Cellular uptake and anti-inflammatory effects of palm oil-derived delta (δ)-tocotrienol in microglia.

Tocopherols long dominated studies on vitamin E, although interest has shifted to tocotrienols. It was previously shown that δ-tocotrienol derived from palm oil reduced nitric oxide released by BV2 microglia as early as 18 h after lipopolysaccharide stimulation. The current study measured δ-tocotrienol uptake by BV2 over a 24 h incubation period and its anti-inflammatory effects on primary microglia. Uptake of 17.5 µg/mL δ-tocotrienol by BV2 microglia began as early as 5 min and rose steeply to 21 ± 3% of the amount administered at 24 h. The amount of δ-tocotrienol retained in the lipopolysaccharide-stimulated microglia at 24 h was 14 ± 2%, with no substantial difference seen in unstimulated microglia. The same δ-tocotrienol regimen reduced nitric oxide levels by 82% at 24 h after lipopolysaccharide stimulation (p < 0.05). This was accompanied by decreased inducible nitric oxide synthase protein expression by 67 ± 5% compared to untreated controls (p < 0.05). In primary microglia, δ-tocotrienol downregulated IL-1ß production, but TNF-α and IL-6 were not affected. δ-Tocotrienol also reduced prostaglandin E2 production by ~78%% and decreased transcription of COX-2 and 5-LOX, but not COX-1. This study showed the anti-inflammatory effects of δ-tocotrienol derived from palm oil and opens up interest for tocotrienol supplementation to reduce the effects of inflammatory conditions.

The influence of serum-supplemented culture media in a transwell migration assay.

Our work cautions against the use of serum-supplemented culture media in a transwell migration assay when using chemoattractants other than FBS. At 24 h, a 5% foetal bovine serum (FBS) gradient caused BV2 microglia to migrate toward the lower compartment of the transwell apparatus. Interestingly, FBS-supplemented media in the absence of a gradient also resulted in notable microglia migration. Serum can therefore confound the interpretation of a transwell migration assay when another chemoattractant is used.

Gene and protein expression profiles of JAK-STAT signalling pathway in the developing brain of the Ts1Cje down syndrome mouse model.

Aims: The JAK-STAT signalling pathway is one of the key regulators of pro-gliogenesis process during brain development. Down syndrome (DS) individuals, as well as DS mouse models, exhibit an increased number of astrocytes, suggesting an imbalance of neurogenic-to-gliogenic shift attributed to dysregulated JAK-STAT signalling pathway. The gene and protein expression profiles of JAK-STAT pathway members have not been characterised in the DS models. Therefore, we aimed to profile the expression of Jak1, Jak2, Stat1, Stat3 and Stat6 at different stages of brain development in the Ts1Cje mouse model of DS. Methods: Whole brain samples from Ts1Cje and wild-type mice at embryonic day (E)10.5, E15, postnatal day (P)1.5; and embryonic cortex-derived neurospheres were collected for gene and protein expression analysis. Gene expression profiles of three brain regions (cerebral cortex, cerebellum and hippocampus) from Ts1Cje and wild-type mice across four time-points (P1.5, P15, P30 and P84) were also analysed. Results: In the developing mouse brain, none of the Jak/Stat genes were differentially expressed in the Ts1Cje model compared to wild-type mice. However, Western blot analyses indicated that phosphorylated (p)-Jak2, p-Stat3 and p-Stat6 were downregulated in the Ts1Cje model. During the postnatal brain development, Jak/Stat genes showed complex expression patterns, as most of the members were downregulated at different selected time-points. Notably, embryonic cortex-derived neurospheres from Ts1Cje mouse brain expressed lower Stat3 and Stat6 protein compared to the wild-type group. Conclusion: The comprehensive expression profiling of Jak/Stat candidates provides insights on the potential role of the JAK-STAT signalling pathway during abnormal development of the Ts1Cje mouse brains.

Lactobacilli-fermented cow's milk attenuated lipopolysaccharide-induced neuroinflammation and memory impairment in vitro and in vivo.

