Identification of differential protein recognition pattern between Naegleria fowleri and Naegleria lovaniensis.

Many pathogenicity factors are involved in the development of primary amoebic meningoencephalitis (PAM) caused by N fowleri. However, most of them are not exclusive for N fowleri and they have not even been described in other nonpathogenic Naegleria species. Therefore, the objective of this work was to identify differential proteins and protein pattern recognition between Naegleria fowleri and Naegleria lovaniensis using antibodies anti-N fowleri as strategy to find vaccine candidates against meningoencephalitis. Electrophoresis and Western blots conventional and 2-DE were performed for the identification of antigenic proteins, and these were analysed by the mass spectrometry technique. The results obtained in 2-DE gels and Western blot showed very notable differences in spot intensity between these two species, specifically those with relative molecular weight of 100, 75, 50 and 19 kDa. Some spots corresponding to these molecular weights were identified as actin fragment, myosin II, heat shock protein, membrane protein Mp2CL5 among others, with differences in theoretical post-translational modifications. In this work, we found differences in antigenic proteins between both species, proteins that could be used for a further development of vaccines against N fowleri infection.

Generation of neuroinflammation in human African trypanosomiasis.

Human African trypanosomiasis (HAT) is caused by infection due to protozoan parasites of the Trypanosoma genus and is a major fatal disease throughout sub-Saharan Africa. After an early hemolymphatic stage in which the peripheral tissues are infected, the parasites enter the CNS causing a constellation of neurologic features. Although the CNS stage of HAT has been recognized for over a century, the mechanisms generating the neuroinflammatory response are complex and not well understood. Therefore a better understanding of the mechanisms utilized by the parasites to gain access to the CNS compartment is critical to explaining the generation of neuroinflammation. Contrast-enhanced MRI in a murine model of HAT has shown an early and progressive deterioration of blood-CNS barrier function after trypanosome infection that can be reversed following curative treatment. However, further studies are required to clarify the molecules involved in this process. Another important determinant of brain inflammation is the delicate balance of proinflammatory and counterinflammatory mediators. In mouse models of HAT, proinflammatory mediators such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and CXCL10 have been shown to be crucial to parasite CNS invasion while administration of interleukin (IL)-10, a counter inflammatory molecule, reduces the CNS parasite burden as well as the severity of the neuroinflammatory response and the clinical symptoms associated with the infection. This review focuses on information, gained from both infected human samples and animal models of HAT, with an emphasis on parasite CNS invasion and the development of neuroinflammation.

Mouse neutrophils release extracellular traps in response to Naegleria fowleri.

Naegleria fowleri is a free-living amoeba, which is able to infect humans through the nasal mucosa causing a disease in the central nervous system known as primary amoebic meningoencephalitis (PAM). Polymorphonuclear cells (PMNs) play a critical role in the early phase of N fowleri infection. Recently, a new biological defence mechanism called neutrophil extracellular traps (NETs) has been attracting attention. These structures represent an important strategy to immobilize and kill invading microorganisms. In this work, we evaluate the capacity of N fowleri to induce the NETs release by PMNs cells in mice in vitro and in vivo. In vitro: Neutrophils from bone marrow were cocultured with N fowleri trophozoites. In vivo: we employed a mouse model of PAM. We evaluated DNA, histone and myeloperoxidase (MPO) and the formation of NETs by confocal microscopy. Our results showed N fowleri induce both NETs and MPO release by PMNs cells in mice after trophozoite exposure, which increased through time, in vitro and in vivo. These results demonstrate that NETs are somehow associated with the amoebas. We suggest PMNs release their traps trying to avoid N fowleri attachment at the apical side of the nasal epithelium.

Report: Effect of macrophage alone or primed with cytokines on Balamuthia mandrillaris interactions with human brain microvascular endothelial cells in vitro.

