Effects of Neospora caninum infection on brain microvascular endothelial cells bioenergetics

Abstract

BackgroundThe brain is the most commonly affected organ during Neospora caninum infection but the mechanisms utilized by this protozoan parasite for traversal of the blood–brain barrier (BBB) are not yet understood. Herein, we investigate the cellular pathogenicity of N. caninum infection on bioenergetics of human brain microvascular endothelial cells (HBMECs), a fundamental component of the BBB.MethodsWe tracked the growth kinetics of N. caninum in HBMECs. Focusing on cell bioenergetics, oxygen consumption rate (OCR) was determined using Clark electrode system and mitochondrial membrane potential (ΔΨm) was evaluated using DePsipher staining by fluorescence microscopy in the presence and absence of infection.ResultsHBMECs provided a receptive environment for parasite proliferation. N. caninum tachyzoites were able to invade and replicate within HBMECs without significantly altering cell proliferation rate, as measured with the MTT assay, up to 24 hr post infection (pi). The oxygen consumption rate (OCR) was significantly inhibited (p < 0.001) by 10 mM glucose [from −2.26±0.23 to −0.6±0.21 nmol 106 cell min-1 and from −0.29±0.09 to −0.16±0.1 nmol 106 cell min-1 for uninfected HBMECs and free N. Caninum tachyzoites, respectively]. After normalization for DNA content the basal OCR did not differ between two host cell types: HBMECs and K562. The OCR of HBMECs was significantly elevated 24 hr pi in the absence of substrate, in 10 mM glucose and in the presence of a tetramethyl-p-phenylenediamine (TMPD)/ascorbate redox shuttle. Although quantitatively similar results were observed for uninfected K562 cells, there was no effect on their OCR 24 hr pi with N. caninum under any of the above substrate conditions. 6mM azide abolished OCR in all situations. Mitochondrial staining with DePsipher indicated no change in their membrane potential (Δψm) up to 24 hr pi.ConclusionsN. caninum is able to grow in HBMECs without markedly disrupting their normal proliferation or mitochondrial integrity. However, it is associated with an increase in infected cell respiration. Whether this increase reflects numeric addition of the parasites own respiration or results from an additional energy demand upon the host cell remains to be elucidated.

Metallome of cerebrovascular endothelial cells infected with Toxoplasma gondii using μ-XRF imaging and inductively coupled plasma mass spectrometry

“…Human brain microvascular endothelial cells (ECs) were used at passage 9 and were maintained in vitro, as described previously. 16,17 Briefly, ECs were propagated in tissue-culture medium composed of RPMI 1640 medium supplemented with 20% (v/v) heat inactivated FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acids, 1% MEM vitamins, and 2% penicillin/streptomycin (Invitrogen, GIBCO, UK). Cells were maintained in an incubator in a humidified atmosphere at 37 1C and 5% CO 2 .…”

Analysis of interaction between the apicomplexan protozoan Toxoplasma gondii and host cells using label-free Raman spectroscopy

“…HBMECs were grown as described previously. 19 Infected host cell monolayers were scraped, parasites were isolated from host cells by passage through 25-and 27-gauge needles and purified by using PD-10 Desalting Columns prepacked with Sephadex G-25 medium as described previously. 20 Purified parasites were centrifuged at 800 × g, washed twice with fresh cRPMI, re-suspended in fresh medium and quantified using a hemacytometer.…”

Adverse effects of antipsychotics on micro-vascular endothelial cells of the human blood–brain barrier

“…Cells were used from passage 21-23 and were grown and maintained as previously described (Elsheikha et al, 2013). When confluent, our HBMECs demonstrated an endothelial morphology with features all typical of a functional endothelium constructed from this cell type (Wong et al, 2004;Colgan et al, 2008;Allen and Bayraktutan, 2009 …”