Developing Cellular & Molecular Biomarkers for Anti-Inflammatory Activities of Probiotic Bacteria in Fermented Foods

Probiotics benefit the host through communicating with many types of cell. Contact between the host and intestinal microbes occur in the intestinal epithelial cells (IECs), where variety of complex interactions between probiotics bacteria and the different constituents of the intestinal ecosystem take place. Additionally, IECs act as the first line of defense against pathogenic bacteria. IECs extensively communicate with commensal microbes and probiotics, and they are also affected by probiotics in various ways, as by enhancing barrier function, inducing antimicrobial and heat shock protein production, which were shown to cause an increase in mucin production, interference with pathogenic organisms, as well as signaling pathways' modulation ⁸.

Probiotics modulations of tight junctions and enhanced mucin production are the most important factors that enhance the Intestinal epithelial cell (IEC) barrier function. By modifying the microbial community within the gut, intestinal disorders such as inflammatory bowel disease (IBD) could be prevented or treated. Moreover, immune responses associated with chronic inflammation were shown to be attenuated by gut microbial strains in experimental animal models ⁸.

Cytokine secretion by IECs and macrophages is regulated (up or down) by probiotics through key signaling pathways, such as the nuclear factor kappa-light-chain-enhancer of activated B cells (Nuclear Factor-κB, NFκB) and mitogen-activated protein kinases (MAPKs), which could alleviate or treat several disorders, such as allergies, diabetes, obesity and cancer ⁸.

The NF-κB pathway is a major signaling pathway for activation of immune responses, secondary to a range of stimuli. NF-κB has been extensively used as an intracellular signaling molecule for hormones, cytokines, chemokines, and growth factors ⁹. NF-κB pathway signifies a connection between probiotics and IECs. Under non-stimulatory conditions, non-activatedNFκB is present in its inactive form in the cytoplasm binding to inhibitory IκB proteins.NF-κB cannot enter the nucleus and activate transcription when it is bound to its IκB inhibitor molecule. Upon stimulation of the pathway by an activation factor, signaling pathways are triggered, resulting in activation of the IκB kinase (IKK), which in turn phosphorylates IκB proteins. Upon phosphorylation by IKK, IκB is targeted for degradation by an ubiquitin–proteasomal pathway, resulting in IkB dissociation from the NF-κB, and release ofNF-κB. Released NF-κB migrates into the nucleus, activates transcription of effector genes and binds target promoters. These stimuli include inflammatory cytokines, such as interleukin-1 (IL-1), or tumor necrosis factor alpha (TNF-α) ¹⁰.

Other probiotic strains can also avert degradation of IκB. Studies that explored the impacts of both variables and also heat-killed Lactobacillus rhamnosus  GG in an epithelial cell model, showed the probiotic's ability to diminish IκB degradation and the consequent NFκB translocation into the nucleus, resulting in decreased TNF-induced IL-8 production ¹⁵. In another study, L. rhamnosus GG has been shown to prevent the adherence of Bacillus vulgatus to epithelial cells, preventing the recurrence of colitis after antibiotic treatment in transgenic rats, suggesting that antibiotics and probiotic agents provide synergistic therapeutic immunomodulation effects ¹⁶. An important factor that is affected by probiotics is the peroxisome proliferator-activated receptor gamma (PPARγ). PPARγ is a nuclear receptor that plays an anti-inflammatory role through the inhibition of nuclear factor-κB (NF-κB) pathway. PPARγ is highly expressed in the colon. The probiotic strain L. caseinhas been reported to suppress the expression of inflammatory mediators in intestinal epithelial cells, which is assumed to be related to Par activation ¹¹. Other studies showed probiotics to have inhibitory effects on the NFκB pathway in IECs, but have been unsuccessful to describe how NFκB activation is prevented. Some probiotic bacteria could excite the NFκB pathway to cause increased cytokine secretion rather than the inactivation of the signaling pathway ¹⁷.

