Classical and Alternative Activation of Cyanobacterium Oscillatoria sp. Lipopolysaccharide-Treated Rat Microglia in vitro.

The purpose of this investigation was to test the hypothesis that an in vitro exposure to cyanobacterium Oscillatoria sp. Lipopolysaccharide (LPS) might result in classical and alternative activation of rat neonatal microglia. Using Escherichia coli LPS-primed microglia as a positive control, this study revealed that treatment of rat microglia with Oscillatoria sp. LPS for 17 h in vitro resulted in both classical and alternative activation as well as concomitant pro-inflammatory and anti-inflammatory mediator release, in a concentration-dependent manner: (1) treatment with 0.1-10 000 ng/ml Oscillatoria sp. LPS resulted in minimal lactic dehydrogenase (LDH) release, induced concentration-dependent and statistically significant O2 (-) generation, matrix metalloproteinase-9 (MMP-9) release, generation of the cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and the chemokines macrophage inflammatory protein-2 (MIP-2/CXCL2), interferon γ-induced protein 10 kDa (IP-10/CXCL-10), (MIP-1α/CCL3), monocyte chemotactic protein-1 (MCP-1/CCL2), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), and the alternative activation cytokine IL-10; (3) in contrast, treatment with 100 000 ng/ml Oscillatoria sp. LPS appeared to damage the microglia cell membrane, because it resulted in minimal O2 (-) generation, statistically significant LDH release, and a decrease in the generation of all the cytokines and chemokines investigated, with the exception of IL-1α and cytokine-induced neutrophil chemoattractant 1 (CINC-1/CXCL1) generation, which was increased. Thus, our results provide experimental support for our working hypothesis, namely that Oscillatoria sp. LPS induces classical and alternative activation of rat brain microglia in vitro in a concentration-dependent manner, namely 0.1-10 000 ng/ml Oscillatoria sp. LPS, when microglia cells were shown to be viable. Furthermore, should cyanobacterium Oscillatoria sp. LPS gain entry into the CNS, our findings suggest that classical and alternative activation of rat brain microglia in vivo, might lead to concomitant mediator release that could result in an interplay between neuroinflammation and neural repair in a concentration-dependent manner.

Momordica charantia (bitter melon) attenuates high-fat diet-associated oxidative stress and neuroinflammation.

BACKGROUND: The rising epidemic of obesity is associated with cognitive decline and is considered as one of the major risk factors for neurodegenerative diseases. Neuroinflammation is a critical component in the progression of several neurological and neurodegenerative diseases. Increased metabolic flux to the brain during overnutrition and obesity can orchestrate stress response, blood-brain barrier (BBB) disruption, recruitment of inflammatory immune cells from peripheral blood and microglial cells activation leading to neuroinflammation. The lack of an effective treatment for obesity-associated brain dysfunction may have far-reaching public health ramifications, urgently necessitating the identification of appropriate preventive and therapeutic strategies. The objective of our study was to investigate the neuroprotective effects of Momordica charantia (bitter melon) on high-fat diet (HFD)-associated BBB disruption, stress and neuroinflammatory cytokines. METHODS: C57BL/6 female mice were fed HFD with and without bitter melon (BM) for 16 weeks. BBB disruption was analyzed using Evans blue dye. Phosphate-buffered saline (PBS) perfused brains were analyzed for neuroinflammatory markers such as interleukin-22 (IL-22), IL-17R, IL-16, NF-κB1, and glial cells activation markers such as Iba1, CD11b, GFAP and S100ß. Additionally, antioxidant enzymes, ER-stress proteins, and stress-resistant transcription factors, sirtuin 1 (Sirt1) and forkhead box class O transcription factor (FoxO) were analyzed using microarray, quantitative real-time RT-PCR, western immunoblotting and enzymatic assays. Systemic inflammation was analyzed using cytokine antibody array. RESULTS: BM ameliorated HFD-associated changes in BBB permeability as evident by reduced leakage of Evans blue dye. HFD-induced glial cells activation and expression of neuroinflammatory markers such as NF-κB1, IL-16, IL-22 as well as IL-17R were normalized in the brains of mice supplemented with BM. Similarly, HFD-induced brain oxidative stress was significantly reduced by BM supplementation with a concomitant reduction in FoxO, normalization of Sirt1 protein expression and up-regulation of Sirt3 mRNA expression. Furthermore, plasma antioxidant enzymes and pro-inflammatory cytokines were also normalized in mice fed HFD with BM as compared to HFD-fed mice. CONCLUSIONS: Functional foods such as BM offer a unique therapeutic strategy to improve obesity-associated peripheral inflammation and neuroinflammation.

Highly brominated antimicrobial metabolites from a marine Pseudoalteromonas sp.

Extracts of a marine Pseudoalteromonas sp. (CMMED 290) isolated from the surface of a nudibranch collected in Kaneohe Bay, Oahu, displayed significant antimicrobial activity against methicillin-resistant Staphylococcus aureus. Bioassay-guided fractionation of the lipophilic extract led to the isolation and structure elucidation of two new highly brominated compounds, 2,3,5,7-tetrabromobenzofuro[3,2-b]pyrrole (1) and 4,4',6-tribromo-2,2'-biphenol (2). In addition, we have identified the known compounds pentabromopseudilin and bromophene. We describe the isolation and structure elucidation of the compounds 1 and 2 together with their antimicrobial activities against methicillin-resistant Staphylococcus aureus.

A 2-substituted prodiginine, 2-(p-hydroxybenzyl)prodigiosin, from Pseudoalteromonas rubra.

In the course of work aimed at the discovery of new pharmaceutical lead compounds from marine bacteria, a lipophilic extract of the bacterium Pseudoalteromonas rubra displayed significant cytotoxicity against SKOV-3, a human ovarian adenocarcinoma cell line. Bioassay-directed fractionation of this extract resulted in the isolation of a series of known and new prodiginine-type azafulvenes. The structure of the major metabolite was elucidated by interpretation of spectroscopic data as a 2-substituted prodigiosin, which we named 2-(p-hydroxybenzyl)prodigiosin (HBPG).

Total RNA-isolation of abdominal hernia of rats for quantitative real-time reverse transcription (RT) PCR assays.

Increasing complications in incisional hernia surgery call for novel treatments. A gene expression analysis of injured tissues displays important parameters for tissue regeneration. Until today, no reliable method has been described for a quantitative gene expression analysis of hernia tissues. In this work, a protocol is described for the isolation of DNA-free total RNA of incisional hernias for the first time. Moreover, real-time RT PCR assays for collagen type I and III and TGF-beta1 are demonstrated for relative gene expression analyses. Both methods enable relative gene expression analyses of hernia tissues for the first time.