Nutritional interventions are now recommended as strategies to delay Alzheimer's disease (AD) progression. The present study evaluated the neuroprotective effect (anti-inflammation) of lactic acid bacteria (either Lactobacillus fermentum LAB9 or L. casei LABPC) fermented cow's milk (CM) against lipopolysaccharide (LPS)-activated microglial BV2 cells in vitro. The ability of CM-LAB in attenuating memory deficit in LPS-induced mice was also investigated. ICR mice were orally administered with CM-LAB for 28 d before induction of neuroinflammation by LPS. Learning and memory behaviour were assessed using the Morris Water Maze Test. Brain tissues were homogenised for measurement of acetylcholinesterase (AChE), antioxidative, lipid peroxidation (malondialdehyde (MDA)) and nitrosative stress (NO) parameters. Serum was collected for cytokine analysis. CM-LAB9 and CM-LABPC significantly (P < 0·05) decreased NO level but did not affect CD40 expression in vitro. CM-LAB attenuated LPS-induced memory deficit in mice. This was accompanied by significant (P < 0·05) increment of antioxidants (SOD, GSH, GPx) and reduction of MDA, AChE and also pro-inflammatory cytokines. Unfermented cow's milk (UCM) yielded greater cytokine lowering effect than CM-LAB. The present findings suggest that attenuation of LPS-induced neuroinflamation and memory deficit by CM-LAB could be mediated via anti-inflammation through inhibition of AChE and antioxidative activities.

Mesenchymal stem cells: From stem cells to sarcomas.

Mesenchymal stem cells (MSCs) have garnered vast interests in clinical settings, especially in regenerative medicine due to their unique properties-they are reliably isolated and expanded from various tissue sources; they are able to differentiate into mesodermal tissues such as bones, cartilages, adipose tissues, and muscles; and they have unique immunosuppressive properties. However, there are some concerns pertaining to the role of MSCs in the human body. On one hand, they are crucial component in the regeneration and repair of the human body. On the contrary, they are shown to transform into sarcomas. Although the exact mechanisms are still unknown, many new leads have pointed to the belief that MSCs do play a role in sarcomagenesis. This review focuses on the current updates and findings of the role of MSCs in their transformation process into sarcomas.

Isolation and characterization of primary microglia from post-natal murine brain tissues: a comparison of two methods.

Microglia are resident macrophages of the central nervous system (CNS). Apart from playing vital roles as sentinel cells, they are crucial in physiological processes such as synaptic pruning during brain development. CNS disorders require an understanding of the contribution of each cellular compartment to the pathogenesis. Elucidating the role of microglia in disease development and progression in the intricate CNS environment is technically challenging and requires the establishment of reliable, reproducible techniques to isolate and culture microglia. A number of different protocols have been developed for isolation of neonatal microglia and here we compare two widely used methods, namely, mild trypsinization and EasySep® magnetic separation. EasySep® magnetic separation provided higher microglia yield, and flow cytometric evaluation of CD11b and F4/80 markers revealed that EasySep® separation method also produced significantly higher purity compared to mild trypsinization. Microglia isolated using EasySep® separation method were functional, as demonstrated by the generation of nitric oxide, IL-6, TNF-α, and MCP-1 in response to lipopolysaccharide stimulation. In summary, this study has revealed that magnetic separation is superior to mild trypsinization in terms of yield and purity of microglia.

Malaysian endophytic fungal extracts-induced anti-inflammation in Lipopolysaccharide-activated BV-2 microglia is associated with attenuation of NO production and, IL-6 and TNF-α expression.

BACKGROUND: Excessive production of inflammatory mediators such as nitric oxide (NO) and proinflammatory cytokines like tumour necrosis factor-alpha (TNF-α) from activated microglia contributes to uncontrolled inflammation in neurodegenerative diseases. This study investigated the protective role of five endophytic extracts (HAB16R12, HAB16R13, HAB16R14, HAB16R18 and HAB8R24) against LPS-induced inflammatory events in vitro. These endophytic extracts were previously found to exhibit potent neuroprotective effect against LPS-challenged microglial cells. METHODS: The effects of these fungal endophytic extracts against nitric oxide (NO), CD40 phenotype and, pro- and anti-inflammatory cytokine production in lipopolysaccharide (LPS)-stimulated BV2 microglia cells were examined using commercially available assay kits, immunophenotyping and flow cytometry, respectively. RESULTS: Microglia pre-treated with the five endophytic extracts (0.1 mg/mL) reduced NO production without compromising cell viability. Whilst CD40 expression in LPS-stimulated microglia was not significantly different with or without the influence of endophytic extracts, expression of the proinflammatory cytokines, IL-6 and TNF-α in LPS-stimulated microglia was significantly (P < 0.05) inhibited by these endophytic extracts. CONCLUSIONS: The outcomes suggest that the neuroprotective effect of the fungal endophytic extracts is likely mediated through supression of neuroinflammation. To our knowledge, this is the first report of the effect of a fungal endophytic extract in controlling inflammation in BV2 microglia cells.

