Neurotoxic reactive astrocytes induce cell death via saturated lipids.

Astrocytes regulate the response of the central nervous system to disease and injury and have been hypothesized to actively kill neurons in neurodegenerative disease1-6. Here we report an approach to isolate one component of the long-sought astrocyte-derived toxic factor5,6. Notably, instead of a protein, saturated lipids contained in APOE and APOJ lipoparticles mediate astrocyte-induced toxicity. Eliminating the formation of long-chain saturated lipids by astrocyte-specific knockout of the saturated lipid synthesis enzyme ELOVL1 mitigates astrocyte-mediated toxicity in vitro as well as in a model of acute axonal injury in vivo. These results suggest a mechanism by which astrocytes kill cells in the central nervous system.

Exploring Antifouling Activity of Biosurfactants Producing Marine Bacteria Isolated from Gulf of California.

Biofouling causes major problems and economic losses to marine and shipping industries. In the search for new antifouling agents, marine bacteria with biosurfactants production capability can be an excellent option, due to the amphipathic surface-active characteristic that confers antimicrobial and antibiofilm activities. The aim of this study was to evaluate the antifouling activity of biosurfactants producing marine bacteria from the Gulf of California. The cell free culture supernatant (CFCS) of Bacillus niabensis (S-69), Ralstonia sp. (S-74) (isolated from marine sediment) and of B. niabensis (My-30) (bacteria associated to the sponge Mycale ramulosa) were screened for production of biosurfactants (using hemolysis and drop collapse test, oil displacement and emulsifying activity). The toxicity and antifouling activity were evaluated against biofoulers (bacteria forming biofilm and macrofoulers) both in laboratory and field assays. The results indicate that all bacteria were biosurfactant producers, but the higher capability was shown by B. niabensis (My-30) with high emulsifying properties (E24) of 71%. The CFCS showed moderate toxicity but were considered non-toxic against Artemia franciscana at low concentrations. In the antifouling assay, the CFCS of both strains of B. niabensis showed the best results for the reduction of the biofilm formation (up 50%) against all Gram-positive bacteria and most Gram-negative bacteria with low concentrations. In the field assay, the CFCS of B. niabensis (My-30) led to the reduction of 30% of biofouling compared to the control. The results indicate that the biosurfactant produced by B. niabensis (My-30) has promising antifouling activity.

Open chromatin landscape of rat microglia upon proinvasive or inflammatory polarization.

Microglia are brain-resident, myeloid cells that play important roles in health and brain pathologies. Herein, we report a comprehensive, replicated, false discovery rate-controlled dataset of DNase-hypersensitive (DHS) open chromatin regions for rat microglia. We compared the open chromatin landscapes in untreated primary microglial cultures and cultures stimulated for 6 hr with either glioma-conditioned medium (GCM) or lipopolysaccharide (LPS). Glioma-secreted factors induce proinvasive and immunosuppressive activation of microglia, and these cells then promote tumor growth. The open chromatin landscape of the rat microglia consisted of 126,640 reproducible DHS regions, among which 2,303 and 12,357 showed a significant change in openness following stimulation with GCM or LPS, respectively. Active genes exhibited constitutively open promoters, but there was no direct dependence between the aggregated openness of DHS regions near a gene and its expression. Individual regions mapped to the same gene often presented different patterns of openness changes. GCM-regulated DHS regions were more frequent in areas away from gene bodies, while LPS-regulated regions were more frequent in introns. GCM and LPS differentially affected the openness of regions mapped to immune checkpoint genes. The two treatments differentially affected the aggregated openness of regions mapped to genes in the Toll-like receptor signaling and axon guidance pathways, suggesting that the molecular machinery used by migrating microglia is similar to that of growing axons and that modulation of these pathways is instrumental in the induction of proinvasive polarization of microglia by glioma. Our dataset of open chromatin regions paves the way for studies of gene regulation in rat microglia.

Inflammation-Induced Photoreceptor Cell Death.

Neuroinflammation is an important aspect of many diseases of the eye, and experimental animal models have been widely used to determine its impact on retinal homeostasis and neuron survival. Physical separation of the neurosensory retina from the underlying retinal pigment epithelium (RPE) results in activation and infiltration of macrophages. Numerous studies have shown the critical role of macrophages in retinal disease processes. In retinal detachment, accumulation of macrophages in the subretinal space is associated with changes in cytokine and chemokine profile which lead to photoreceptor cell death. Targeted disruption of macrophage chemotaxis significantly reduces retinal detachment-induced photoreceptor degeneration. Apoptosis is the predominant mechanism of cell death; however regulated necrosis is also a contributor of photoreceptor loss. Therefore, effective neuroprotective approaches could integrate combined inhibition of both apoptotic and regulated necrosis pathways.

