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.

Label-free chemical profiling identifies macromolecules significantly associated with Toxoplasma gondii infection in cerebrovascular endothelial cells.

BACKGROUND. The p16 INK4a locus encodes two distinct proteins, p16 INK4a and p14 ARF . Although p16 INK4a and p15 INK4b are involved in the phosphorylation of the retinoblastoma (Rb) protein, p14 ARF interacts with the MDM-2 oncoprotein antagonizing its function as a suppressor of p53. The role of deletions of p16 INK4a /p14 ARF and p15 INK4b and expressions of MDM-2 in myeloid leukemias and its influence on prognosis remain unclear. METHODS. The authors analyzed deletions of p16 INK4 /p14 ARF and p15 INK4b in 74 adults with acute myeloid leukemia (AML) by Southern blotting. Western blotting was used to determine Rb protein phosphorylation in patients with deletions of p16 INK4 /p14 ARF and p15 INK4b . Then, they analyzed the levels of MDM-2 protein expression and correlated it with prognosis in an expanded population of 79 adults with AML by immunoblot analysis and solid-phase radioimmunoassay. RESULTS. Deletions of p16INK4a /p14 ARF and/or p15 INK4b occurred in 4 of 74 patients (5%) (hemizygous in 3, homozygous in 1 patient). Although the complete remission (CR) rate was similar (79% vs. 50%; P ϭ 0.187), CR duration (10 vs. 46 weeks; P Ͻ 0.001), event free survival rate (EFS; 6 vs. 85 weeks; P Ͻ 0.004) and overall survival rate (11 vs. 86 weeks; P ϭ 0.001) were significantly shorter in patients with deletions of p16 INK4a /p14 ARF and/or p15 INK4b . Thirty-seven (47%) of 79 patients studied for MDM-2 showed increased MDM-2 expression. These patients had a significantly shorter EFS rate (50 vs. 64 weeks; P ϭ 0.023) and a trend for shorter CR duration (24 vs. 53 weeks; P ϭ 0.07). Overall survival rate was not significantly different (50 vs. 84 weeks; P ϭ 0.136). CONCLUSIONS.The authors concluded that 1) deletions of p16 INK4a /p14 ARF and/or p15 INK4b occur with low incidence in patients with AML; 2) patients with deletions of p16 INK4a /p14 ARF and/or p15 INK4b have a significantly shorter CR duration, EFS rate, and overall survival rate than do patients without deletions; (3) overexpression of MDM-2 is common in AML and is associated with shorter CR duration and EFS rate. Mechanisms other than p14 ARF deletion are responsible for MDM-2 overexpression, and this overexpression may play a role in the biology of the disease.

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In this work, the effects of the protozoan Neospora caninum on the bioenergetics, chemical composition, and elemental content of human brain microvascular endothelial cells (hBMECs) were investigated. We showed that N. caninum can impair cell mitochondrial (Mt) function and causes an arrest in host cell cycling at S and G2 phases. These adverse effects were also associated with altered expression of genes involved in Mt energy metabolism, suggesting Mt dysfunction caused by N. caninum infection. Fourier Transform Infrared (FTIR) spectroscopy analysis of hBMECs revealed alterations in the FTIR bands as a function of infection, where infected cells showed alterations in the absorption bands of lipid (2924 cm−1), amide I protein (1649 cm−1), amide II protein (1537 cm−1), nucleic acids and carbohydrates (1092 cm−1, 1047 cm−1, and 939 cm−1). By using quantitative synchrotron radiation X-ray fluorescence (μSR-XRF) imaging and quantification of the trace elements Zn, Cu and Fe, we detected an increase in the levels of Zn and Cu from 3 to 24 h post infection (hpi) in infected cells compared to control cells, but there were no changes in the level of Fe. We also used Affymetrix array technology to investigate the global alteration in gene expression of hBMECs and rat brain microvascular endothelial cells (rBMVECs) in response to N. caninum infection at 24 hpi. The result of transcriptome profiling identified differentially expressed genes involved mainly in immune response, lipid metabolism and apoptosis. These data further our understanding of the molecular events that shape the interaction between N. caninum and blood-brain-barrier endothelial cells.