Acanthamoeba interactions with human brain microvascular endothelial cells

Alsam, Selwa; Kim, Kwang Sik; Stins, Monique F.; Rivas, Antonio Ortega; Sissons, James; Khan, Naveed Ahmed

doi:10.1016/j.micpath.2003.07.001

Cited by 102 publications

(95 citation statements)

References 22 publications

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“…Consistent with these findings, we observed that the initial binding of Acanthamoeba to both cell types was mediated by MBP. Moreover, we have recently shown that Acanthamoeba isolates exhibiting higher levels of MBP expression showed increased cytotoxicity to host cells (Alsam et al, 2003), indicating that MBP is a potential marker for the differentiation of pathogenic Acanthamoeba. As indicated above, Acanthamoeba-mediated host cell cytotoxicity did not exhibit tissue specificity.…”

Section: Acanthamoeba Inhibits Prb Phosphorylation Inducing Cell-cycmentioning

confidence: 97%

“…For adhesion assays, Acanthamoeba were metabolically labelled by culturing 5 3 10 6 amoebae ml À1 in PYG medium containing 100 ìCi [ 35 S]methionine (Pharmacia Biotech) at 30 8C for 18 h as described previously (Alsam et al, 2003). Radiolabelled amoebae (.95 % trophozoites) were collected by centrifugation at 750 g for 10 min and resuspended in 20 ml PBS containing 0 .…”

Section: Human Brain Microvascular Endothelial Cells (Hbmec)mentioning

confidence: 99%

“…Primary BMEC from a human origin were isolated and cultured as described previously (Alsam et al, 2003). Briefly, HBMEC were purified by fluorescence-activated cell sorting and their purity was tested using endothelial markers such as expression of F-VIII, carbonic anhydrase IV and uptake of acetylated low-density lipoprotein, resulting in .99 % pure cultures.…”

Section: Human Brain Microvascular Endothelial Cells (Hbmec)mentioning

confidence: 99%

“…Briefly, HBMEC were purified by fluorescence-activated cell sorting and their purity was tested using endothelial markers such as expression of F-VIII, carbonic anhydrase IV and uptake of acetylated low-density lipoprotein, resulting in .99 % pure cultures. HBMEC were routinely grown on rat tail collagen-coated dishes in RPMI 1640 containing 10 % heat-inactivated fetal bovine serum, 10 % Nu-Serum, 2 mM glutamine, 1 mM pyruvate, 100 U penicillin ml À1 , 100 ìg streptomycin ml À1 , non-essential amino acids and vitamins (Alsam et al, 2003).Human corneal epithelial cells (HCEC). Immortalized HCEC were routinely cultured as described previously (Araki-Sasaki et al, 2000).…”

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confidence: 99%

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Acanthamoeba induces cell-cycle arrest in host cells

