사의인사드립니다. 한편인생의많은이정표가되어주시는홍성노교수님, 정하승교수님, 김대식교수님, 조윤경교수님모두감사의인사를드립니다. 또한자주찾아뵙지는못하지만인생의전환기에많은도움을주시고계신최돈찬교수님, 김세화교수님, 이웅빈교수님, 김판기교수님, 최성부교수님감사의인사를드립니다. 서라벌대임상병리과를위해많이힘써주시는정옥교학장님, 동경이연구를함께하는최석규교수님, 성기창교수님, 이은우교수님, 박순태교수님께도감사드립니다. 그리고항상저의주변에서아껴주시는지영진교수님, 박진건교수님, 백형기교수님, 김대중실장님, 박동엽실장님, 변영길부장님, 김주원교수님, 정종현교수님, 권수자교수님, 김동현교수님, 민만기교수님모두감사드립니다. 고마움을주시고도움을주시는많은분들을다언급할수없지만아무쪼록감사의인사를드립니다. 이자리에오기까지는무엇보다도가족들의이해와협조가없었으면힘들었을것입니다. 많은뒷받침에고마운마음을전합니다. 언제나저의편이되어주시는어머니윤영희여사님께도진심으로머리숙여감사인사드립니다. 묵묵히뒤에서지켜봐주고힘이되어주는내인생의영원한반려자, 수빈이엄마인정숙이에게도고마운마음을전합니다. 함께하는시간이적어서늘미안한마음뿐인우리딸, 수빈이에게도고마움을전합니다. 마지막으로학위논문을받을수있도록도와주신모든분들께감사인사드리며, 내가이세상에서살아갈수있도록해주신하늘나라에계시는아버님, 어머님께감사드립니다.

6 - ABSTRACT - Cell Death and Pro-inflammatory Cytokine Release of Rat Microglial Cells by Naegleria fowleri Naegleria fowleri, a free-living amoeba, causes fatal primary amoebic meningoencephalitis in humans and experimental animals. In previous study, N. fowleri trophozoites showed the highly cytopathic activity and cytotoxicity against rat microglial cells by the morphological observation and 51 Cr release assay. In the present study, to determine whether a pathogenic N. fowleri lysate shows the cytopathic effects against primary cultured rat microglial cells, the morphological changes of microglial cells was observed by a light, scanning and transmission electron microscopes. And then, the cytotoxicity of N. fowleri lysate against rat microglial cells was also observed by 51 Cr release assay. In addition, the pro-inflammatory cytokine release from microglial cells in co-culture system was estimated. As results with a light and electron microscopes, most of microglial cells were severely destroyed by N. fowleri lysate, showing the necrotic (above 85%) and apoptotic cell death (below 15%) in a time- and dose dependent manner. As the results of 51 Cr release assay, the cytotoxicity of N. fowleri lysate against microglial cells were 14.6, 21.9, 38.5 and 71.5% at 3, 6, 12 and 24 h post incubation, respectively. And then, the amount of cytokines released from microglial cells in co-culture system at 3, 6 and 12 hr were 121.6, 90.4 and 81.0 pg/ml of TNF-α, 88.5, 92.7 and pg/ml of IL-1β, and 298.8, and pg/ml of IL-6, respectively. i

7 Keywords: Naegleria fowleri, amoebic meningoencephalitis, microglial cells, cytopathic effect, cytotoxicity, pro-inflammatory cytokine ii

8 TABLE OF CONTENTS ABSTRACT ⅰ TABLE OF CONTENTS ⅲ LIST OF FIGURES ⅴ LIST OF TABLES ⅵ ABBREVIATION ⅶ Ⅰ. INTRODUCTION 1 A. NAEGLERIA FOWLERI 1 1. Life cycle 1 2. Incidence 2 3. Symptoms 2 4. Association with Water 3 5. Pathogenesis 4 B. Background 5 C. Purpose 8 Ⅱ. MATERIALS AND METHODS 9 A. Amoeba and lysate 9 B. Preparation of microglial cells 9 C. Light microscopy 10 D. Scanning electron microscopy 10 E. Transmission electron microscopy 11 iii

9 F. DNA fragmentation 11 G. Flow cytometry analysis 12 H. In vitro cytotoxicity by chromium release assay 13 I. ELISA for measurement of pro-inflammatory cytokines 14 Ⅲ. RESULTS 15 A. Cytopathic changes of microglial cells by a light microscope 15 B. SEM and TEM findings for cytopathic changes of microglial cells 19 C. Apoptotic process of microglial cells by N. fowleri lysate 22 D. Cytotoxicity of N. fowleri lysate on microglial cells 25 E. Cytokines released from microglial cells treated with N. fowleri lysate 27 Ⅳ. DISCUSSION 29 Ⅴ. CONCLUSION 35 REFERENCES 36 국문요약 51 iv

10 LIST OF FIGURES Fig. 1. Primary cultured rat microglial cells 16 Fig. 2. Light microscopic findings of microglial cells treated with the N. fowleri lysate in a dose-dependent manner 17 Fig. 3. Light microscopic findings of microglial cells treated with the N. fowleri lysate in a time-dependent manner 18 Fig. 4. SEM and TEM findings of microglial cells 20 Fig. 5. TEM and SEM findings of microglial cells treated with N. fowleri lysate 21 Fig. 6. DNA fragmentation of microglial cells treated with N. fowleri lysate 23 Fig. 7. FACS analysis of microglial cells stained with PI and Anexin-V in treated with N. fowleri lysate 24 Fig. 8. Amounts of TNF-α, IL-1β and IL-6 secreted from microglial cells treated with N. fowleri lysate for 3, 6 and 12 h were determined by ELISA 28 v