Balamuthia mandrillaris is well known to cause fatal Balamuthia amoebic encephalitis (BAE). Amoebic transmission into the central nervous system (CNS), haematogenous spread is thought to be the prime step, followed by blood-brain barrier (BBB) dissemination. Macrophages are considered to be the foremost line of defense and present in excessive numbers during amoebic infections. The aim of the present investigation was to evaluate the effects of macrophages alone or primed with cytokines on the biological characteristics of Balamuthia in vitro. Using human brain microvascular endothelial cells (HBMEC), which constitutes the BBB, we have shown that Balamuthia demonstrated <90% binding and <70% cytotoxicity to host cells. However, macrophages further increased amoebic binding and Balamuthia-mediated cell cytotoxicity. Furthermore macrophages exhibited no amoebicidal effect against Balamuthia. Zymography assay demonstrated that macrophages exhibited no inhibitory effect on proteolytic activity of Balamuthia. Overall we have shown for the first time macrophages has no inhibitory effects on the biological properties of Balamuthia in vitro. This also strengthened the concept that how and why Balamuthia can cause infections in both immuno-competent and immuno-compromised individuals.

Detection of serum antibodies in children and adolescents against Balamuthia mandrillaris, Naegleria fowleri and Acanthamoeba T4.

The presence of free-living amoebae of the genera Naegleria, Acanthamoeba and Balamuthia, which contain pathogenic species for humans and animals, has been demonstrated several times and in different natural aquatic environments in the northwest of Mexico. With the aim of continuing the addition of knowledge about immunology of pathogenic free-living amoebae, 118 sera from children and adolescents, living in three villages, were studied. Humoral IgG response against B. mandrillaris, N. fowleri and Acanthamoeba sp. genotype T4, was analyzed in duplicate to titers 1: 100 and 1: 500 by enzyme-linked immunosorbent assay (ELISA). Children and adolescents ages ranged between 5 and 16 years old, with a mean of 9 years old, 55% males. All tested sera were positive for the 1: 100 dilution, and in the results obtained with the 1: 500 dilution, 116 of 118 (98.3%) were seropositive for N. fowleri, 101 of 118 (85.6%) were seropositive for Acanthamoeba sp. genotype T4, and 43 of 118 (36.4%) were seropositive for B. mandrillaris. The statistical analysis showed different distributions among the three communities and for the three species of pathogenic free-living amoebae, including age. Lysed and complete cells used as Balamuthia antigens gave differences in seropositivity.

Excretory and Secretory Proteins of Naegleria fowleri Induce Inflammatory Responses in BV-2 Microglial Cells.

Naegleria fowleri, a free-living amoeba that is found in diverse environmental habitats, can cause a type of fulminating hemorrhagic meningoencephalitis, primary amoebic meningoencephalitis (PAM), in humans. The pathogenesis of PAM is not fully understood, but it is likely to be primarily caused by disruption of the host's nervous system via a direct phagocytic mechanism by the amoeba. Naegleria fowleri trophozoites are known to secrete diverse proteins that may indirectly contribute to the pathogenic function of the amoeba, but this factor is not clearly understood. In this study, we analyzed the inflammatory responses in BV-2 microglial cells induced by excretory and secretory proteins of N. fowleri (NfESP). Treatment of BV-2 cells with NfESP induced the expression of various cytokines and chemokines, including the proinflammatory cytokines IL-1α and TNF-α. NfESP-induced IL-1α and TNF-α expression in BV-2 cells were regulated by p38, JNK, and ERK MAPKs. NfESP-induced IL-1α and TNF-α production in BV-2 cells were effectively downregulated by inhibition of NF-kB and AP-1. These results collectively suggest that NfESP stimulates BV-2 cells to release IL-1α and TNF-α via NF-kB- and AP-1-dependent MAPK signaling pathways. The released cytokines may contribute to inflammatory responses in microglia and other cell types in the brain during N. fowleri infection.

Primary Amoebic Meningoencephalitis: Neurochemotaxis and Neurotropic Preferences of Naegleria fowleri.