MicroRNAs (miRNAs) are a small non-coding RNA molecules ^{18, 19, 20, 21, 22, 23, 24} (inucleotide long), which are involved in transcriptional and post-transcriptional regulation of gene expression by inhibiting gene translation ¹⁸. MiRNAs silence gene expression through inhibiting mRNA translation to protein or by promoting the degradation of mRNA. Since first reported in the literature in 1993¹⁹, the number of identified miRNAs in 2009 was more than 700 according to miRbase 13.0, March 2009, http://microrna.sanger.ac.uk/; in 2010 the number of identified miRNAs has been reported to be 500–1500 miRNAs per genome, in total, over 15000 microRNAloci, expressing more than 17000 different mature sequences from 142 species ²⁰. Recently miRNA count is 24521 entries according to the last release of miRBase in June 2013 (http://www.mirbase.org/).MiRNAs are processedby RNA polymerase II to form a precursor step which is a long primary transcript. Pri-miR is converted to miRNA by sequential cutting with two enzymes belonging to a class of RNA III endonucleases, Drosha and Dicer. Drosha converts the a long primary transcripts to about ~70 nt long primary miRNAs (pri-miR) which are then exported to the cytoplasm by Exportin 5, converted to mature miRNA (~22 net) by Dicer ²¹. Each miRNA may control multiple genes, and one or more miRNAs regulate a large proportion of human protein-coding genes, whereas each single gene may be regulated by multiple miRNAs ²². MicroRNAs inhibit gene expression through interaction with 3-untranslated regions (3 UTRs) of target messenger RNAs (mRNAs) carrying complementary sequences ²³. Recently, miRNAs have been reported to regulate both innate and adaptive immune responses in addition to inflammations in various cell and tissues.

Our recent study that investigating the effect of dietary intervention with probiotic foods on changes in the intestinal microflora among healthy Egyptians, twenty eight adolescent human males, who were randomly assigned to one of four groups consuming regular yogurt, synbiotic yogurt (combining probiotic & prebiotic: inulin), sobya (fermented rice), or unfermented rice milk placebo. The supplement was served daily for three weeks. Before starting and at the end of the intervention, the intestinal permeability was assessed by measuring the urinary lactose mannitol dual test (LMDT). Microbial examination, hydrolytic enzymes and short chain fatty acids (SCFA), mainly acetic, butyric and propionic acids, are the products of the bacterial fermentation of undigested carbohydrates, which were measured in feces. Urinary lactose/mannitol ratios were reduced only after dietary interventions with regular yogurt (P<0.05), or with combining data from symbiotic yogurt plus sobya (P<0.05). The fecal Lactocilli counts increased (p<0.05) in those receiving the fourth dietary treatments (P<0.05) compared to the preintervention levels. Similar trends were noticed for the bifido bacteria strains. On the other hand, enterobacteriaceae counts were reduced in the three groups consuming fermented supplements. Tendencies for increase in concentrations of fecal butyric, propionic and total SCAF were found among the groups consuming symbiotic yogurt or soybean compared to preintervention values. Intervention did not affect the activities of fecal hydrolytic enzymes (β-galactosidase, β-glucosidase and β-glucuronidase). Therefore, we concluded that consumption of fermented food supplements increased bifidobacteria and lactobacilli populations, and also decreased the pathogenic bacteria, which could be a potential health-promoting supplement for adolescents ²⁴.

The innovation of our multifaceted proposal lies in using in vitro and in vivo approaches in cell lines and mice, respectively, to study effect of probiotic conditional media & heat killed bacterial strains with anti-inflammatory effect to elucidate the mechanism by which probiotics affect signaling pathways, as well as use global cytokine and miRNA gene expression analyses approaches to develop biomarkers for the different pro- & anti-inflammatory activities. The novel contribution of this work is two folds: 1) Developing specific disease biomarkers that could be used for early diagnosis of certain pathogenic states. Moreover, revealing signaling pathway mechanisms may fill up the gap in our understanding of the initiation and development processes of related diseases, which could move the research from diagnosis to treatment state. 2) Evaluating the effect of different nutrients and dietary components on developed biomarkers in health states will promote health status, and prevent health problems. During the course of the experiments, combing in vivo and in vitro approaches will enable us to screen a large number of probiotic strains and their secreted factors in vitro. However, because in vitro results do not always coincide with in vivo data, this finding raise the importance of confirming the correspondence of the in vivo activity to the in vitro findings, in addition to complementing the in vitro results by using different mice models. Moreover, this study will be the first to screen for several cytokines along with a large number of miRNA gene expressions, which enables the application and validation of principal component analysis (PCA) statistical multivariate method, in which the patterns in the data could be predicted by similarities or differences of the tested components. Finally, although it has been shown that miRNAs have a prominent role in the regulation of cancerous and inflammation pathways, the literature is still in the early stage of elucidating the role of miRNAs and their respective targets in inflammation. The proposed study will pave the research to diagnosis, to reach the stage of treating the pathogenic states.

1.The resistance to gastric acid to ensure that the administered probiotic bacteria survive transit to intestinal environment through the stomach, and that they are functioning effectively there ²⁵. 

2.The resistance to bile acids’ antibacterial effects ²⁶.

3.The adherence (binding) to human gut epithelial tissues in order to colonize the gastrointestinal tract ²⁶.

Subsequently, in vitro assessment to evaluate the anti-inflammatory effect of the conditional media (probiotics) and the heat killed bacteria (secreted probiotics factors) of each strain which passes the above mentioned criteria are employed as follow:

Macrophages activated with bacterial lipopolysaccharide (LPS) express inducible nitric oxide (NO) synthase (a short-lived radical that is produced by enzymatic oxidation of the amino acid L-arginine), and produce large amounts of NO, which has been reported to be an effector molecule for the cytostatic/cytotoxic properties of the activated macrophages ²⁷.