Sissons

Alsam

Jayasekera

et al. 2004

Journal of Medical Microbiology

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Acanthamoeba can cause fatal granulomatous amoebic encephalitis (GAE) and eye keratitis. However, the pathogenesis and pathophysiology of these emerging diseases remain unclear. In this study, the effects of Acanthamoeba on the host cell cycle using human brain microvascular endothelial cells (HBMEC) and human corneal epithelial cells (HCEC) were determined. Two isolates of Acanthamoeba belonging to the T1 genotype (GAE isolate) and T4 genotype (keratitis isolate) were used, which showed severe cytotoxicity on HBMEC and HCEC, respectively. No tissue specificity was observed in their ability to exhibit binding to the host cells. To determine the effects of Acanthamoeba on the host cell cycle, a cell-cycle-specific gene array was used. This screened for 96 genes specific for host cell-cycle regulation. It was observed that Acanthamoeba inhibited expression of genes encoding cyclins F and G1 and cyclin-dependent kinase 6, which are proteins important for cell-cycle progression. Moreover, upregulation was observed of the expression of genes such as GADD45A and p130 Rb, associated with cell-cycle arrest, indicating cell-cycle inhibition. Next, the effect of Acanthamoeba on retinoblastoma protein (pRb) phosphorylation was determined. pRb is a potent inhibitor of G 1 -to-S cell-cycle progression; however, its function is inhibited upon phosphorylation, allowing progression into S phase. Western blotting revealed that Acanthamoeba abolished pRb phosphorylation leading to cell-cycle arrest at the G 1 -to-S transition. Taken together, these studies demonstrated for the first time that Acanthamoeba inhibits the host cell cycle at the transcriptional level, as well as by modulating pRb phosphorylation using host cell-signalling mechanisms. A complete understanding of Acanthamoeba-host cell interactions may help in developing novel strategies to treat Acanthamoeba infections. INTRODUCTIONAcanthamoeba are the causative agents of life-threatening granulomatous amoebic encephalitis (GAE) in immunocompromised patients (Jones et al., 1975;Martinez, 1987;Di Gregorio et al., 1992;Friedland et al., 1992;Gonzalez et al., 1986;Gordon et al., 1992) and a common amoebic keratitis, a sight-threatening disease of the eye, which is mostly associated with contact-lens use (Chynn et al., 1995;Moore & McCulley, 1989;Wright et al., 1985; Mathers et al., 1996;Niederkorn et al., 1999;Marciano-Cabral & Cabral, 2003;Khan, 2003). In addition, Acanthamoeba have been associated with cutaneous lesions and sinusitis in immunocompromised patients (reviewed by Marciano-Cabral & Cabral, 2003). Over the past few decades, Acanthamoeba infections have remained significant and are on the rise. This is due to increasing populations of contact-lens wearers and immunocompromised patients, as well as improved awareness and detection of these emerging diseases. However, the precise mechanisms associated with the pathogenesis and pathophysiology of Acanthamoeba infection remain unclear. The clinical outcome of Acanthamoeba infection is dependent on the virulence of...

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Section: Acanthamoeba Inhibits Prb Phosphorylation Inducing Cell-cycmentioning

confidence: 97%

Section: Human Brain Microvascular Endothelial Cells (Hbmec)mentioning

confidence: 99%

Section: Human Brain Microvascular Endothelial Cells (Hbmec)mentioning

confidence: 99%

mentioning

confidence: 99%

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Acanthamoeba induces cell-cycle arrest in host cells

Sissons

Alsam

Jayasekera

et al. 2004

Journal of Medical Microbiology

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“…these fatal diseases. Recently, we have developed an in vitro model of the blood-brain barrier by isolating human brain microvascular endothelial cells (HBMECs) (Alsam et al, 2003). Using this model, we have isolated B. mandrillaris from the brain necropsy as well as the post-mortem cerebrospinal fluid (CSF) of an immunocompetent GAE patient.…”

Section: Introductionmentioning

confidence: 99%

Post-mortem culture of Balamuthia mandrillaris from the brain and cerebrospinal fluid of a case of granulomatous amoebic meningoencephalitis, using human brain microvascular endothelial cells

Jayasekera

Sissons

Tucker

et al. 2004

Journal of Medical Microbiology

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The first isolation in the UK of Balamuthia mandrillaris amoebae from a fatal case of granulomatous amoebic meningoencephalitis is reported. Using primary cultures of human brain microvascular endothelial cells (HBMECs), amoebae were isolated from the brain and cerebrospinal fluid (CSF). The cultures showed a cytopathic effect at 20-28 days, but morphologically identifiable B. mandrillaris amoebae were seen in cleared plaques in subcultures at 45 days. The identification of the organism was later confirmed using PCR on Chelex-treated extracts. Serum taken while the patient was still alive reacted strongly with slide antigen prepared from cultures of the post-mortem isolate, and also with those from a baboon B. mandrillaris strain at 1 : 10 000 in indirect immunofluorescence, but with Acanthamoeba castellanii (Neff) at 1 : 160, supporting B. mandrillaris to be the causative agent. If the presence of amoebae in the post-mortem CSF reflects the condition in life, PCR studies on CSF and on biopsies of cutaneous lesions may also be a valuable tool. The role of HBMECs in understanding the interactions of B. mandrillaris with the blood-brain barrier is discussed.

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