Naegleria fowleri causes one of the most devastating necrotic meningoencephalitis in humans. The infection caused by this free-living amoeba is universally fatal within a week of onset of the signs and symptoms of the disease called primary amoebic meningoencephalitis (PAM). In all the affected patients, there is always a history of entry of water into the nose. Even though the diagnostic and treatment protocols have been revised and improved, the obstinate nature of the disease can be gauged by the fact that the mortality rate has persisted around ∼95% over the past 60 years. Some of the unanswered questions regarding PAM are is there a neurochemical basis of the chemotaxis of N. fowleri to the brain? What immune evasion means occurs preceding the neurotropic invasion? What is the contribution of the acute inflammatory response in the fatal cases? Can a combination of anti-amoebic drugs with antagonism of the acute inflammation help save the patient's life? As prevention remains the most valuable safeguard against N. fowleri, a quicker diagnosis, better understanding of the pathogenesis of PAM coupled with testing of newer and safer drugs could improve the chances of survival in patients affected with PAM.

Primary Amoebic Meningoencephalitis.

Primary amoebic meningoencephalitis due to free living amoeba, also called 'brain eating amoeba', Naegleria fowleri, was detected in retroviral disease patient of 40 years who has history of using well water. Patient was admitted with severe headache, fever intermittent, nausea, vomiting and slurring of speech. CT scan and MRI scan findings were normal. CSF examination showed increased protein, low sugar and predominant lymphocytes. CSF was negative for cryptococcal antigen but wet mount preparation showed highly motile free living amoeba Naegleria fowleri. Patient was put on Amphotericin B, Metronidazole, Rifampicin in addition to ART and ATT and other supportive medications. His headache was relieved and patient improved and was discharged on request. Earlier eight cases have been reported from India of which four cases survived the acute episode.

Pathogenesis of amoebic encephalitis: Are the amoebae being credited to an 'inside job' done by the host immune response?

Pathogenic free living amoeba like Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris are known to cause fatal "amoebic meningoencephalitis" by acquiring different route of entries to the brain. The host immune response to these protist pathogens differs from each another, as evidenced by the postmortem gross and microscopic findings from the brains of the affected patients. Cited with the expression of 'brain eating amoeba' when the infection is caused by N. fowleri, this expression is making its way into parasitology journals and books. The impression that it imparts is, as if the brain damage is substantially due to the enzymes and toxins produced by this amoeba. A detailed review of the literature, analysis of archived specimens and with our experimental assays, here we establish that with N. fowleri, Acanthamoeba and Balamuthia spp., the infections result in an extensive brain damage that in fact is substantially caused by the host immune response rather than the amoeba. Due to the comparatively larger sizes of these pathogens and the prior exposure of the amoebal antigen to the human body, the host immune system launches an amplified response that not only breaches the blood brain barrier (BBB), but also becomes the major cause of brain damage in Amoebic meningoencephalitis. It is our understanding that for N. fowleri the host immune response is dominated by acute inflammatory cytokines and that, in cases of Acanthamoeba and Balamuthia spp., it is the type IV hypersensitivity reaction that fundamentally not only contributes to disruption and leakiness of the blood brain barrier (BBB) but also causes the neuronal damage. The further intensification of brain damage is done by toxins and enzymes secreted by the amoeba, which causes the irreversible brain damage.

Protective immunity against Naegleria fowleri infection on mice immunized with the rNfa1 protein using mucosal adjuvants.