11 LIST OF TABLES Table 1. The cytotoxicity of N. fowleri lysate against microlglial cells by 51 Cr release assay 26 vi

12 ABBREVIATION anti-nfa1 antibody: anti-naegleri Fowleri 1 antibody CNS: Central Nervous System CPE : CytoPathic Effect DMEM: Dulbecco's Modified Eagle's Medium ED 1 antibody: FITC-conjugated mouse anti-rat CD68 ELISA: Enzyme-Linked Immunosorbent Assay FACS: Fluorescence Activated Cell Sorter FBS: Fetal Bovine Serum IL-1: Interleukin-l IL-1β: Interleukin-1 beta IL-6: Interleukin-6 LPS: Lipopolysaccharide PAME: Primary Amoeba MeningoEncephalitis PBS : Phosphate Buffered Saline PI : Propidium Iodide SEM: Scanning Electron Microscopes TEM: Transmission Electron Microscopes TNF-α: Tumor Necrosis Factor alpha vii

13 I. INTRODUCTION A. NAEGLERIA FOWLERI 1. Life Cycle Naegleria fowleri are three stages to the life cycle: the trophozoite, the flagellate, and the cyst (Visvesvara, 1993). Trophozoites are active and usually elongated with broadly rounded processes called lobopodia. Their cytoplasm is granular and contains vacuoles, and they feed on bacteria such as Escherichia coli (Martinez and Visvesvara, 1991; Martinez, 1993; Visvesvara, 1993). The flagellate stage is pear shaped and motile and eventually reverts to the trophic stage (Martinez and Visvesvara, 1991; Bottone, 1993; Martinez, 1993; Visvesvara, 1993). Cysts are usually spherical, smooth, double walled, and refractile, measuring about 10 mm in diameter (Martinez and Visvesvara, 1991; Visvesvara, 1993). Adverse environmental conditions cause the organisms to encyst (Martinez, 1993). The portals of entry for human infection are the olfactory neuroepithelium and nasal passages, which are usually exposed to the flagellate stage during periods of swimming or bathing in hot baths or hot springs (Martinez, 1993; Visvesvara, 1993; Kilvington and Beeching, 1995). Infection can also occur by breathing infectious cysts present in dust or soil particles (Martinez and Visvesvara, 1991; Bottone, 1993; Martinez, 1993; Visvesvara, 1993). Once the organism has been inhaled, excystation occurs and the trophozoite penetrates the nasopharyngeal mucosa, migrates to the olfactory nerves, and invades the brain through the - 1 -

14 cribriform plate (Bottone, 1993). 2. Incidence While there are six species in the genus, N. fowleri is the primary human pathogen, producing primary amebic meningoencephalitis (PAME), a rapidly progressing meningoencephalitis which is almost always fatal. N. australiensis may be pathogenic to a lesser extent than N. fowleri (Martinez, 1993; Visvesvara, 1993; Kilvington and Beeching, 1995). In 1997 years, there have been more than 192 reported cases of disease worldwide and more than 64 cases in the United States (Marshall et al., 1997). While the numbers of cases may appear low, exposure to the organisms may be relatively common since antibodies to Naegleria spp. are widespread in human sera (Bottone, 1993). 3. Symptoms No predisposing factors are necessary for human infections to occur (Martinez, 1993; Visvesvara, 1993). After a 2- to 7-day incubation period, the symptoms of PAME are evident. Onset is abrupt, with rapidly progressive headaches, fever, nausea, vomiting, pharyngitis, and nasal obstruction or discharge (Martinez and Visvesvara, 1991; Martinez, 1993). As the symptoms persist, lethargy, confusion, and stiff neck develop. Convulsions may also occur, with progressive deterioration to coma and death within 1 to 14 days. The mean time interval from onset to death is 6.4 days (Ma et al., 1990; Visvesvara and Stehr-Green, 1990; Martinez, 1993; Visvesvara, 1993; Kilvington and Beeching, 1995)

15 Other symptoms include abnormalities of taste and smell; seizures; cerebellar ataxia; nuchal rigidity; photophobia; palsies of the third, fourth, and sixth cranial nerves; and increased intracranial pressure. Cardiac abnormalities may also develop (Ma et al., 1990). Subclinical infections are possible in healthy people when these protozoa colonize the nose and throat (Ma et al., 1990; Martinez and Visvesvara, 1991). 4. Association with water The preferred environment for N. fowleri is the soil; however, heavy rains and runoffs introduce this organism into lakes, ponds, and surface waters (Marciano-Cabral, 1988; Elder et al., 1994). Naegleria spp. Are distributed worldwide in thermally polluted streams, and they tolerate temperatures of 40 to 458 (Bottone, 1993; Martinez, 1993; Sparagano, 1993; Visvesvara, 1993). They can also be found in coastal water, freshwater, sewage, heating and ventilation units, poorly chlorinated swimming pools, artificial lakes, and warm water near discharge outlets of power plants (Martinez, 1993). In Australia, one fatal case of PAME led to the detection of Naegleria spp. in the household water supply. This case emphasizes that PAME may be associated with washing and bathing as well as with swimming (Marciano-Cabral, 1988). Vertical distribution in freshwater has been correlated with physical, chemical, and biological parameters. Significant numbers of Naegleria spp. were found in water layers containing filamentous cyanobacteria and eubacteria, which serve as food sources. In addition, large numbers of organisms have been isolated from water with increased iron and manganese concentrations. As expected, Naegleria spp. was found in increased - 3 -