The murine macrophage cell line J774.2 from mouse BALB/C monocyte macrophagewill be used (Sigma, St. Louis, MO). These cell lines will be grown and maintained in 100 mm plastic culture dishes containing Dulbecco's modified Eagle's medium (Gibco) (Sigma, St. Louis, MO) at 37°C in an atmosphere of air and5% CO₂. Cells will be passaged every 3-6 days by diluting a suspension of the cells 1:10 in fresh medium to maintain the cultures between 3-9x10⁵cells/ml. These cells will be used to determine NO production. All selected probiotics strains will be assessed as previously described ²⁸ in the presence or absence of LPS from E. coli serotype O55:B5.

The production of nitric oxide is measured using Griess test where Nitrite is detected and analyzed by formation of a red pink color upon treatment of a NO₂-containing sample with the Gris reagents previously described ²⁸. Briefly, the murine macrophage J774 cells will be removed from culture dishes by pipetting vigorously. Cells will be precipitated by centrifugation followed by resuspension in the medium to a concentration of 1 x10⁶cells/ml. Cells are plated in 24 well culture plates and allowed to adhere for 2h. Cells are activated by LPS (0.1 µg/ml) after replacing the old with a fresh medium. They will then be incubated at 37°C for 72h in the presence or absence of probiotic strains. After appropriate incubation time, supernatants from the plated cells (300 µl) are mixed with equal volumes of Griess reagent (1% sulphanilamide (SA)/0.1%naphthylethylenediamine dihydrochloride (NEA)/ 2.5% phosphoric acid). After 10 min incubation at room temperature, the absorbance of the formed chromophore is measured at 550 nm using a spectrophotometer, and NO₂ measured using NaN0₂ as a standard. The Griess test reaction is summarized in Figure 1.

Figure 1.Illustration of the Griess Test Reaction.

Pro-inflammatory cytokines such as interleukin-1β (IL-1β), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-12 (IL-12) and tumor necrosis factor-α (TNF-α), as well as anti-inflammatory cytokines  such as Interleukin-4 (IL-4), Interleukin-10 (IL-10), IL-1 RA and sTNF RI and RII, are measured using flow cytometry multiplex immune assay (luminex) and mouse cytokine ELISA kits as follows:

The enzyme-linked immunosorbent assay (ELISA) is the most common method utilized in research laboratories for the detection and quantification of secreted cytokines, due to its having a number of advantages such as its accuracy, specificity, sensitivity, low cost, ease of implementation, wide availability of cytokines kits for human, mouse and rats, and use of non radioactive agents. Generally, ELISA detects a specific sequence of amino acids (epitope) that binds to an antibody of interest ³⁰. ELISA capture assay format for cytokines analysis is mostly the sandwich assay. The sandwich format is used because it is sensitive and robust. In this type of assay the analyte to be measured is bound between two antibodies – the capture antibody and the detection antibody (either monoclonal or polyclonal) that bind to target sites on the cytokine. The first ‘capture’ antibody is attached to a solid support. The second ‘detection’ antibody that links the captured cytokine to a common detection system is either biotinylated, or is conjugated to a detector enzyme, most commonly horseradish peroxidase, which produces spectrophotometrically measured color that is proportional to the concentration of the tested cytokine ³¹. A drawback of the ELISA method is that it only detects one allergen per test, requiring a large sample volumes, and an extensive labor, which could limit its effectiveness ³².

In order to partially overcome this drawback, sequential ELISA methods have been introduced in which the same sample is repeatedly employed in different detection cycles. Depending on the high binding specificity between the antibody and the antigen, in each cycle only the selected marker (cytokine) will bind to its specific antibody, leaving the other cytokines in the sample, which will be selected during other cycles. Recently, Multi-Analyte ELIS Array Kit has been available for multiple cytokines detection, using the same ELISA Kit ^{33, 34}.

In this proposed research, mouse Inflammatory Cytokines Multi-Analyte ELISArray Kit from Qiagen (Valencia, CA) will be employed. The kit is designed to survey a specific panel of 12 pro- and anti- inflammatory cytokines at once, using a conventional ELISA protocol under uniform conditions. The cytokines & chemokines represented by this array are IL1A, IL1B, IL2, IL4, IL6, IL10, IL12, IL17A, IFNγ, TNFα, G-CSF, and GM-CSF. Cytokines analyses will be carried out according to the manufacturer’s instructions. Quantitative data are obtained by measuring the optical density at wavelength (λ) 450 nm, using a microplate spectrophotometer. All standard curves are generated with the standard solutions provided with the test kits. Data analysis is carried out using data collection analysis software Gen5 (Biotek, Winooski, Vermont, USA). Calibration curves are constructed from three replicates ateach point of the standard curves. ELISA measurements is obtained in duplicates for each sample, and all experiments will be performed twice.