The free-living amoeba, Naegleria fowleri, causes a fatal disease called primary amoebic meningoencephalitis (PAM) in humans and experimental animals. Of the pathogenic mechanism of N. fowleri concerning host tissue invasion, the adherence of amoeba to hose cells is the most important. We previously cloned the nfa1 gene from N. fowleri. The protein displayed immunolocalization in the pseudopodia, especially the food-cups structure, and was related to the contact-dependent mechanism of the amoebic pathogenicity in N. fowleri infection. The cholera toxin B subunit (CTB) and Escherichia coli heat-labile enterotoxin B subunit (LTB) have been used as potent mucosal adjuvants via the parenteral route of immunization in most cases. In this study, to examine the effect of protective immunity of the Nfa1 protein for N. fowleri infection with enhancement by CTB or LTB adjuvants, intranasally immunized BALB/c mice were infected with N. fowleri trophozoites for the development of PAM. The mean time to death of mice immunized with the Nfa1 protein using LTB or CTB adjuvant was prolonged by 5 or 8 days in comparison with that of the control mice. In particular, the survival rate of mice immunized with Nfa1 plus CTB was 100% during the experimental period. The serum IgG levels were significantly increased in mice immunized with Nfa1 protein plus CTB or LTB adjuvants. These results suggest that the Nfa1 protein, with CTB or LTB adjuvants, induces strong protective immunity in mice with PAM due to N. fowleri infection.

IL-33 mediates the expressions of IL-5 and IL-13 in Angiostrongylus cantonensis-infected mice.

Angiostrongylus cantonensis is the major cause of human eosinophilic meningoencephalitis. C57BL/6 mice were experimentally infected with 35 infectious larvae. Two groups of infected mice received intraperitoneal injections of mouse IL-33 (1µg) or anti-IL-33 monoclonal antibody (mAb) (10µg) 3days post infection (dpi) and subsequent booster shots of the same dose at 5day intervals. Blood samples from each group were collected weekly for assays. IgE levels were significantly increased in all infected mice. The eosinophil percentage and levels of IL-5 and IL-13 significantly increased in the IL-33-treated group relative to infected but non-treated animals. The level of IL-5 decreased in the mAb-treated group. The severity of eosinophilic meningitis was exacerbated in the IL-33 injected group. Taken together, these results suggest that IL-33 mediates the expressions of IL-5 and IL-13, and plays a crucial role in the pathogenesis of angiostrongylosis.

Vaccination with lentiviral vector expressing the nfa1 gene confers a protective immune response to mice infected with Naegleria fowleri.

Naegleria fowleri, a pathogenic free-living amoeba, causes fatal primary amoebic meningoencephalitis (PAM) in humans and animals. The nfa1 gene (360 bp), cloned from a cDNA library of N. fowleri, produces a 13.1-kDa recombinant protein which is located on pseudopodia, particularly the food cup structure. The nfa1 gene plays an important role in the pathogenesis of N. fowleri infection. To examine the effect of nfa1 DNA vaccination against N. fowleri infection, we constructed a lentiviral vector (pCDH) expressing the nfa1 gene. For the in vivo mouse study, BALB/c mice were intranasally vaccinated with viral particles of a viral vector expressing the nfa1 gene. To evaluate the effect of vaccination and immune responses of mice, we analyzed the IgG levels (IgG, IgG1, and IgG2a), cytokine induction (interleukin-4 [IL-4] and gamma interferon [IFN-γ]), and survival rates of mice that developed PAM. The levels of both IgG and IgG subclasses (IgG1 and IgG2a) in vaccinated mice were significantly increased. The cytokine analysis showed that vaccinated mice exhibited greater IL-4 and IFN-γ production than the other control groups, suggesting a Th1/Th2 mixed-type immune response. In vaccinated mice, high levels of Nfa1-specific IgG antibodies continued until 12 weeks postvaccination. The mice vaccinated with viral vector expressing the nfa1 gene also exhibited significantly higher survival rates (90%) after challenge with N. fowleri trophozoites. Finally, the nfa1 vaccination effectively induced protective immunity by humoral and cellular immune responses in N. fowleri-infected mice. These results suggest that DNA vaccination using a viral vector may be a potential tool against N. fowleri infection.

Incidental consequences of antihelmintic treatment in the central nervous system.