16 numbers in waters contaminated with coliforms. In addition, some species of Naegleria interact with Legionella spp. and are thought to play a possible role in the dissemination of Legionella in water (Ma et al., 1990). 5. Pathogenesis Naegleria species typically cause PAME in children and healthy adults who have been swimming in polluted pond water or inadequately chlorinated swimming pools, as well as man-made or natural freshwater lakes. Organisms enter through the olfactory neuroepithelium at the level of the cribriform plate and invade the amyelinic submucosal nervous plexus (Culbertson, 1971, Carter, 1972). Symptoms begin after a 3-7 day incubation period, and infections caused by N. fowleri tend to be fulminant, with rapid progression to death in most cases (John, 1993). Pathological changes include acute hemorrhagic necrotizing meningoencephalitis with purulent exudates in the brain, brainstem and cerebellum (John, 1993). Patients who develop N. fowleri meningoencephalitis may have an immunoglobulin IgA deficiency, which would imply weaker defenses at the mucus membrane level (Reilly, 1983). Acanthamoeba and Hartmanella are similar organisms that cause a more subacute form of illness in immunocompromised, debilitated or malnourished individuals, including those undergoing suppressive therapy for organ transplant and HIV/AIDS patients (Gonzalez, 1986). These organisms enter the body through the respiratory tract or skin ulcerations, reaching the central nervous system (CNS) by hematogenous spread (Martinez and Visvesvara, 1997), causing a patchy, chronic granulomatous encephalitis with trophozoites - 4 -

17 and cysts in the lesions. The incubation period is unknown but is thought to be more than 10 days (Gonzalez, 1986). B. Background Pathogenic N. fowleri, a free-living amoeba found in widespread environment, causes fatal PAME in experimental animal and humans. PAME occurs most commonly in healthy, young adults and non-immunocompromised children. It has been associated with swimming or bathing in contaminated warm waters (John, 1982; Im and Shin, 2004; Schuster and Visvesvara, 2004). The infection results from the introduction of water which contains amoebae into the nasal cavity and the subsequent passage of these organisms to the CNS via the olfactory apparatus (Ma et al., 1990; Carter, 2001). CNS inflammation occurs in both disease and trauma, and is mediated in part by microglial cell, the resident immune cells of the CNS. Microglial cells originate from bone marrow and migrate into the CNS during early stages of development (Shuman et al., 1997). Microglial cell display graded levels of activation in the CNS, from resting, highly ramified microglial cell, to phagocytic macrophages (Streit et al., 1988). microglial cell react quickly in response to CNS injury or disease (Kreutzberg, 1996), migrating into an injury site (Carbonell et al., 2005) and secreting a wide array of molecules that can be toxic to oligodendrocyte progenitor cells (OPCs) and oligodendrocytes, including tumor necrosis factor-α (TNF-α) (Selmaj and Raine, 1988; Dasgupta et al., 2003; Jana et al., 2003), glutamate (Nakamura et al., 2003), and free radicals (Benveniste, 1997). Furthermore, - 5 -

18 molecules that induce oligodendrocyte death can also lead to microglial cell activation, such as glutamate (Christensen, 2006) and proinflammatory cytokines (Thery and Mallat, 1993). In vitro, microglial cell is capable of inducing OPC death even without the two cell populations being in direct contact (Li et al., 2005). However, in vivo microglial cell has been observed in close proximity to dying oligodendrocytes after spinal cord injury (Shuman et al., 1997). This proximity after injury suggests a mechanism by which microglial cell may influence oligodendrocyte and OPC survival, as it has been shown in vitro that microglial cell in contact with oligodendrocytes can induce oligodendrocyte death via membrane-bound TNF-α which is more potent than soluble TNF-α (Zajicek et al., 1992). Additionally, any soluble factors secreted by microglial cell could have a higher effective concentration if secreted into a small space between cells. Additionally, cytokines produced by microglial cell may aid in repair after injury, as mice lacking TNF-α undergo delayed remyelination (Arnett et al., 2004). Even the observations of Shuman and colleagues (Shuman et al., 1997) that activated microglial cells are found in contact with apoptotic oligodendrocytes after spinal cord injury, raises the question of whether microglial cell destroy oligodendrocytes that would otherwise survive after injury, or are simply phagocytosing oligodendrocytes already destroyed by other toxins in the damaged CNS. Some data suggest that microglial cell play a dual role in CNS injury, exacerbating damage in some instances or at some times, and promoting repair or regeneration at others (Popovich et al., 2002). Shuman and colleagues (Shuman et al., 1997) also reported that microglial cell undergo apoptosis after spinal cord injury. Regarding the host-tissue invasion, the adherence of the amoeba to host cells is the most - 6 -