Recently, flow cytometry technique has been introduced for cytokines' detection. One of its key advantages is the potential to simultaneously detect multiple cytokines. It is an automated fluorescent microsphere-based multiplex immunoassay that employs Multi-Analyte Profiling (xMAP) technology. The xMAP technology, in theory, enables multiple (up to100) microsphere sets to be distinguished simultaneously as each bead set is encoded by two fluorescent dyes, red and orange. The flow cytometer has a dual laser, red and green. The system’s red laser is used to excite the bead fluorescent in order to identify the bead set. Each bead set capture a specific cytokine antibody. The captured cytokines are detected through an immunoassay where a detector antibody (secondary antibody) conjugated with phycoerthrine (PE), which emits at λ 585 nm, and that will be measured using the system’s green laser. Coupled beads will be analyzed using a Luminex analyzer according to the system manual, in which each tested cytokine will be identified through distinctive fluorescence of the coupled bead, and the quantity of each cytokines is determined from the phycoerthrine fluorescence intensity. Flow cytometry, enhances ease-of-use and automation, and markedly reduces the time and labor required for cytokines detection and quantification ^{31, 35, 36}. In the proposed research, flow cytometry are the primary assay platform for cytokines detection, using the Bio-Plex Pro™ mouse cytokine 23-plex immunoassay kit (Bio Rad, Herculese, CA) that simultaneously detects the following 23 cytokines (see Table 2) in the same sample.

Among these measured cytokines, TNF-α and IL-1β are particularly relevant. Several literatures have related these two cytokines to the activation of NF-kB pathway ^{37, 38, 39}.

NF-κB signal transduction pathways have different nodes which may be regulated by the probiotics preventing the activation of NFκB and influencing downstream cytokine secretion. In the NF-κB reporter gene cell line, four copies of the NF-κB consensus transcriptional response element are in conjunction with minimal cytomegalovirus (mCMV) promoter. The green fluorescent protein (GFP) and the luciferase reporter genes are under the regulation of a NF-kB response element. Activation of NF-κB pathway leads to the activation of the promoter genes, which in turn activates the expression of reporter genes (firefly luciferase or GFP) as illustrated in Figure 1. The NF-κB pathway will be monitored in vitro using NF-κB/293/ GFP-Luc cell line, System Biosciences (SBI). Addition to the reaction catalyzed by Luciferase reporter gene with luciferin to produce light. Relative light intensity is measured for quantitative transcription activation reporter assays ^{40, 41}.

Expression of GFP reporter gene activation will be measured by flow cytometry through the detection of GFP reporter gene fluorescence. The activities of firefly luciferases will be measured by adding Luciferase Assay Reagent II (LAR II) to generate a stabilized luminescent signal, which is then measured by a luminometer plate reader according to the manufacturers’ instructions.

The change in the activity of NF-κB signaling pathway is determined by comparing the GFP and luciferase activities in treated versus untreated transfectants. The identically treated negative control transfectants serve as a specificity control. The positive control serves as a control for transfection efficiency. The three most powerful bacterial strains presenting highest inhibition of LPS-induced NO production, along with the lowest activation of NFκB, will be selected for in-vivo animal studies.

NF-κB/293/GFP-Luc™ Transcriptional Reporter Cell Line. (http://www.systembio.com/downloads/ TR860_Web_Manual.pdf). Figure 2

Figure 2.Activation of NF-κB pathway leads to the activation of the promoter genes, which in turn activates the expression of reporter genes (firefly luciferase or GFP).

Evaluate the selected probiotics by employing in vivo studies in mice models to elucidate the mechanism by which probiotics affect signaling pathways, and using gene expression analysis to develop microRNA biomarkers for the different anti-inflammatory activities

1.The 1^st part of this objective has been to employ in vivo studies in mice to elucidate the mechanism by which probiotics affect signaling pathways. Specifically, this involves studying the effect of probiotics on the nuclear factor NFκB in response to different inducing agents such as TNF-α or LPS, MAPKs, and transcriptional regulators such as heat shock transcription factor 1 and PPARγ.

All studies are approved by the local animal ethics committee. Mice strain C57BL/6J (C57 black 6) will be used. It's genome was initially published in 2002 ⁴² while its finished genome assembly was published in 2009 ⁴³ making it the second mammalian species to get its entire genome sequenced after publishing the initial sequence of the human genome in 2001 ⁴⁴ and the complete human genome sequence in2004 ⁴⁵. This strain is recommended to for explore human disease providing the most powerful model for studying gene expression regulation ⁴². Moreover, it has the advantage of strain stability and easy breeding.