Teriflunomide and monomethylfumarate target HIV-induced neuroinflammation and neurotoxicity.

HIV-associated neurocognitive disorders (HAND) affect about 50% of infected patients despite combined antiretroviral therapy (cART). Ongoing compartmentalized inflammation mediated by microglia which are activated by HIV-infected monocytes has been postulated to contribute to neurotoxicity independent from viral replication. Here, we investigated effects of teriflunomide and monomethylfumarate on monocyte/microglial activation and neurotoxicity. Human monocytoid cells (U937) transduced with a minimal HIV-Vector were co-cultured with human microglial cells (HMC3). Secretion of pro-inflammatory/neurotoxic cytokines (CXCL10, CCL5, and CCL2: p < 0.001; IL-6: p < 0.01) by co-cultures was strongly increased compared to microglia in contact with HIV-particles alone. Upon treatment with teriflunomide, cytokine secretion was decreased (CXCL10, 3-fold; CCL2, 2.5-fold; IL-6, 2.2-fold; p < 0.001) and monomethylfumarate treatment led to 2.9-fold lower CXCL10 secretion (p < 0.001). Reduced toxicity of co-culture conditioned media on human fetal neurons by teriflunomide (29%, p < 0.01) and monomethylfumarate (27%, p < 0.05) indicated functional relevance. Modulation of innate immune functions by teriflunomide and monomethylfumarate may target neurotoxic inflammation in the context of HAND.

A novel, potent dual inhibitor of Arg-gingipains and Lys-gingipain as a promising agent for periodontal disease therapy.

The periodontal pathogen Porphyromonas gingivalis produces a unique class of cysteine proteinases termed gingipains that comprises Arg-gingipain (Rgp) and Lys-gingipain (Kgp). Growing evidence indicates that these 2 types of gingipains synergistically contribute to the entire virulence of the organism and increase the risk of periodontal disease (PD) by disrupting the host immune system and degrading the host tissue and plasma proteins. Therefore, a dual inhibitor of both gingipains would have attractive clinical potential for PD therapy. In this study, a novel, potent, dual inhibitor of Rgp and Kgp was developed through structure-based drug design, and its biological potency was evaluated in vitro and in vivo. This inhibitor had low nanomolar inhibitory potency (Ki=40 nM for Rgp, Ki=0.27 nM for Kgp) and good selectivity for host proteases and exhibited potent antibacterial activity against P. gingivalis by abrogating its manifold pathophysiological functions. The therapeutic potential of this inhibitor in vivo was also verified by suppressing the vascular permeability that was enhanced in guinea pigs by the organism and the gingival inflammation in beagle dog PD models. These findings suggest that a dual inhibitor of Rgp and Kgp would exhibit noteworthy anti-inflammatory activity in the treatment of PD.

Crosstalk Between Co-cultured A549 Cells and THP1 Cells Exposed to Cigarette Smoke.

Cigarette smoke (CS) is considered as a major etiological factor in the pathogenesis of chronic obstructive pulmonary disease. In this study we used A549 cells and THP-1 cells grown for 24 h in monoculture or in co-culture in CS-conditioned media and changes in their proliferation, viability, acetylated histone H3 levels and expression of extracellular antigens CD14, HLA-DR, CD11a, and CD11b were assessed. CS was highly toxic to A549 cells but not to THP1 cells. In A549 cells, oxidative stress reached the highest values after 1 h of CS exposure and then decreased. In THP1 cells oxidative stress was lower and increased progressively with time. CS decreased proliferation of A549 and THP1 cells by about 80% and 21%, respectively. CS did not alter acetylated histone H3 levels in A549 cells, while in THP1 cells the levels were reduced by about 35%. CS significantly increased expression of CD14, HLA-DR, CD11a, and CD11b in THP1 cells. In co-culture, naïve or CS-pretreated THP1 cells significantly protected A549 cells against CS toxicity but had higher death rates. These results show that epithelial cells are more fragile to CS than monocytes and that CS-activated monocytes may protect epithelial cells against CS-induced cytotoxicity.

Calpain-mediated degradation of MDMx/MDM4 contributes to HIV-induced neuronal damage.