19 important step in the mechanism of pathogenicity of N. fowleri, and a specific pseudopodia projection, called an amoebastome, is formed. Invasive amoeba capable of entering the nervous system usually digests neuronal tissue and other mammalian cells by effective cytolysis and phagocytosis, as observed in culture or in infected sections of brain tissue (Martinez, 1985; Marciano-Cabral, 1988). Rat microglial cells exist in three morphological forms following cell differentiation, i.e., an amoeboid form during embryogenesis, a ramified shape in the mature brain, and a rodshaped morphology around inflammatory lesions in the CNS (Giulian and Baker, 1986; Suzumura et al., 1991). Moreover, they function as phagocytotic cells and produce cytokines such as interleukin-l (IL-1), IL-6, and TNF-α (Chao et al., 1994; Oh et al., 2005). Thus, it has been suggested that microglial cells play important roles as inflammatory cells or as immunoregulatory cells in the protective immune system of the CNS (Suzumura et al., 1993). In the previous study, it was seen that N. fowleri trophozoites in contact with microglial cells produced vigorous pseudopodia and a food-cup structure. Microglial cells were destroyed by N. fowleri trophozoites as seen from necrotic and apoptotic cell death in a timedependent manner (Oh et al., 2005). As the results of 51 Cr release assay, N. fowleri showed increasing cytotoxicity against microglial cells in a culture-time dependent manner. And then, microglial cells co-cultured with N. fowleri trophozoites secreted the proinflammatory cytokines, TNF-α, IL-1β and IL-6 (Oh et al., 2005). In addition, When an anti- Nfa1 antibody which was made with a recombinant Nfa1 protein expressed nfa1 gene cloned from a cdna library of pathogenic N. fowleri was treated in a co-culture system, N. fowleri showed decreasing cytotoxicity against microglial cells, and the secretion of TNF-α from - 7 -

20 microglial cells was inhibited (Cho et al., 2003; Jeong et al., 2004; Oh et al., 2005). A few attempts to study the cytopathic effects of pathogenic N. fowleri lysate against actual target cells of PAME, rat microglial cells, have been poorly reported. C. Purpose To determine whether pathogenic N. fowleri lysate showed cytopathic effects against primary cultured rat microglial cells, the morphological changes of microglial cells cultured with N. fowleri lysate were observed with light, scanning and transmission electron microscopes (SEM and TEM). The in vitro cytotoxicity of N. fowleri lysate against rat microglial cells was also subsequently observed. Additionally, the pro-inflammatory cytokines that are released from microglial cells in a culture system were estimated

21 II. MATERIALS AND METHODS A. Amoeba and lysate The trophozoites of N. fowleri (Cater NF69 strain, ATCC NO ) were axenically cultured at 37 C in Nelson's medium (Shin et al., 2001). Amoebic lysate was prepared according to the previous paper (Kang et al., 2005). After freezing and thawing of trophozoites, the soluble proteins (whole lysate) was filtered with 0.22-μm-pore-size disk filters, and the protein concentration was determined by the method of Bradford assay (Kang et al., 2005). B. Preparation of microglial cells Rat microglial cells were prepared using a modified method of Giulian and Baker (Giulian and Baker, 1986). Mainly, the cortexes of the brain were obtained from newborn rats (Sprague-Dawley, purchased from KIST in Daejeon, Korea) and homogenized by pumping with 21-gauge syringe. The mixture was centrifuged at 300 g for 5 min and suspended in Dulbecco's modified Eagle's medium (DMEM; Sigma) with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. The suspension was put into 75 cm 3 tissue culture flasks. The flasks were then incubated for 14 days at 37 C, 5% CO 2 humidified atmosphere. Upon 14 days of culture, microglial cells were harvested by vigorous shaking of each culture flask. They were then filtered with nylon wool to remove - 9 -

22 any remaining astrocytes and were centrifuged at 300 g for 5 min. The pellets were suspended in DMEM with 10% FBS, and the mixtures were incubated at 37 C for 2 h. After the supernatant was removed, the attached microglial cells were harvested and counted at a concentration of per well in a 24-well culture plate with subjected to subsequent experiments. The purity of the microglial cells was determined by indirect immunofluorescent staining with a FITC-conjugated mouse anti-rat CD68 (ED 1 antibody) (Serotec, Bicester, United Kingdom) to be nearly 95%, as shown (Fig. 1). C. Light microscopy Microglial cells ( ) were cultured with N. fowleri lysate (0.1, 0.5 or 1 mg/ml) in 24-well culture plates for 3, 6 or 12 h. After co-incubation, samples were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (ph 7.4) and post-fixed with 1% osmium tetroxide-1.5% potassium for 1 h. The cells were then examined with a light microscope. D. Scanning electron microscopy Microglial cells ( ) were seeded onto the Lab-tek II chamber slide system (Nunc A/S, Roskilde, Belgium) and the lysate of N. fowleri were added to the monolayer. After incubation for 3, 6 and 12 h, samples were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (ph 7.4) and were dehydrated with increasing concentrations of ethanol. Samples were vacuum-dried and coated with ultra-thin layers (300 Å) of gold/pt in an ion

23 sputter (E-1010, Hitachi, Tokyo, Japan). An image analyzer program (Escan 4000, Bumi-Mi Universe Co., Ltd., Ansan, Korea) was used to capture the images of cells and modified surfaces. Samples were characterized using an SEM (S-800, Hitachi, Tokyo, Japan). E. Transmission electron microscopy After microglial cells were incubated with amoebic lysate in 24-well culture plates for 3, 6, or 12 h. They were then fixed in modified Karnovsky's fixative solution in cacodylate buffer (ph 7.4) and post-fixed in 1% osmium tetroxide-1.5% potassium ferrocyanide. The cells were stained en bloc in 0.5% uranyl acetate, dehydrated through a graded ethanol series, and embedded in resin (Polyscience, Warrington, Pa.). Then, the blocks were sectioned with Ultrostain 1H and 2 (Leica, Vienna, Austria). Specimens were observed and photographed with a Zeiss EM 902A TEM (Leo, Oberkohen, Germany). F. DNA fragmentation DNA extractions and agarose-gel electrophoresis were performed to observe DNA fragmentation of microglial cells treated with N. fowler lysate. Microglial cells were harvested by sterile cell scrapers after cultivation with an amoeba lysate (1 mg/ml) for 3, 6, 12 and 24 h. Then, microglial cells were washed with phosphate-buffered saline (PBS) (ph 7.4) and suspended in 0.5 ml of TBE buffer (45 mm Tris-borate buffer, 1 mm EDTA [ph 8.0]) containing 0.25% Nonidet P-40 and 1 mg of RNase A per ml. After the mixture was incubated at 37 C for 30 min, 1 mg of proteinase K per ml was added. The mixture was