In this research different mouse models are used, in which each group of mice contains equal numbers of males and females as has been recommended by Mogil and Chanda, 2005 ⁴⁶. Mice are housed in plastic cages (5 or 6 mice/cage). The mean initial body weight at approximately 4 weeks of age is recorded and then mice will be divided into many dietary groups, and fed either a control diet or one of probiotics enriched diets. A standard diet is used. Diets and water will be provided daily and body weights and food intake will be recorded weekly. The animals are kept in cages under standard conditions (21–25^oC, 50±10% humidity with a 12 h day–night cycle). After a 6 week feeding period, mice are anesthetized with CO₂ following an overnight fast. Following this general procedure different model of animal trials are used.

From both economical and ethical point of view, the experiments are designed in order to obtain significant results using the minimum number of animals. This will reduce the cost of the animals and their care, allowing for an efficient use of the resources, in addition to being scientifically and ethically justifiable. A recent study concluded that a comparable and statistically powerful result could be obtained using one third of the number of the animal that have been used in some literature. The results highlight the importance of using the correctly identified experimental unit (number of mice for each group) in a well designed bias free study ⁴⁷. As such, for each experiment the group size is determined using power analysis ⁴⁸. Power analysis has been reported to be the most favored method for calculating the sample size in animal studies ⁴⁹. Using power analysis, the number of mice required for each group will be calculated from the literature depending on the factor to be measured and the expected variance in this measure (noise), which ensures that the animals are capable of responding, giving a good measurement of analyzed factor “signal” so that there is a high signal/noise ratio.

The formula used to calculate the sample size is: Sample size = 2 SD2 (1.96 + 0.842) 2/d2, where:

SD = Standard deviation (from previous studies, or pilot study).

1.96 obtained from Z table at type 1 error of 5%.

0.842 obtained from Z table for 80% power.

d = effect size = difference between mean values representing the signal ⁴⁹.

The dextran sulfate sodium (DSS)-induced colitis model is used. DSS animal model has been reported as a powerful model for investigating colitis-related colon carcinogenesis pathogenesis and chemoprevention ⁵⁰. DSS will be introduced to the mice in their drinking water to induce colitis which is a chronic condition marked by an inflammation of the and a significant increase of the inflammatory cytokines and macrophages ⁵¹. DSS induce a defect in epithelial barrier function, due to its cytotoxic effect. This defect in the colonic mucosal barrier subsequently leads to emergence of various inflammatory cells, macrophage activation and pro-inflammatory cytokine production ⁵². The effect of each selected probiotics strain on the induction of colonic inflammation is then investigated.

Sample Size

In literature where DSS-induced colitis model were used ^{51, 52, 53}, applying power analysis using disease activity index as a signal factor, showed that the number of mice to be used in each group should be between 4 -6 using 80% power factor assuming a 5% significance level and a two sided test, after considering 10% loss of the animals during the course of the experiment due to dying or for any other reason. Indeed, literature have quoted use of 6 mice ⁵¹, 8 mice ⁵², or 10 ⁵³. As such, 30 mice of each sex will be divided to 5 groups of 6 mice each/group/sex.

Duration of the study: The study lasted 3 weeks in addition to one week acclimatization to allow animals to habituate to the animal unit in which they are housed before initiation of experiments.

The model used is described by Osman et al ⁵³. Briefly, six- to 8-week-old C57BL/6J mice weighing from 20 to 25 g are obtained from The Jackson Laboratory (Bar Harbor, ME). Mice are divided into five groups. One group of animals will be a negative control (does not receive either DSS or probiotics), and the rest four group swill receive colitis. The colitis control group will receives DSS without probiotics administration. One of the three tested probiotics strains will be administered for each group (except for the negative control and the colitis control as mentioned before).

Normal saline (vehicle) in the colitis control group or the tested bacterial strain (3 ml; 3x 10⁹ CFU of each probiotic strain per animal will be administered orally (once/day) for 7 days before starting DSS and continued for 7 days after DSS induction. DSS of molecular weight 36 000 to 50 000 Da will be obtained from MP Biomedicals (Aurora, OH, USA). Colitis will be induced by 5% (w/v) dissolved in drinking water for 7 days.

The severity of colitis is assessed daily using a disease activity index (DAI) according to which scores changes in body weight, stool consistency, and evidence of intestinal bleeding are used ⁵¹. A scoring scale from 0-4 will be used for the weight loss for 0% change, <5% change, <10% change, <20% change, and>20% change, respectively. Stool consistency, however, is scored as 0 for normal, 2 for loose stool and 4 for diarrhea. Finally, gross bleeding will be scored as 0 for negative, 2 for hemoccult and 4 for gross bleeding. DAI will be calculated as the third of the combined score of the three parameters; this method is reported to correlate well with the inflammation level, crypt score and inflammatory bowel disease (IBD) ⁵¹.