Functional transcriptome analysis of the postnatal brain of the Ts1Cje mouse model for Down syndrome reveals global disruption of interferon-related molecular networks.

BACKGROUND: The Ts1Cje mouse model of Down syndrome (DS) has partial triplication of mouse chromosome 16 (MMU16), which is partially homologous to human chromosome 21. These mice develop various neuropathological features identified in DS individuals. We analysed the effect of partial triplication of the MMU16 segment on global gene expression in the cerebral cortex, cerebellum and hippocampus of Ts1Cje mice at 4 time-points: postnatal day (P)1, P15, P30 and P84. RESULTS: Gene expression profiling identified a total of 317 differentially expressed genes (DEGs), selected from various spatiotemporal comparisons, between Ts1Cje and disomic mice. A total of 201 DEGs were identified from the cerebellum, 129 from the hippocampus and 40 from the cerebral cortex. Of these, only 18 DEGs were identified as common to all three brain regions and 15 were located in the triplicated segment. We validated 8 selected DEGs from the cerebral cortex (Brwd1, Donson, Erdr1, Ifnar1, Itgb8, Itsn1, Mrps6 and Tmem50b), 18 DEGs from the cerebellum (Atp5o, Brwd1, Donson, Dopey2, Erdr1, Hmgn1, Ifnar1, Ifnar2, Ifngr2, Itgb8, Itsn1, Mrps6, Paxbp1, Son, Stat1, Tbata, Tmem50b and Wrb) and 11 DEGs from the hippocampus (Atp5o, Brwd1, Cbr1, Donson, Erdr1, Itgb8, Itsn1, Morc3, Son, Tmem50b and Wrb). Functional clustering analysis of the 317 DEGs identified interferon-related signal transduction as the most significantly dysregulated pathway in Ts1Cje postnatal brain development. RT-qPCR and western blotting analysis showed both Ifnar1 and Stat1 were over-expressed in P84 Ts1Cje cerebral cortex and cerebellum as compared to wild type littermates. CONCLUSIONS: These findings suggest over-expression of interferon receptor may lead to over-stimulation of Jak-Stat signaling pathway which may contribute to the neuropathology in Ts1Cje or DS brain. The role of interferon mediated activation or inhibition of signal transduction including Jak-Stat signaling pathway has been well characterized in various biological processes and disease models including DS but information pertaining to the role of this pathway in the development and function of the Ts1Cje or DS brain remains scarce and warrants further investigation.

Mesenchymal stem cells exert anti-proliferative effect on lipopolysaccharide-stimulated BV2 microglia by reducing tumour necrosis factor-α levels.