BACKGROUND: Neurocysticercosis (NCC) is the most common parasitic infection in the central nervous system and the most common cause of acquired neurological symptoms in young adults living in developing countries. Many "asymptomatic" patients begin experiencing neurological symptoms after the use of antiparasitic drugs for gastrointestinal treatment. Patients who are previously diagnosed with NCC require special care during cysticidal treatment because of the inflammatory effects caused by the interaction between the drug, the parasite, and the host. CASE DESCRIPTION: Of a series of 46 cases, we selected five patients with a history of being "asymptomatic" and who began experiencing neurologic symptoms after the use of albendazole, which led to a diagnosis of cysticercosis. Another case of the patient, who already had been diagnosed of ventricular cysticercosis, was given a drug treatment without consulting the neurosurgeon and had a fatal outcome attributable to secondary meningoencephalitis. RESULTS: In the first five cases, with new neurological symptoms after antihelmintic treatment, the self-prescription is remarkable. The symptoms appear between the third and fifth day of treatment. All of them had a clinical course without complications. Only two cases minimally invasive techniques were required. The case who had been already diagnosed developed meningoencephalitis and died after eight days of antihelmintic treatment. CONCLUSIONS: Anthelminthic drug treatment requires tailor-based prescription considering risk-benefit ratio with the drug-parasite-host interaction in mind. Treatment is not harmless so patients have to be closely watched. In select cases, medical treatment cannot replace surgical procedures, which can be the primary approach with drug treatment as a complement.

Effects of immunization with the rNfa1 protein on experimental Naegleria fowleri-PAM mice.

Free-living Naegleria fowleri causes primary amoebic meningoencephalitis (PAM) in humans and animals. To examine the effect of immunization with Nfa1 protein on experimental murine PAM because of N. fowleri, BALB/c mice were intra-peritoneally or intra-nasally immunized with a recombinant Nfa1 protein. We analysed Nfa1-specific antibody and cytokine induction, and the mean survival time of infected mice. Mice immunized intra-peritoneally or intra-nasally with rNfa1 protein developed specific IgG, IgA and IgE antibodies; the IgG response was dominated by IgG1, followed by IgG2b, IgG2a and IgG3. High levels of the Th1 cytokine, IFN-γ, and the regulatory cytokine, IL-10, were also induced. The mean survival time of mice immunized intra-peritoneally with rNfa1 protein was prolonged compared with controls, (25.0 and 15.5 days, respectively). Similarly, the mean survival time of mice immunized intra-nasally with rNfa1 protein was 24.7 days, compared with 15.0 days for controls.

Acute granulomatous acanthamoeba encephalitis in an immunocompetent patient.

BACKGROUND: Acanthamoeba sp. are known to cause fatal granulomatous Acanthamoeba encephalitis (GAE) in immunocompromised patients. METHODS: The case of a 17 year old immunocompetent patient with acute purulent meningoencephalitis is reported. RESULTS: After substantial improvement of cerebrospinal fluid under conventional antimicrobial treatment the patient was suffering several relapses. The causative agent was identified as Acanthamoeba lenticulata T5 which entered the CNS through a bone dehiscence of ethmoidal cells. Due to severe vasospasm the patient developed multiple strokes, which led to marked neurologic sequels. CONCLUSIONS: This case report demonstrates successful treatment of usually lethal GAE in an immunocompetent patient with high dose meropenem, linezolid, moxifloxacin and fluconazole followed by a partially adapted antimicrobial combination therapy.

African trypanosome infections of the nervous system: parasite entry and effects on sleep and synaptic functions.

The extracellular parasite Trypanosoma brucei causes human African trypanosomiasis (HAT), also known as sleeping sickness. Trypanosomes are transmitted by tsetse flies and HAT occurs in foci in sub-Saharan Africa. The disease, which is invariably lethal if untreated, evolves in a first hemo-lymphatic stage, progressing to a second meningo-encephalitic stage when the parasites cross the blood-brain barrier. At first, trypanosomes are restricted to circumventricular organs and choroid plexus in the brain outside the blood-brain barrier, and to dorsal root ganglia. Later, parasites cross the blood-brain barrier at post-capillary venules, through a multi-step process similar to that of lymphocytes. Accumulation of parasites in the brain is regulated by cytokines and chemokines. Trypanosomes can alter neuronal function and the most prominent manifestation is represented by sleep alterations. These are characterized, in HAT and experimental rodent infections, by disruption of the sleep-wake 24h cycle and internal sleep structure. Trypanosome infections alter also some, but not all, other endogenous biological rhythms. A number of neural pathways and molecules may be involved in such effects. Trypanosomes secrete prostaglandins including the somnogenic PGD2, and they interact with the host's immune system to cause release of pro-inflammatory cytokines. From the sites of early localization of parasites in the brain and meninges, such molecules could affect adjacent brain areas implicated in sleep-wakefulness regulation, including the suprachiasmatic nucleus and its downstream targets, to cause the changes characteristic of the disease. This raises challenging issues on the effects of cytokines on synaptic functions potentially involved in sleep-wakefulness alterations.