After sacrifice, the mice colon (approximately 10 cm long) is harvested from the colocecal junction to the anal verge. The colon is opened and cleaned from fecal material by thorough rinsing in 0·9% saline. Gross morphological changes are observed in the opened colon in addition to the presence of blood. The colon will be divided into three equal parts presenting the proximal, middle and distal portion of the colons for histopathological analysis, cytokines measurements and RNA isolation, respectively. Each segment measures about 3 centimeters long. The distal portion of colons is fixed in neutral buffered formalin and then evaluated histopathologically. Colonic histological evaluation is carried out as described by Fitzpatrick et al ⁵². Briefly, histological damage is determined based on inflammation severity and extent, in addition to crypt damage and epithelial erosion. For the assessment of the severity of colitis, histological scores of the proximal, middle and distal portion of the colons is determined, and the average calculated (see Table 3).

Because the presence of stromal cells in excised inflamed tissue might obscure differential cell-specific gene expression profile, we will employ GEM Tox Arcturus Pix Cell II LCM System^ (Applied Biosystems, Foster City, CA), which produces 5-50 ms infrared laser pulses, 2-30 µm from above, allowing selective adherence of certain cells to a vinyl acetate thermoplastic polymer film that is bound to a sterile plastic cap ^{54, 55, 56}.

Tissue specimens that have been embedded in Tissue Tek OCT before freezing will be longitudinally sectioned at 7 m in a cryostat to capture bottom mature crypt colon cells. Sections are picked up on a supporting poly-1-lysine foil mounted on non-charged microscope slides to ensure that tissue stays on during staining. The target area selected by our Collaborating Pathologist is chosen for LCM. The slides will be fixed for 1 min in 75% ethanol, dipped in nuclease free water for 30 s, stained with 1% Crystal Violet Acetate (Sigma, St. Louis, MO) for 30 s, rinsed in 75%, 95% and 100% ethanol for 30 s each, and air dried. We found this procedure to result in less RNA degradation than traditional H&E staining.

The second segment of the excised colon is snap frozen in liquid nitrogen and stored at -80°C until used for cytokines levels measurement. The last segment will be stored by submerging in RNAlater^® solution (Invitrogen, Life Technologies) to maintain the quality and integrity of subsequently isolated RNA for molecular studies.

For serum biochemical analyses, the blood is also collected in EDTA tubes and centrifuged to generate plasma. Cytokines level measurement will be determined in both colon tissue and blood as mentioned above.

The second model of animal trials is mice strain C57BL/6J with a deficiency in low density lipoprotein (LDL) receptor (B6.129S7-Ldlr^tm1Her/J). Mice with such mutation have an elevated serum cholesterol level of 200-400 mg/dl, and have very high cholesterol levels (>2,000 mg/dl) when fed a high fat diet, compared to a serum cholesterol level of 80-100 mg/dl for normal mice. This strain has been widely used as a model to mimic human atherosclerosis. Using this model, the prevention effect of probiotics on diet-induced fatty liver disease in hyperlipidemia mice fed a highly palatable energy-rich obesogenic diet will be evaluated. Duration of the study: The study lasted for 6 weeks in addition to one week to allow animals to habituate to the animal unit before initiation of the experiments. During the six weeks of study, the animals are fed ad-libitum (free feeding) fed the diets described below.

Many miRNAs have been reported to be related to inflammation such as miRNA-155 during the macrophage inflammatory response ^{58, 59}; miR-146a was found to be a NF-κB-dependent gene induced by LPS in an NF-κB-dependent manner ⁶⁰. Moreover, miR-155, miR-21, and miR-126 have been reported to contribute to vascular inflammation and diseases ⁶¹. With regard to endothelial cell function regulation, a few specific miRNAs have been validated. Cell-specific targeting with miRNAs is an important area of investigation to be developed. Further studies need to analyze the complex interactions between endothelial-specific miRNAs and their targets during angiogenesis. Identification of miRNAs and their targets and better understanding of in vivo mechanisms is critical for the development of miRNA drugs of specific molecular clinical therapy targets ⁶².