BACKGROUND: Progression of neurodegenerative diseases occurs when microglia, upon persistent activation, perpetuate a cycle of damage in the central nervous system. Use of mesenchymal stem cells (MSC) has been suggested as an approach to manage microglia activation based on their immunomodulatory functions. In the present study, we describe the mechanism through which bone marrow-derived MSC modulate the proliferative responses of lipopolysaccharide-stimulated BV2 microglia. METHODS: BV2 microglia were cultured with MSC and stimulated with 1 µg/ml lipopolysaccharide. Using an inducible nitric oxide synthase inhibitor, tritiated thymidine (3H-TdR) incorporation assay was performed to determine the role of nitric oxide in the anti-proliferative effect of MSC. We also studied apoptosis and the cell cycle of both cell types using flow cytometry and explored their cytokine profile using protein and cytometric arrays. Moreover, the role of IL-6 and TNF-α in immunomodulation was deduced using specific blocking antibodies and recombinant proteins. RESULTS: MSC reduces microglia proliferation upon lipopolysaccharide stimulation by 21 to 28% and modulates the levels of nitric oxide, IL-6 and TNF-α. The role of nitric oxide in conferring the anti-proliferative effect of MSC was ruled out. Furthermore, we found that MSC exert their anti-proliferative effect by restoring the percentage of BV2 cells at S and G2/M phase to levels similar to unstimulated cells. MSC undergo a G0/G1 arrest while exerting this effect. We have also identified that MSC-mediated modulation of microglia is independent of IL-6, whilst reduction of TNF-α in co-culture is critical for inhibition of microglia proliferation. CONCLUSIONS: Our study demonstrates that MSC inhibit microglia proliferation independent of nitric oxide and IL-6, although reduction of TNF-α is critical for this effect. The inhibition of proliferation is through cell cycle modulation. These findings shed light on the mechanisms of microglial immunomodulation by MSC.

A three-dimensional collagen construct to model lipopolysaccharide-induced activation of BV2 microglia.

BACKGROUND: We report a novel method of culturing microglia in three dimension (3D) using collagen as a substrate. By culturing microglia within a matrix, we aim to emulate the physical state of microglia embedded within parenchyma. METHODS: BV2 microglia cell suspensions were prepared with type I collagen and cast into culture plates. To characterise the BV2 microglia cultured in 3D, the cultures were evaluated for their viability, cell morphology and response to lipopolysaccharide (LPS) activation. Conventional monolayer cultures (grown on uncoated and collagen-coated polystyrene) were set up concurrently for comparison. RESULTS: BV2 microglia in 3D collagen matrices were viable at 48 hrs of culture and exhibit a ramified morphology with multiplanar cytoplasmic projections. Following stimulation with 1 µg/ml LPS, microglia cultured in 3D collagen gels increase their expression of nitric oxide (NO) and CD40, indicating their capacity to become activated within the matrix. Up to 97.8% of BV2 microglia grown in 3D cultures gained CD40 positivity in response to LPS, compared to approximately 60% of cells grown in a monolayer (P<.05). BV2 microglia in 3D collagen gels also showed increased mRNA and protein expression of inflammatory cytokines IL-6, TNF-α and the chemoattractant MCP-1 following LPS stimulation. CONCLUSIONS: In summary, BV2 microglia cultured in 3D collagen hydrogels exhibit multiplanar cytoplasmic projections and undergo a characteristic and robust activation response to LPS. This culture system is accessible to a wide range of analyses and provides a useful new in vitro tool for research into microglial activation.

Cytoprotective Effect of Tocotrienol-Rich Fraction and α-Tocopherol Vitamin E Isoforms Against Glutamate-Induced Cell Death in Neuronal Cells.

Glutamate is the major mediator of excitatory signals in the mammalian central nervous system. Extreme amounts of glutamate in the extracellular spaces can lead to numerous neurodegenerative diseases. We aimed to clarify the potential of the following vitamin E isomers, tocotrienol-rich fraction (TRF) and α-tocopherol (α-TCP), as potent neuroprotective agents against glutamate-induced injury in neuronal SK-N-SH cells. Cells were treated before and after glutamate injury (pre- and post-treatment, respectively) with 100-300 ng/ml TRF/α-TCP. Exposure to 120 mM glutamate significantly reduced cell viability to 76% and 79% in the pre- and post-treatment studies, respectively; however, pre- and post-treatment with TRF/α-TCP attenuated the cytotoxic effect of glutamate. Compared to the positive control (glutamate-injured cells not treated with TRF/α-TCP), pre-treatment with 100, 200, and 300 ng/ml TRF significantly improved cell viability following glutamate injury to 95.2%, 95.0%, and 95.6%, respectively (p<0.05).The isomers not only conferred neuroprotection by enhancing mitochondrial activity and depleting free radical production, but also increased cell viability and recovery upon glutamate insult. Our results suggest that vitamin E has potent antioxidant potential for protecting against glutamate injury and recovering glutamate-injured neuronal cells. Our findings also indicate that both TRF and α-TCP could play key roles as anti-apoptotic agents with neuroprotective properties.