24 incubated at 37 C for 30 min and resuspended in 0.1 ml of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 30% glycerol). The suspended volume of 25 ml was put on 1.5% agarose gel containing 10 ml of ethidium bromide per ml. Electrophoresis was carried out at 2 V/cm for 6 h. A 123-bp DNA ladder and PCR marker containing fragments of 1,000, 750, 500, 300, 150, and 50 bp (Promega Corporation, Madison, Wis.) were used as molecular size standards. G. Flow cytometry analysis The apoptotic cell death from microglial cells treated with N. fowleri lysate were measured with the PI and Annexin V end labeling assay (Promega Corporation). Briefly, microglial cells cultured with 1 mg/ml of each amoeba lysate for 6 or 18 h were harvested and washed twice with PBS (ph 7.4). The cells were resuspended in 0.5 ml of PBS. The cells were fixed by adding 5 ml of 1% methanol-free formaldehyde for 20 min on ice, centrifuged, and resuspended in 0.5 ml of PBS. The cell suspension was mixed with 5 ml of 70% ice-cold ethanol and kept at - 20 C for 4 h. The mixture was centrifuged and resuspended in 1 ml of PBS. The suspended cells were adjusted to a concentration of and transferred into a 1.5-ml microcentrifuge tube. After centrifugation, cells were resuspended in 80 ml of equilibration buffer (200 mm potassium cacodylate, 24 mm Tris- HCl, 0.2 mm dithiothreitol, 0.25 mg of bovine serum albumin/ml, 2.5 mm cobalt chloride [ph 6.6]). After centrifugation, the cells were resuspended in 50 ml of Annexin V incubation buffer. The suspended cells were incubated in a water bath for 60 min at 37 C. The reaction was terminated by adding 1 ml of 20 mm EDTA. Following centrifugation after the reaction,

25 the pelleted cells were resuspended in 0.5 ml of PBS containing Triton X-100 and 5 mg of bovine serum albumin/ml. The cells were washed twice with PBS, centrifuged, and resuspended in 0.5 ml of propidium iodide (PI) solution (freshly diluted to 5 mg/ml in PBS) containing 250 mg of DNase-free RNase A. After the cells were incubated for 30 min in the dark, the green fluorescence of fluorescein-annexin V at 520 nm and the red fluorescence of PI at 620 nm were measured by FACScan flow cytometry (Becton Dickinson, Paramus, N.J.). H. In vitro cytotoxicity by chromium release assay Using the methods of a previous study (Oh et al., 2005), a 51 Cr (chromium) release assay was performed to determine the cytopathic effects of N. fowleri lysate. Target microglial cells were labeled with 100 µci of [Na] 51 2 CrO 4 per 10 5 cells for 60 min at 37 C. The cells were washed to remove unbound radioisotope. Labeled microglial cells ( ) were added to each well of a 96-well culture plate. They were then cultured with N. fowleri lysate (0.1, 0.5 or 1 mg/ml) in 5% CO 2 for 3, 6 or 12 h. Spontaneous release from labeled microglial cells was determined by acquiring the counts per min (cpm) in the supernatant fluid without amoebic lysate. All assays were performed in triplicate. At the end of the experimental incubation period, plates were centrifuged at 300 g for 3 min and the supernatant from each well was harvested. For maximal release, each of these was lysed with 5% (vol/vol) Triton X-100 and harvested. Following 3, 6, or 12 h, the supernatant fluid and lysed cells were counted in a gamma counter. The percentage of the radioisotope released from target microglial cells was determined to be the index of lysis using the following formula:

26 experimental release spontaneous release Cytotoxicity (%) = 100 maximum release spontaneous release I. ELISA for measurement of pro-inflammatory cytokines To determine whether microglial cells released the pro-inflammatory cytokines as a result of a cytopathic effect (CPE) induced by pathogenic N. fowleri lysate, the amounts of tumor necrosis factor (TNF)-α, Interleukin (IL)-1β, and IL-6 released from microglial cells in the co-culture system were measured using enzyme-linked immunosorbent assay (ELISA) kits (BioSource International, California, USA). The amounts of cytokines produced by the microglial cells were estimated by generating a standard curve according to the instructions of the manufacturer

27 III. RESULTS A. Cytopathic changes of microglial cells by a light microscope Light microscopic findings of primary cultured rat microglial cells showing three types; amoeboid form, rod form and rammified form (Fig. 1A). For the identification of microglial cells, fluorescent microscopic findings of microglial cells immunostained with a FITC conjugated mouse anti-rat CD68 was carried out (Fig. 1B). The microglial cells treated with N. fowleri lysate showed morphological changes such as the cell membrane destruction and reduction in number (Fig. 2). They were proceed in a dose- and time-dependent manner (Fig. 2, 3)

28 ra a r A No stained B ED1(anti-rat CD68) stained Fig. 1. Primary cultured rat microglial cells. A. Light microscopic findings of primary cultured rat microglial cells showing three types; amoeboid form (a), rod form (r) and rammified form (ra). (x200). B. Fluorescent microscopic findings of microglial cells immunostained with a FITC conjugated mouse anti-rat CD68. (x400)