Neuronal damage in HIV-associated Neurocognitive Disorders (HAND) has been linked to inflammation induced by soluble factors released by HIV-infected, and non-infected, activated macrophages/microglia (HIV M/M) in the brain. It has been suggested that aberrant neuronal cell cycle activation determines cell fate in response to these toxic factors. We have previously shown increased expression of cell cycle proteins such as E2F1 and phosphorylated pRb in HAND midfrontal cortex in vivo and in primary neurons exposed to HIV M/M supernatants in vitro. In addition, we have previously shown that MDMx (also referred to as MDM4), a negative regulator of E2F1, was decreased in the brain in a primate model of HIV-induced CNS neurodegeneration. Thus, we hypothesized that MDMx provides indirect neuroprotection from HIV-induced neurodegeneration in our in vitro model. In this report, we found significant reductions in MDMx protein levels in the mid-frontal cortex of patients with HAND. In addition, treatment of primary rat neuroglial cultures with HIV M/M led to NMDA receptor- and calpain-dependent degradation of MDMx and decreased neuronal survival, while overexpression of MDMx conferred partial protection from HIV M/M toxicity in vitro. Further, our results demonstrate that MDMx is a novel and direct calpain substrate. Finally, blocking MDMx activity led to neuronal death in vitro in the absence of toxic stimulus, which was reversed by calpain inhibition. Overall, our results indicate that MDMx plays a pro-survival role in neurons, and that strategies to stabilize and/or induce MDMx can provide neuroprotection in HAND and in other neurodegenerative diseases where calpain activation contributes to neuropathogenesis.

Mutant SOD1-expressing astrocytes release toxic factors that trigger motoneuron death by inducing hyperexcitability.

Amyotrophic lateral sclerosis (ALS) is a devastating paralytic disorder caused by dysfunction and degeneration of motoneurons starting in adulthood. Recent studies using cell or animal models document that astrocytes expressing disease-causing mutations of human superoxide dismutase 1 (hSOD1) contribute to the pathogenesis of ALS by releasing a neurotoxic factor(s). Neither the mechanism by which this neurotoxic factor induces motoneuron death nor its cellular site of action has been elucidated. Here we show that acute exposure of primary wild-type spinal cord cultures to conditioned medium derived from astrocytes expressing mutant SOD1 (ACM-hSOD1(G93A)) increases persistent sodium inward currents (PC(Na)), repetitive firing, and intracellular calcium transients, leading to specific motoneuron death days later. In contrast to TTX, which paradoxically increased twofold the amplitude of calcium transients and killed motoneurons, reduction of hyperexcitability by other specific (mexiletine) and nonspecific (spermidine and riluzole) blockers of voltage-sensitive sodium (Na(v)) channels restored basal calcium transients and prevented motoneuron death induced by ACM-hSOD1(G93A). These findings suggest that riluzole, the only FDA-approved drug with known benefits for ALS patients, acts by inhibiting hyperexcitability. Together, our data document that a critical element mediating the non-cell-autonomous toxicity of ACM-hSOD1(G93A) on motoneurons is increased excitability, an observation with direct implications for therapy of ALS.

Dual mechanism for Mycobacterium tuberculosis cytotoxicity on lung epithelial cells.

Mycobacterium tuberculosis strains CDC1551 and Erdman were used to assess cytotoxicity in infected A549 human alveolar epithelial cell monolayers. Strain CDC1551 was found to induce qualitatively greater disruption of A549 monolayers than was strain Erdman, although total intracellular and cell-associated bacterial growth rates over the course of the infections were not significantly different. Cell-free culture supernatants from human monocytic cells infected with either of the 2 M. tuberculosis strains produced a cytotoxic effect on A549 cells, correlating with the amount of tumor necrosis factor alpha (TNF-α) released by the infected monocytes. The addition of TNF-α-neutralizing antibodies to the supernatants from infected monocyte cultures did prevent the induction of a cytotoxic effect on A549 cells overlaid with this mixture but did not prevent the death of epithelial cells when added prior to infection with M. tuberculosis bacilli. Thus, these data agree with previous observations that lung epithelial cells infected with M. tuberculosis bacilli are rapidly killed in vitro. In addition, the data indicate that some of the observed epithelial cell killing may be collateral damage; the result of TNF-α released from M. tuberculosis-infected monocytes.

Oral toxicity of Photorhabdus culture media on gene expression of the adult sweetpotato whitefly, Bemisia tabaci.