Mesenchymal stem cells of human placenta and umbilical cord suppress T-cell proliferation at G0 phase of cell cycle.

Mesenchymal stem cells (MSC) generated from human umbilical cord (UC-MSC) and placenta (PLC-MSC) were assessed and compared for their immunomodulatory function on T cells proliferation by analysis of the cell cycle. Mitogen stimulated or resting T cells were co-cultured in the presence or absence of MSC. T-cell proliferation was assessed by tritiated thymidine ((3) H-TdR) assay and the mechanism of inhibition was examined bycell cycle and apoptosis assay. Both UC-MSC and PLC-MSC profoundly inhibited the proliferation of T-cell, mainly via cell-to-cell contact. MSC-mediated anti-proliferation does not lead to apoptosis,but prevented T cells from entering S phase and they therefore accumulated in the G(0) /G(1) phases. The anti-proliferative activity of MSC was related to this cell cycle arrest of T-cell. UC-MSC produced a greater inhibition than PLC-MSC in all measured parameters.

Alterations in neuronal morphology and synaptophysin expression in the rat brain as a result of changes in dietary n-6: n-3 fatty acid ratios.

BACKGROUND: Polyunsaturated fatty acids (PUFA) play important roles in brain fatty acid composition and behavior through their effects on neuronal properties and gene expression. The hippocampus plays an important role in the formation of memory, especially spatial memory and navigation. This study was conducted to examine the effects of PUFA and specifically different dietary n-6: n-3 fatty acid ratios (FAR) on the number and size of hippocampal neurons and the expression of synaptophysin protein in the hippocampus of rats. METHODS: Forty 3-week old male Sprague-Dawley rats were allotted into 4 groups. The animals received experimental diets with different n-6: n-3 FAR of either 65:1, 26.5:1, 22:1 or 4.5:1 for 14 weeks. RESULTS: The results showed that a lowering dietary n-6: n-3 FAR supplementation can increase the number and size of neurons. Moreover, lowering the dietary n-6: n-3 FAR led to an increase in the expression of the pre-synaptic protein synaptophysin in the CA1 hippocampal subregion of the rat brain. CONCLUSIONS: These findings support the notion that decreasing the dietary n-6: n-3 FAR will lead to an intensified hippocampal synaptophysin expression and increased neuron size and proliferation in the rat brain.

Response Profiles of BV2 Microglia to IFN-γ and LPS Co-Stimulation and Priming.

(1) Background: The latest research illustrates that microglia phenotype is not the binary 'resting' and 'activated' profiles. Instead, there is wide diversity in microglia states. Similarly, when testing different stimulation protocols for BV2 microglia, we discovered differences in the response of the cells in terms of the production of intracellular ROS (iROS), nitric oxide (NO), CD40 expression, and migratory capacity. (2) Methods: BV2 microglia were treated with single interferon gamma (IFN-γ) stimulation, LPS/IFN-γ co-stimulation, and priming with IFN-γ followed by stimulation with LPS for 24 h. The responses of BV2 microglia were then assessed using the H2DCFDA test for iROS, the Griess assay for NO, immunophenotyping for CD40/CD11b/MHC II, and migration using a transwell apparatus. (3) Results: Single stimulation with IFN-γ induced NO but not ROS in BV2 microglia. Co-stimulation with LPS200IFN-γ2.5 induced a higher iROS production (a 9.2-fold increase) and CD40 expression (28031 ± 8810.2 MFI), compared to priming with primedIFN-γ50LPS100 (a 4.0-fold increase in ROS and 16764 ± 1210.8 MFI of CD40). Co-stimulation also induced cell migration. On the other hand, priming BV2 microglia (primedIFN-γ50LPS100) resulted in a higher NO production (64 ± 1.4 µM) compared to LPS200IFN-γ2.5 co-stimulation (44 ± 1.7 µM). Unexpectedly, priming inhibited BV2 migration. (4) Conclusions: Taken together, the findings from this project reveal the ability of co-stimulation and priming in stimulating microglia into an inflammatory phenotype, and the heterogeneity of microglia responses towards different stimulating approaches.

Regulatory role of the endocannabinoid system on glial cells toward cognitive function in Alzheimer's disease: A systematic review and meta-analysis of animal studies.