29 A B C D E F Fig. 2. Light microscopic findings of microglial cells treated with the Naegleria fowleri lysate in a dose-dependent manner. Microglial cells were cultured for 12 h in DMEM media only (A), treated with PBS (B) or LPS (C), and the lysate of N. fowleri (0.1, 0.5 and 1 mg/ml) (D, E and F), respectively. Microglial cells showed severe destruction in a dosedependent manner. ( 200)

30 A B C Fig. 3. Light microscopic findings of microglial cells treated with the Naegleria fowleri lysate in a time-dependent manner. Microglial cells were cultured in DMEM media only (A), and treated with a lysate of N. fowleri (1 mg/ml) for 6 h(b) and 12 h (C). Microglial cells showed severe destruction in a time-dependent manner. ( 200)

31 B. SEM and TEM findings for cytopathic changes of microglial cells By SEM and TEM findings, a primary culture microglial cells show numerous cytoplasmic projections, and scattered chromatin materials in large nucleus were showed (Fig. 4). In a co-culture system at 6 h, most of microglial cells were destroyed by N. fowleri lysate, which showed the necrotic process characterized by the swelling of the membrane and the bursting of the nuclear membrane and nucleus (Fig. 5A, C). And some of them showed an apoptotic feature such as blebing and chromatin condensation in nucleus (Fig. 5B, D)

32 Fig. 4. SEM and TEM findings of microglial cells. A. Microglial cell shows numerous cytoplasmic projections by SEM. B. Scattered chromatin materials in large nucleus were showed by TEM. Bars, 5um

33 N C N A B C D Fig. 5. TEM and SEM findings of microglial cells treated with Naegleria fowleri lysate. Microglial cells were cultured with N. fowleri lysate (1 mg/ml) for 12 hr. Microglial cells showed a necrotic process (A, C) and produced apoptotic bodies (B, D). Bars, 5 µm

34 C. Apoptotic process of microglial cells by N. fowleri lysate In the experiments to identify the apoptotic process of microglial cells by N. fowleri lysate, DNA fragmentation of microglial cells in a co-culture system was observed (Fig. 6). DNA fragmentation was not observed with total genomic DNA of microglial cells treated with PBS, but the development of DNA ladders was shown in microglial cells treated with N. fowleri lysate (1 mg/ml) for 3, 6 and 12h, respectively. PI staining demonstrated that some microglial cells cultured with the N. fowleri lysate (1 mg/ml) for 6 h underwent apoptosis. Additionally, apoptotic cells stained with Annexin-V were observed about 22% at 3 h and 17% at 6 h post-incubation, as which showed increased intensity of Annexin-V stained apoptotic cells (Fig. 7D, E). By contrast, microglial cells cultured with N. fowleri lysate for 12 h did not show apparent changes in the intensity of fluorescence of the Annexin-V stained apoptotic cells (Fig. 7F)

35 Fig. 6. DNA fragmentation of microglial cells treated with Naegleria fowleri lysate. Lane 1, 1kb Plus DNA Ladder marker; Lane 2, intact genomic DNA of microglial cells; Lanes 3, 4, 5 and 6, microglial cells treated with N. fowleri lysate for 3, 6, 12 and 24h, respectively

36 A B C D E F Fig. 7. FACS analysis of microglial cells stained with PI and Annexin-V in treated with Naegleria fowleri lysate. A and B, unstained and microglial cells only, respectively; C, D, E and F, stained microglial cells cultured with N. fowleri lysate for 1, 3, 6 and 12 h, respectively. Maximum number of stained microglial cells showing the apoptotic process was observed at 3~6 hr post incubation

37 D. Cytotoxicity of N. fowleri lysate on microglial cells To determine whether N. fowleri lysate showed the cytotoxicity to microglial cells, 51 Cr release assay was carried out. When microglial cells were cultured with N. fowleri lysate (0.5 mg/ml), the cytotoxicity of amoeba on microglial cells was increased in a time-dependent manner, 7.9, 14.4, 23.8 and 51.5 % at 3, 6, 12, and 24 h, respectively (Table 1). When microglial cells were cultured with N. fowleri lysate (1 mg/ml), the cytotoxicity of amoeba on microglial cells was increased in a time-dependent manner, 14.6, 21.9, 38.5 and 71.5 % at 3, 6, 12, and 24 h, respectively (Table 1)

38 Table 1. The cytotoxicity of Naegleria fowleri lysate against microlglial cells by 51 Cr release assay Times of co-cultivation Groups 3 h 6 h 12 h 24 h Microglia + lysate (0.5 mg/ml) 7.9%* 14.4% 23.8% 51.5% Microglia + lysate (1.0 mg/ml) 14.6% 21.9% 38.5% 71.5% * % cytotoxicity calculated by the 51 Cr amount released from radiolabelled microglial cells (mean ± standard variation)

39 E. Cytokines released from microglial cells treated with N. fowleri lysate To determine whether microglial cells showed the release of cytokines induced by pathogenic N. fowleri lysate, pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, were measured with enzyme-linked immunosorbent assay kits. In a co-culture system with N. fowleri lysate (1 mg/ml), the amount of TNF-α released from microglial cells was 121.6, 90.4 and 81.0 pg/ml at 3, 6 and 12 h, respectively, which was peaked at 3 h and decreased during the period of co-incubation (Fig. 8A). The amount of IL-1β was 88.5, 92.7 and pg/ml at 3, 6 and 12 h, respectively, which was increased during the period of co-incubation (Fig. 8B). The amount of IL-6 was 298.8, and pg/ml at 3, 6 and 12 h, respectively, which was peaked at 6 h post co-incubation (Fig. 8C)