The oral toxicity of culture media of the symbiotic bacteria, Photorhabdus temperata, mutually associated with entomopathogenic nematode Heterorhabditis megidis and Photorhabdus luminescens ssp. laumondii (TT01) mutually associated with Heterorhabditis bacteriophora, were investigated in the adults of Bemisia tabaci. The oral ingestion of sucrose diet solutions (20%) containing bacteria-free supernatant of the culture media from symbiotic bacteria gradually increased mortalities and was completely lethal at 60 h after the treatments, whereas the mortalities of the controls, sucrose solutions with or without media that uncultured with bacteria, were less than 17% up to 84 h of incubation. The effects of oral ingestion of symbiont culture media were demonstrated on the expression rates of several genes of B. tabaci using quantitative real-time RT-PCR analysis. Genes associated with immunity (knottin) and nervous system (acetylcholine receptor, acetylcholine esterase and sodium channel) were up-regulated while genes involved in metabolism (cytochromep450 and carboxylesterase) were down-regulated, but genes involved in development (ecdysone receptor), reproduction (vitellogenin) and stress (hsp70, hsp90 and shsp) did not change transcription rates. Our results provide information for the understanding of the mechanism of symbiont pathogenic factors for the manipulation of host physiology at the transcription level.

In vitro effects of Choukroun's platelet-rich fibrin conditioned medium on 3 different cell lines implicated in dental implantology.

OBJECTIVE: The aim of this work was to determine the relevance of Choukroun's platelet-rich fibrin (PRF) in dental implantology by determining the in vitro effects of soluble factors released by PRF clot. We used 3 different cell lines implicated in dental implantology: osteoblast, keratinocyte, and fibroblast. METHODS: Cellular viability, cell proliferation, and gene expression were analyzed using PRF conditioned medium. Three different cells lines were used: SaOS2 (osteoblast), MRC5 (fibroblast), and KB (epithelial cell). RESULTS: The sulforhodamine B assay showed a significant increase in cell number for the undiluted and 1:3 diluted conditioned medium after 24 and 48 hours. There was no effect for the 1:9 dilution. Cell cycle analysis by flow cytometry confirmed the viability test results. After 48 hours, PRF conditioned medium induced gene expression in osteoblasts. Expression of osteopontin and osteocalcin, late osteogenic markers, was observed using reverse transcriptase-polymerase chain reaction (RT-PCR). CONCLUSIONS: This study establishes a model to evaluate, in vitro, the effects of soluble growth factors released by PRF clot. Our work confirmed PRF is useful in stimulating tissue healing and bone regeneration. This work should recommend Choukroun's PRF in numerous implantology clinical applications.

Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons.

Mutations in superoxide dismutase-1 (SOD1) cause a form of the fatal paralytic disorder amyotrophic lateral sclerosis (ALS), presumably by a combination of cell-autonomous and non-cell-autonomous processes. Here, we show that expression of mutated human SOD1 in primary mouse spinal motor neurons does not provoke motor neuron degeneration. Conversely, rodent astrocytes expressing mutated SOD1 kill spinal primary and embryonic mouse stem cell-derived motor neurons. This is triggered by soluble toxic factor(s) through a Bax-dependent mechanism. However, mutant astrocytes do not cause the death of spinal GABAergic or dorsal root ganglion neurons or of embryonic stem cell-derived interneurons. In contrast to astrocytes, fibroblasts, microglia, cortical neurons and myocytes expressing mutated SOD1 do not cause overt neurotoxicity. These findings indicate that astrocytes may play a role in the specific degeneration of spinal motor neurons in ALS. Identification of the astrocyte-derived soluble factor(s) may have far-reaching implications for ALS from both a pathogenic and therapeutic standpoint.

[Effects of Staphylococcus aureus supernatant on expression of various cytokines by neutrophils and retinal pigment epithelium cells].