Objective: Over the last decade, researchers have sought to develop novel medications against dementia. One potential agent under investigation is cannabinoids. This review systematically appraised and meta-analyzed published pre-clinical research on the mechanism of endocannabinoid system modulation in glial cells and their effects on cognitive function in animal models of Alzheimer's disease (AD). Methods: A systematic review complying with PRISMA guidelines was conducted. Six databases were searched: EBSCOHost, Scopus, PubMed, CINAHL, Cochrane, and Web of Science, using the keywords AD, cannabinoid, glial cells, and cognition. The methodological quality of each selected pre-clinical study was evaluated using the SYRCLE risk of bias tool. A random-effects model was applied to analyze the data and calculate the effect size, while I2 and p-values were used to assess heterogeneity. Results: The analysis included 26 original articles describing (1050 rodents) with AD-like symptoms. Rodents treated with cannabinoid agonists showed significant reductions in escape latency (standard mean difference [SMD] = -1.26; 95% confidence interval [CI]: -1.77 to -0.76, p < 0.00001) and ability to discriminate novel objects (SMD = 1.40; 95% CI: 1.04 to 1.76, p < 0.00001) compared to the control group. Furthermore, a significant decrease in Aß plaques (SMD = -0.91; 95% CI: -1.55 to -0.27, p = 0.006) was observed in the endocannabinoid-treated group compared to the control group. Trends were observed toward neuroprotection, as represented by decreased levels of glial cell markers including glial fibrillary acid protein (SMD = -1.47; 95% CI: -2.56 to -0.38, p = 0.008) and Iba1 (SMD = -1.67; 95% CI: -2.56 to -0.79, p = 0.0002). Studies on the wild-type mice demonstrated significantly decreased levels of pro-inflammatory markers TNF-α, IL-1, and IL-6 (SMD = -2.28; 95% CI: -3.15 to -1.41, p = 0.00001). Despite the non-significant decrease in pro-inflammatory marker levels in transgenic mice (SMD = -0.47; 95% CI: -1.03 to 0.08, p = 0.09), the result favored the endocannabinoid-treated group over the control group. Conclusion: The revised data suggested that endocannabinoid stimulation promotes cognitive function via modulation of glial cells by decreasing pro-inflammatory markers in AD-like rodent models. Thus, cannabinoid agents may be required to modulate the downstream chain of effect to enhance cognitive stability against concurrent neuroinflammation in AD. Population-based studies and well-designed clinical trials are required to characterize the acceptability and real-world effectiveness of cannabinoid agents. Systematic Review Registration: [https://inplasy.com/inplasy-2022-8-0094/], identifier [Inplasy Protocol 3770].

Omega 3 polyunsaturated fatty acid improves spatial learning and hippocampal peroxisome proliferator activated receptors (PPARα and PPARγ) gene expression in rats.

BACKGROUND: This study examined the effects of dietary polyunsaturated fatty acids (PUFA) as different n-6: n-3 ratios on spatial learning and gene expression of peroxisome- proliferator-activated receptors (PPARs) in the hippocampus of rats. Thirty male Sprague-Dawley rats were randomly allotted into 3 groups of ten animals each and received experimental diets with different n-6: n-3 PUFA ratios of either 65:1, 22:1 or 4.5:1. After 10 weeks, the spatial memory of the animals was assessed using the Morris Water Maze test. The expression of PPARα and PPARγ genes were determined using real-time PCR. RESULTS: Decreasing dietary n-6: n-3 PUFA ratios improved the cognitive performance of animals in the Morris water maze test along with the upregulation of PPARα and PPARγ gene expression. The animals with the lowest dietary n-6: n-3 PUFA ratio presented the highest spatial learning improvement and PPAR gene expression. CONCLUSION: It can be concluded that modulation of n-6: n-3 PUFA ratios in the diet may lead to increased hippocampal PPAR gene expression and consequently improved spatial learning and memory in rats.

Gender effect on in vitro lymphocyte subset levels of healthy individuals.