Systematic investigation of miRNA for a universal coverage using microarray expression profiling ⁶⁵ that employ Affymetrix GeneChip miRNA 3.0 array (Affymetrix, Inc, Santa Clara, CA), which provides for 100% miRBase v17 coverage (www.mirbase.org) by a one-color approach, is carried out as we described before ⁶⁶. Briefly, the enriched fraction for miRNA profiling studies is obtained by passing 10 μg of high quality, undegraded total RNA through a flash PAGETM Fractionator apparatus (Invitrogen). RNA molecules will be tailed and labeled using a 3DNA Array Detection Flash Tag Biotin HSR microarray technology (Genisphere LLC, Hatfield, PA). The fluorescence on the array is scanned using an Affymetrix GCS3000 Gene Array Scanner with a high resolution 6g patch. Thresholding and signal is generated using appropriate algorithms. The background adjusted fluorescent values generated by the scanner is normalized for each miRNA using a variation stabilization transformation method such as cyclic LOWESS, as we detailed earlier ⁶⁶. Hypothetical testing with one-way analysis of variance (ANOVA) or Student’s t-test is employed for statistical analysis of miRNA arrays. P-values < 0.05 are considered significant. Reproducibility and linearity are evaluated using ANOVA or Pearson correlation coefficients. Pair-wise comparisons is carried out on differentially expressed genes identified by ANOVA. For each pair of treatments, a two-sample t-test is carried out for every gene & multiplicity correction is followed to control the false discovery rate (FDR) using a step-up approach known as protected least significant difference, LSD. Pairwise volcano plots will be generated for measuring over- or under-expressed miRNAs ⁶⁷.

A few microliters of ss-cDNA is subsequently amplified by PCR, employing a two-step format, permitting reverse transcription and amplification to be performed separately under optimal conditions. We will then use modified Taxman-based semi-quantitative(q) real-time PCR assay that includes mi RNA-specific tailed stem-loop forward primer that bind at the 3’ portion of miRNA molecules, reverse primer and a dye-labeled hydrolysis Tasman®minor groove binding (MG) probe (Applied Bio systems) ⁶⁸, which collectively improve the thermal stability (Tm) depending on the sequence composition of the assayed miRNA molecule, extends the effective footprint of RT primer/RNA duplex that may be required for effective RT from relatively shorter RT primers, and prevents them from binding double strand (dd) genomic DNA molecules, and therefore prevent amplification of any potential genomic DNA contaminants that may be present in the preparation ⁶⁹.

Figure 3. Activation of NF-κB pathway leads to the activation of the promoter genes, which in turn activates the expression of reporter genes (firefly luciferase or GFP). For analysis of crap results, we employed the comparative cross point (CO) value (or E-method) ⁷⁰ using the Highlighter (LC) Quantification Software™, Version 3-5 ⁷¹ for a Roche 480 PCR instrument (Mannheim, Germany). In that method, standard concentrations are plotted versus the threshold cycle to calculate the expression of miRNA genes automatically without user’s input, with high sensitivity and specificity calculations ⁷². A CO value corresponds to the cycle number at which each well has the same kinetic properties. The CO method corresponds to the 2^-TACT method ⁷³ used by other PCR instruments, although the latter method produces reliable quantitative results only if the efficiency ^{E= 10} of the PCR assay for both target and reference genes are identical and equal to 2 (i.e., doubling of molecules in each amplification cycle) ⁷⁴; for example if well A1 has a CP value of 15 and well A2 has a CP value of 16, we deduce that there is twice as much of the gene of interest in well A1. A 10-fold difference is shown by a difference of ~ 3.3 CP value.

Figure 3.Real-time quantification of microRNA byStem-loop RT-PCR (from Reference 68).

Normalization of PCR data is carried out against endogenous housekeeping internal standards such, as 18S rRNA gene, RNU6 and any of three stably-expressed human miRNA (let-7a, miR-16 and miR-103) ^{75, 76}, to ascertain unbiased normalization and elimination of spurious data, and to give consistent and reliable results for normalizing of PCR expression of miRNAs ⁷⁷.

The following three nutrients have been tested: n-3 poly unsaturated fatty acids (PUFA), vitamin D and selenium. The long chain n-3 PUFA of marine origin, specially EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid), have been reported to have anti-inflammatory properties that reverse and prevent the adipose tissue inflammation and insulin resistance induced by a high fat by reducing plasma triglycerides and exhibiting antiobesity effects. The mode of action of PUFA is through affecting the adipose tissue function increasing the secretions of hormones such as adiponectin, leptin and visfatin. These hormones regulating energy intake, energy expenditure, glucose levels and fatty acid breakdown.These fatty acids increase the production of adiponectin through PPARу dependent mechanism. Moreover, EPA and DHA modulate cytokines production through aNF-kB dependent mechanism. This modulation includes reducing the production of pro-inflammatory cytokines including TNFα, IL-6, MCP-1, and PAI-1; and increase anti-inflammatory cytokine such as IL-10. Finally, EPA and DHA increase the oxidation of fatty acid and lower lipid accumulation in adipocytes ⁷⁸. Vitamin D has been reported to reduce insulin resistance and affect the promoter region of cytokine genes. Such effect regulates glucose homeostasis, stimulates lipogenesis and reduces systemic inflammation induced by TNFα and IL6 ⁷⁹. In addition, vitamin D have an anti-inflammatory effect as it suppress the release of TNFα and up-regulates the synthesis of the anti-inflammatory cytokine IL-10 and induces IL-10 receptor expression ⁸⁰. Selenium deficiency has been reported to affect inflammation contributing to higher cancer risk in selenium deficiency states due to its incorporated into selenoproteins and its effect on the expression of non-selenoprotein ⁸¹. Interestingly, moderate deficiency of selenium has been reported to down regulate inflammation related genes inducing an impaired NF-κB signaling ⁸².