40 Fig. 8. Amounts of TNF-α (A), IL-1β (B) and IL-6 (C) secreted from microglial cells treated with Naegleria fowleri lysate for 3, 6 and 12 h were determined by ELISA. M, microglia; L, N. fowleri lysate

41 IV. DISCUSSION N. fowleri is the principal protozoa, commonly referred to as pathogenic free-living amoebae that cause PAM (Matinez, 1997; Parija and Jayakeerthee, 1999). PAME occurs very rarely but is usually fatal. Since the disease was first identified in 1965 (Fowler and Carter, 1965), fewer than 200 cases have been identified worldwide (Ockert, 1993; Matinez, 1997; Parija and Jayakeerthee, 1999). N. fowleri are ubiquitous in the environment, in soil, water, and air (Rodriguez-Zaragoza, 1994). Infections in humans are acquired through water entering the nasal passages (usually during swimming) and by inhalation. Most human victims of PAME are exposed to free-living amoebae while swimming in warm surface water. This may include ponds, lakes, streams, rivers, and improperly maintained swimming pools. The risk of acquiring PAME increases as water temperatures rise (Rodriguez-Zaragoza, 1994; Matinez, 1997). Transmission to humans occurs when the organism gains access to brain tissues through the nasal passages. Invasion of the CNS and development of PAME are prominent features in fatal human and animal cases of N. fowleri infection. As the major route of invasion for N. fowleri infection, amoebas enter the nasal cavity, attach, and invade the nasal mucosa and olfactory nerve (Culbertson, 1971; Chang, 1979). The organism can enter the nasal passages when water containing the organism is forced up the nose through activities such as diving, jumping into water, and underwater swimming. However, PAM is not transmitted from person to person (Ockert, 1993; Matinez, 1997;

42 Parija and Jayakeerthee, 1999). N. fowleri, the causative agent of primary amoebic meningoencephalitis in humans, is cytopathogenic for a variety of cultured mammalian cells (Chang, 1974; Visvesvara and Callaway, 1974; Brown, 1978; Cursons and Brown, 1978; Brown, 1979; Marciano-Cabral et al., 1982). The proposed mechanism(s) of the cytopathic action of this organism for mammalian cells include active phagocytosis of cells by pseudopod formation (Visvesvara and Callaway, 1974), trogocytosis or repeated nibbling by trophozoites (Brown, 1978; Brown, 1979), and secretion of cytolytic substances by N. fowleri (Chang, 1974). In the previous transmission electron microscopic study established that amoebae ingest portions of mammalian cells without cell lysis at 12 h of cocultivation with N. fowleri (Marciano-Cabral et al., 1982). Several investigators have suggested that N. fowleri releases cytolytic substances, phospholipolytic enzymes and acid phosphatase. These substances account for the invasiveness and tissue damage in vivo and the cytopathogenicity in vitro (Feldman, 1977; Ferrante and Bates, 1988). It has been proposed that cytotoxic toxins and cytolytic proteases are influential in the destruction of target cells. In another free-living amoeba, Acanthamoeba spp., its cytopathogenic effects on host cells require the adhesion of amoeba to the host cell (Moore et al., 1991; Yang et al., 1997; Shin et al., 2001), phagocytosis, and amoebic proteolytic enzymes, including serine proteases (Mitro et al., 1994), contact-dependent metalloproteases (Hadas and Mazur, 1993), elastases (Ferrante and Bates, 1988), cysteine proteases (Khan et al., 2000), and cytotoxic proteinases induced by mannose-mediated adhesion (Leher et al., 1988)

43 The lysate of N. fowleri has been observed to cause tissue destruction in vitro (Fulford et al., 1985; Marciano-Cabral and Fulford, 1986). Proteinases in N. fowleri could also be involved in tissue destruction and in its pathogenesis since this enzymes present in the cell lysate (Mat, 2004). The involvements of proteinases in the pathogenecity of some parasitic protozoa have been proven by Keene et al., (1990) and Robertson and Coombs (1992). Proteinases in N. folweri observed in this study appear to be high molecular weight enzymes whereas proteinases reported from other protozoa generally have their apparent molecular weight in the range of 20 kda to 96 kda (Keene et al., 1990; North et al., 1990; Robertson and Coombs, 1992). Despite high molecular weight, the proteinases of this amoeba might be the cysteine proteinase. It is interesting to speculate why there are not many forms of proteinases present in pathogenic N. fowleri compared with other parasitic protozoa such as Trichomonas spp. (Lockwood et al., 1987) and Leishmania mexicana mexicana (Robertson and Coombs, 1992). Details on the functional aspects of proteinases in N. fowleri however, need to be carried out to further verify if these enzymes are related to the amoeba s pathogenesis as have been observed in Entamoeba histolytica. In this study, to determine whether N. foweri lysate has cytopathic effects, microglial cells cultured from the brains of newborn rats were used as target cells. As determined by morphological findings by a light microscope, an SEM and a TEM, microglial cells in coculture systems with N. fowleri lysate were destroyed by showing necrotic and apoptotic process. In previous reports, pathogenic A. culbertsoni lysate induced cytopathic effects in primary-culture rat microglial cells, with the effects characterized by necrosis and apoptosis