Naegleria fowleri induces MUC5AC and pro-inflammatory cytokines in human epithelial cells via ROS production and EGFR activation.

Naegleria fowleri is an amoeboflagellate responsible for the fatal central nervous system (CNS) disease primary amoebic meningoencephalitis (PAM). This amoeba gains access to the CNS by invading the olfactory mucosa and crossing the cribriform plate. Studies using a mouse model of infection have shown that the host secretes mucus during the very early stages of infection, and this event is followed by an infiltration of neutrophils into the nasal cavity. In this study, we investigated the role of N. fowleri trophozoites in inducing the expression and secretion of airway mucin and pro-inflammatory mediators. Using the human mucoepidermal cell line NCI-H292, we demonstrated that N. fowleri induced the expression of the MUC5AC gene and protein and the pro-inflammatory mediators interleukin-8 (IL-8) and interleukin-1 beta (IL-1 beta), but not tumour necrosis factor-alpha or chemokine c-c motif ligand 11 (eotaxin). Since the production of reactive oxygen species (ROS) is a common phenomenon involved in the signalling pathways of these molecules, we analysed if trophozoites were capable of causing ROS production in NCI-H292 cells by detecting oxidation of the fluorescent probe 2,7-dichlorofluorescein diacetate. NCI-H292 cells generated ROS after 15-30 min of trophozoite stimulation. Furthermore, the expression of MUC5AC, IL-8 and IL-1 beta was inhibited in the presence of the ROS scavenger DMSO. In addition, the use of an epidermal growth factor receptor inhibitor decreased the expression of MUC5AC and IL-8, but not IL-1 beta. We conclude that N. fowleri induces the expression of some host innate defence mechanisms, such as mucin secretion (MUC5AC) and local inflammation (IL-8 and IL-1 beta) in respiratory epithelial cells via ROS production and suggest that these innate immune mechanisms probably prevent most PAM infections.

Astrocytic and microglial response and histopathological changes in the brain of horses with experimental chronic Trypanosoma evansi infection.

This study aimed to characterize astrocytic and microglial response in the central nervous system (CNS) of equines experimentally infected with T. evansi. The experimental group comprised males and females with various degrees of crossbreeding, ages between four and seven years. The animals were inoculated intravenously with 10(6) trypomastigotes of T. evansi originally isolated from a naturally infected dog. All equines inoculated with T. evansi were observed until they presented symptoms of CNS disturbance, characterized by motor incoordination of the pelvic limbs, which occurred 67 days after inoculation (DAI) and 124 DAI. The animals in the control group did not present any clinical symptom and were observed up to the 125th DAI. For this purpose the HE histochemical stain and the avidin biotin peroxidase method was used. Lesions in the CNS of experimentally infected horses were those of a wide spread non suppurative meningoencephalomyelitis.The severity of lesions varied in different parts of the nervous system, reflecting an irregular distribution of inflammatory vascular changes. The infiltration of mononuclear cells was associated with anisomorphic gliosis and reactive microglia was identified. The intensity of the astrocytic response in the CNS of the equines infected by T. evansi characterizes the importance of the performance of these cells in this trypanosomiasis. The characteristic gliosis observed in the animals in this experiment suggests the ability of these cells as mediators of immune response. The parasite, T. evansi, was not identified in the nervous tissues.