OBJECTIVE: To evaluate the effects of Staphylococcus aureus supernatant on the levels of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in co-cultures of neutrophils and retinal pigment epithelium (RPE) cells. METHODS: The co-culture system was established by co-culturing of human RPE cell line D407 and human peripheral blood neutrophils. Bacterium-free supernatant of Staphylococcus aureus ATCC29213 was added to the co-culture system for studying its effects. The volume of bacterium-free supernatant was divided into six groups: negative control, brain-heart infusion control, 50 µl, 100 µl, 250 µl and 500 µl group. The number of neutrophils was divided into four groups: negative control, 1 × 10(4), 5 × 10(4) and 5 × 10(5) group. The supernatants was collected at 6 h, 12 h, 24 h, 48 h and 72 h later and the levels of IL-1ß, TNF-α and IL-6 were measured by ELISA kits. RESULTS: When RPE cells were cultured with different doses of bacterium-free supernatant (0, 50, 100, 250 and 500 µl), the levels of IL-1ß was positively correlated with the volume of bacterium-free supernatant and the duration. The levels of IL-6 were significantly higher than that of the control group in the 500 µl group at 24 h [(23.17 ± 3.16) ng/L vs (7.61 ± 1.53) ng/L] and 48 h [(35.00 ± 4.37) ng/L vs (13.17 ± 3.27) ng/L] duration (P = 0.001 and P = 0.026, respectively). When RPE cells were co-cultured with the bacterium-free supernatant and the neutrophils (1 × 10(4), 5 × 10(4) and 5 × 10(5) cells) for 6 and 12 h, the levels of IL-1ß in the 5 × 10(5) group at both 6 h [(236.62 ± 8.20) ng/L] and 12 h [(447.42 ± 35.13) ng/L] was statistically higher than that in other groups (6 h: P = 0.000, P = 0.000, P = 0.002; 12 h: P = 0.000, P = 0.000, P = 0.000, respectively). The levels of IL-6 in the 5 × 10(5) group [(46.96 ± 2.72) ng/L] was significantly higher than that in the other groups at 12 h (P = 0.000, P = 0.000, P = 0.000, respectively). TNF-α could not be detected in the conditioned media from all cultures. CONCLUSIONS: Both IL-1ß and IL-6 are expressed in the co-cultures of neutrophils and RPE cells with Staphylococcus aureus supernatant. IL-1ß is upregulated at the early stage and the high level is maintained longer than that of IL-6. The virulence factor of bacteria and the neutrophil may play a role in the expression of IL-1ß and IL-6 in the RPE cells.

Conditioned Medium from Cells Overexpressing TDP-43 Alters the Metabolome of Recipient Cells.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the progressive death of both upper and lower motor neurons. The disease presents a poor prognosis, and patients usually die 2-5 years after the onset of symptoms. The hallmark of this disease is the presence of phosphorylated and ubiquitinated aggregates containing trans-active response DNA-binding protein-43 (TDP-43) in the cytoplasm of motor neurons. TDP-43 pathology has been associated with multiple pathways in ALS, such as metabolic dysfunction found in patients and in in vivo models. Recently, it has been described as a "prion-like" protein, as studies have shown its propagation in cell culture from ALS brain extract or overexpressed TDP-43 in co-culture and conditioned medium, resulting in cytotoxicity. However, the cellular alterations that are associated with this cytotoxicity require further investigation. Here, we investigated the effects of conditioned medium from HEK293T (Human Embryonic Kidney 293T) cells overexpressing TDP-43 on cellular morphology, proliferation, death, and metabolism. Although we did not find evidence of TDP-43 propagation, we observed a toxicity of TDP-43-conditioned medium and altered metabolism. These results, therefore, suggest (1) that cells overexpressing TDP-43 produce an extracellular environment that can perturb other cells and (2) that TDP-43 propagation alone may not be the only potentially cytotoxic cell-to-cell mechanism.

Conditioned medium from mesenchymal stem cells induces cell death in organotypic cultures of rat hippocampus and aggravates lesion in a model of oxygen and glucose deprivation.

Cell therapy using bone marrow-derived mesenchymal stem cells (MSC) seems to be a new alternative for the treatment of neurological diseases, including stroke. In order to investigate the response of hippocampal tissue to factors secreted by MSC and if these factors are neuroprotective in a model of oxygen and glucose deprivation (OGD), we used organotypic hippocampal cultures exposed to conditioned medium from bone marrow-derived MSC. Our results suggest that the conditioned medium obtained from these cells aggravates lesion caused by OGD. In addition, the presence of the conditioned medium alone was toxic mainly to cells in the CA1, CA2 and CA3 areas of the hippocampal organotypic culture even in basal conditions. GABA stimulation and NMDA and AMPA receptors antagonists were able to reduce propidium iodide staining, suggesting that the cell death induced by the toxic factors secreted by MSC could involve these receptors.

Culture supernatants from V. cholerae O1 El Tor strains isolated from different geographic areas induce cell vacuolation and cytotoxicity.