Differences in gender immune response have resulted in differences in immune protection and susceptibility to inflammatory diseases. Cultured peripheral blood mononuclear cells (PBMC) are widely used in immunomodulation studies, yet the influence of gender is usually not considered. We examined the effect of in vitro culture and phytohaemagglutinin (PHA) stimulation on PBMC lymphocyte subsets using flowcytometry. Full blood counts of whole blood showed higher levels of lymphocyte in male subjects. Lymphocyte subsets enumeration revealed higher NK cell counts in males and higher B cells in females. Cultured PBMC resulted in significant increases in B and total T cell percentages among females and NK cells among males. PHA stimulated significantly increased percentages of NK and total T cells in males and total activated T cells (CD69+) in females. Our results showed significant gender differences in lymphocyte subsets in cultured conditions. This may affect experimental outcome.

Inhibitory effects of palm α-, γ- and δ-tocotrienol on lipopolysaccharide-induced nitric oxide production in BV2 microglia.

Anti-inflammatory actions of the vitamin E fragment tocotrienol have not been described for microglia. Here, we screened palm α-, γ- and δ-tocotrienol isoforms and Tocomin® 50% (contains spectrum of tocotrienols and tocopherols) for their ability to limit nitric oxide (NO) production by BV2 microglia. Microglia were treated with varying doses of tocotrienols for 24h and stimulated with 1 µg/ml lipopolysaccharide (LPS). All tocotrienol isoforms reduced NO release by LPS-stimulated microglia, with 50 µM being the most potent tocotrienol dose. Of the isoforms tested, δ-tocotrienol lowered NO levels the most, reducing NO by approximately 50% at 48 h post-LPS treatment (p<.05). None of the tocotrienol doses tested affected microglia viability.

Human mesenchymal stem cells protect neutrophils from serum-deprived cell death.

We have previously shown that human MSC (mesenchymal stem cells) inhibit the proliferation of most of the immune cells. However, there are innate immune cells such as neutrophils and other PMN (polymorphonuclear) cells that do not require an extensive proliferation prior to their effector function. In this study, the effect of MSC on neutrophils in the presence of complete and serum-deprived culture media was investigated. In the presence of MSC, the viability of neutrophils increase as measured in 24 h of incubation at various supplementation of serum concentration. We have utilized Annexin V and PI (propidium iodide) staining to confirm whether the enhancement of neutrophil's viability is due to a reduction in PCD (programmed cell death). MSC significantly rescue neutrophils from apoptosis at 1, 5 and 10% of FBS (fetal bovine serum) supplementation. The fractions of viable and dead cells were increased and decreased respectively in the presence of MSC. Our results indicate MSC rescue neutrophils from nutrient- or serum-deprived cell death. However, whether this effect is exerted through a specific signalling pathway or confining neutrophils in resting state by MSC requires further investigation.

Generation of mesenchymal stem cell from human umbilical cord tissue using a combination enzymatic and mechanical disassociation method.

MSCs (mesenchymal stem cells) promise a great potential for regenerative medicine due to their unique properties of self-renewal, high plasticity, modulation of immune response and the flexibility for genetic modification. Therefore, the increasing demand for cellular therapy necessitates a larger-scale production of MSC; however, the technical and ethical issues had put a halt on it. To date, studies have shown that MSC could be derived from human UC (umbilical cord), which is once considered as clinical waste. We have compared the two conventional methods which are classic enzymatic digestion and explant method with our newly tailored enzymatic-mechanical disassociation method to generate UC-MSC. The generated UC-MSCs from the methods above were characterized based on their immunophenotyping, early embryonic transcription factors expression and mesodermal differentiation ability. Our results show that enzymatic-mechanical disassociation method increase the initial nucleated cell yield greatly (approximately 160-fold) and maximized the successful rate of UC-MSC generation. Enzymatic-mechanical disassociation-derived UC-MSC exhibited fibroblastic morphology and surface markers expression of CD105, CD73, CD29, CD90 and MHC class I. Furthermore, these cells constitutively express early embryonic transcription factors (Nanog, Oct-4, Sox-2 and Rex-1), as confirmed by RT-PCR, indicating their multipotency and high self-renewal capacity. They are also capable of differentiating into osteoblasts and adipocytes when given an appropriate induction. The present study demonstrates a new and efficient approach in generating MSC from UC, hence serving as ideal alternative source of mesenchymal stem cell for clinical and research use.