Duration of the study: The study lasted for 6 weeks in addition to one week to allow animals to habituate to the animal unit before initiation of the experiments. During the 6 weeks of study, the animals will be ad-libitum (free feeding) fed the diets described below.

The statistical methodology for the analysis of study specific aims has been as follows:

We have proposed the most practical, least labor-intensive and economical approach to accomplish study aims. However, in a few problematic samples (< 5% based on our experience) in control or diseased cases, it may be necessary to use other methods. However, because the error rate is so small and would occur in control and cases, adopting different extraction/analyses methods will not bias results.

In some cases, measurement repetition is required to account for the variation in the level of one or more cytokines with %CV more than 15%. The mouse cytokine 23-plex immunoassay kit in not economically practical to be used for such purpose. To overcome this difficulty, a single cytokine flow cytometry analysis will be developed as follows: Anti-cytokine specific antibody pair (monoclonal antibody for capture reporter, polyclonal antibody for detection) will be obtained from Abcam (Cambridge, MA).The capture antibody will be coupled to magnetic COOH flow cytometry beads using protein coupling kit (Bio-Rad, Hercules, CA). Detection (reporter) antibodies will be obtained by conjugating the pre-mentioned capture antibodies to phycoerythrin (PE) using a Phycolink R-Phycoerythrin (RPE). Cytokine flow cytometry measurement will be performed using the prepared antibodies as described before.

For the same reason detailed in 1 above, a single cytokine commercial ELISA kit (Endogen, Inc., Woburn, MA) will be used to measure the concentrations of the specific cytokine.

In very few samples, inhibitors present in stool may make it difficult to isolate RNA using Qiagen kits that provide the advantage of manufacturer's established validation and QC standards. In such cases we will manually isolate RNA by a modification of the classical acid guanidinium thiocyanate-phenol-chloroform (AGPC) method ⁸⁴ using chaotropic guanidinium thiocyanate (GSC) that inactivates ribonucleases and most microorganisms.

Qiagen (SABioscience, Frederick, MD) introduced a focused human PCR array in a 96 well plate containing 88 cancer-related mRNA or miRNA genes, 4 normalization housekeeping synthetic miRNA genes, 2 RT controls and 2 controls to test the efficiency of the qPCR reaction. These focused arrays could be used to study miRNA expressions by a universal multiplex qPCR assay, in which a single cDNA preparation can quantitatively assay 88 miRNA genes with high specificity due to the use of universal primers containing a modified oligonucleotide ⁸⁵.

LNAs^TM are conformationally restricted nucleotide analogs in which the ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge "locks" the ribose in the 3'-endo conformation, which enhances base stacking & backbone preorganization, significantly increasing T_m of nucleotide capture probes & improving mismatch discrimination of the target leading to better hybridization properties ⁸⁶. Exiqon’s miRCURY LNA^TM Universal RT miRNA PCR System (Exiqon A/S, Vedbaek, Denmark) contains two miRNA specific primers, allowing accurate quantification of individual miRNAs from a 1 pg total RNA.

Signosis, Inc., Sunnyvale, CA (www.signosisinc.com) introduced high throughput plate assay for monitoring individual miRNAs, without the need to carry out a RT reaction. In that assay one of the bridge oligos partially hybridizes with the miRNA molecule and the capture oligo, and another bridge forms a hybrid between the miRNA molecule and the detection oligo. The hybrid that is sensitive to the miRNA sequence is immobilized onto a plate and detected by a streptavidin-horse radish peroxidase conjugate and chemiluminescent substrate using a plate reader. One oligonucleotide difference prevents hybrid formation; thus miRNA isoform could be differentiated.

If needed, this aim using alternate approaches will be carried out during months ^{12, 36, 54} and ^{58, 59, 60}.

The expected clinical outcomes of this study include: Identifying cytological and molecular biomarkers that could be used as screening tools to promote health and/or prevent disease, and providing genetic information on how probiotics and nutrients may affect the host metabolism and influence the development of obesity, diabetes, and specific cancers. In addition, results from this study will increase the understanding of the regulatory effect of nutrients and dietary factors at the molecular level.