OBJECTIVE: To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. MATERIAL AND METHODS: Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. RESULTS: All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+) and a non-toxigenic Mexican strain (CM 91-3, ctxAB-). Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. CONCLUSION: The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.

Cytotoxicity of orthodontic temporary anchorage devices on human periodontal ligament fibroblasts in vitro.

Objectives: The objective of this study is to test cytotoxicity of four brands of commercially available orthodontic temporary anchorage devices (TADs). Setting and sample population: Twenty-four (six for each brand, i.e., Aarhus [AO]; Dual top [RMO]; Vector TAS [ORMCO]; and Unitek TAD [3M UNITEK]) TADs were tested. Materials and methods: Twenty-four (six for each brand, i.e., Aarhus [AO]; Dual top [RMO]; Vector TAS [ORMCO]; and Unitek TAD [3M UNITEK]) TADs were individually incubated in complete cell culture medium and shaken at a rate of 1.5 rpm at 37°C for 30 days to extract possible toxic substances in conditioned media (CM). To test cytotoxicity, human periodontal ligament fibroblasts were cultured and exposed to the CM for 24 hr, followed by the examinations of morphological changes, cell viability (MTT assay), and cell damage (lactate dehydrogenase [LDH] assay). Results: No morphological changes were observed in any of the four brands of TADs compared with the negative control. LDH assay showed that none of the four brands of TADs caused significant cell damage after CM treatment compared with the negative control (P > .05). No significant differences were found between any of the four brands of TADs (P > .05). MTT assay showed similar results as did the LDH assay, except for a statistically significant difference found in the TADs from 3M UNITEK compared with the negative control (P = .047). Conclusions: According to the International Standard Organization standards, except for the TAD from 3M, none of the other three brands of commercially available TADs (from AO, RMO, and ORMCO) exhibited significant cytotoxicity, suggesting their safe clinical applications.

Mechanisms of whole smoke conditioned media induced cytotoxicity to human aortic endothelial cells.

Chronic exposure to cigarette smoke can lead to endothelial dysfunction and potentially endothelial cell death. Here, we exposed Human Aortic Endothelial Cells (HAECs) to whole smoke conditioned media (WSCM) over a range of nicotine equivalence (n.e.) concentrations (0-8000 ng/mL n.e.). After 24 h, Neutral Red Uptake (NRU) and reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan was determined for each exposure concentration and compared to control. IC50 values in the NRU assay were: 4582 ng/mL n.e. ± 1074, 4587 ng/mL n.e. ± 951, 4993 ng/mL n.e. ± 1239 and 4691 ng/mL n.e. ± 402 for four HAEC donors. IC50 values in the MTT assay were: 4885 ng/mL n.e. ± 1341, 4584 ng/mL n.e. ± 806, 5749 ng/mL n.e. ± 783 and 5228 ng/mL n.e. ± 593 for the four donors. To examine the mechanism responsible for WSCM-induced cytotoxicity in HAECs, flow cytometry using necrosis (Propidium Iodide) and apoptosis (Annexin V) markers were used. Annexin V-positive cell populations increased in a dose dependent manner while increases in PI-positive cell populations occurred at the highest doses of WSCM (5000-8000 ng/mL n.e.). Western blotting for cleaved caspase-3 confirmed that apoptosis occurs at >5000 ng/mL n.e. WSCM, coinciding with reduced HAEC survival.

Oligomers of the amyloid-beta protein disrupt working memory: confirmation with two behavioral procedures.

Converging lines of evidence suggest that oligomers of amyloid-beta play a role in the cognitive impairment characteristic of Alzheimer's disease, but only three studies have provided experimental evidence of such impairment. To provide additional information about the effects of these oligomers on memory, the present study examined the memory of groups of rats exposed to ICV injections of the culture media (CM) of Chinese Hamster Ovary cells that were (7PA2) and were not (CHO-) transfected with a human mutation of amyloid precursor protein that appears to cause early-onset Alzheimer's disease. The 7PA2 CM, which contained concentrations of soluble amyloid-beta oligomers physiologically relevant to those found in human brain, significantly disrupted working memory in rats tested in a radial-arm maze. In contrast, CHO- CM, which did not contain such oligomers, had no effect on memory. The disruptive effects of 7PA2-derived amyloid-beta oligomers, evident 2h after exposure, disappeared within a day. These findings are compared to results from 7PA2 CM tested under a complex procedure thought to measure aspects of executive function. The results confirm the disruptive effects of low-n amyloid-beta oligomers and extend them to a well-established rat model of memory.