44 of microglial cells (Shin et al., 2000; Shin et al., 2001; Shin et al., 2001(a)). A. culbertsoni lysate also showed stronger destruction than Acanthamoeba castellanii (Marciano-Cabral et al., 2004). Activated macrophages are efficient cytotoxic cells as well as the producers of over 100 secretory molecules (Nathan, 1987). Effector functions of activated macrophages are tightly regulated by a complex network of cytokines. Mouse peritoneal macrophages may be activated in vivo by infection with microorganisms or in vitro by exposure to lymphokines. Macrophages activated for cytolysis of one target cell are not necessarily cytolytic for other target cells (Wing, 1977; Nacy et al., 1984; LeBlanc et al., 1990). Current studies have focused on identifying the effector molecules that mediate the direct, nonspecific cytotoxicity of activated macrophages. Reactive oxygen intermediates produced during the respiratory burst have been implicated as one of the major mechanisms of macrophage microbicidal effector functions (Ding et al., 1988; Schwamberger et al., 1991). More recently, the cytotoxic capacity of nitric oxide produced by nitrogen oxidation of L-arginine has been demonstrated for parasites (Adams et al., 1990; James and Hibbs, 1990; Liew et al., 1990; Green et al., 1991; Vincendeau and Daulouede, 1991; Lin et al., 1992). Also, pro-inflammatory cytokines are produced by activated macrophages, including tumor necrosis factor alpha (TNF-α), interleukin-1 alone or in combination with TNF-α, and cytolytic protease (Adams, 1980; Onozaki et al., 1985; Lachman et al., 1986; Ichinose, 1988; Last-Barney et al., 1988). TNF-α effects tumoricidal activity but has no direct cytolytic activity for a number of parasitic protozoa (DeTitto et al., 1986; Clark et al., 1990). Up to date, it has been demonstrated that TNF-α may indirectly affect cytolysis of

45 parasitic organisms by serving as an inducer of the L-arginine dependent cytolytic pathway (Liew et al., 1990). TNF-α has also been shown to synergize with gamma interferon in the presence of lipopolysaccharide (LPS) and induce the production of nitric oxide in the target cells themselves (Amber et al., 1991). On the other hands, necrosis characterized by the disruption of cell membrane integrity and apoptosis by the membrane blebing and apoptotic bodies formation were determined to be two fundamental mechanisms in related to the cytolysis of target microglial cells by N. fowleri trophozoites (Oh et al., 2005). In this study, N. fowleri lysate induced cytopathic effects in primary-culture rat microglial cells, with the effects characterized by necrosis and apoptosis of microglial cells. More detailed studies are necessary in the future to elucidate what the cell death signaling pathways due to N. fowleri lysate are induced, and what the pathogenic elements produced from N. fowleri lysate are present. It is well known that microglial cells produce pro-inflammatory cytokines, such as TNFα, IL-1β, IL-3, IL-6, IL-8, IL-12 and IL-15, as well as anti-inflammatory cytokines, such as IL-10 and TGF-β, for defense against parasites and brain injury (Benedetto et al., 2001; Marciano-Cabral et al., 2004). In a previous study, to determine whether microglial cells release pro-inflammatory cytokines as a protective mechanism induced by N. fowleri trophozoites, the ELISA assay for cytokines, such as TNF-α, IL-1β and IL-6, was performed with culture supernatants (Oh et al., 2005). TNF-α and IL-6 peaked at 6 h post co-incubation and then decreased, whereas IL-1β peaked at 12 h and continued to accumulate in the medium through 24 h. In addition,

46 when microglial cells were co-cultured with N. fowleri trophozoites and an anti-nfal antibody for 3 and 6 h, the amount of TNF-α secreted from microglial cells was inhibited about 20.3% and 14.1%, respectively. But the amount of IL-1β and IL-6 was not decreased (Oh et al., 2005). In present study, to determine whether N. fowleri lysate induce the releasing of proinflammatory cytokines from microglial cells, amounts of TNF-α, IL-1β and IL-6 were checked. Secretion of TNF-α and IL-6 peaked at early periods of co-incubation and subsequently decreased, whereas IL-1β was increased during the periods of cultivation and continued to accumulate in the medium during the following 24 h (data not shown). More extensive studies on the cytokine responses of microglial cells due to N. fowleri are necessary in the future study

47 V. CONCLUSION In the present study, to determine whether a pathogenic N. fowleri lysate shows the cytopathic effects against primary culture rat microglial cells, the morphological changes of microglial cells was observed by a light, scanning and transmission electron microscope. And then, the cytotoxicity of N. fowleri lysate against microglial cells was also observed by 51 Cr release assay. In addition, the pro-inflammatory cytokine release from microglial cells in the co-culture system was estimated. As results with a light and electron microscopes, most of microglial cells were severely destroyed by N. fowleri lysate, showing the necrotic (above 85%) and apoptotic cell death (below 15%) in a time- and dose dependent manner. As the results of 51 Cr release assay, the cytotoxicity of N. fowleri lysate against microglial cells were 14.6, 21.9, 38.5 and 71.5% at 3, 6, 12 and 24 hr post incubation, respectively. And then, the amount of cytokines released from microglial cells in co-culture systems at 3, 6 and 12 hr were 121.6, 90.4 and 81.0 pg/ml of TNF-α, 88.5, 92.7 and pg/ml of IL-1β, and 298.8, and pg/ml of IL-6, respectively. Thus, this present study shows that the microglial cells may be involved in the inflammation stage of N. fowleri infection as secreting various inflammatory cytokines

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