The authors have declared that no competing interests exist.
We present below a mechanistic cellular and molecular approaches for the development of Anti-Inflammatory biomarkersof Probiotic Bacteria in Fermented Foods. Probiotics are live microorganisms that promote human health by counteracting the noxious toxic gut microflora in human intestine, by modulating of the tight junctions, and by increasing mucin production, enforcing intestinal epithelial cell barrier function, modifying microbial community within the gut intestinal disorders, and improving immune responses associated with chronic inflammation in experimental animal models, collectively enhancing human health. Cytokine secretion by intestinal epithelial cells and macrophages are regulated by probiotics through key signaling pathways such as nuclear factor-κB and mitogen-activated kinases, resulting in alleviation of several disorders such as allergies, diabetes, obesity, heart diseases and cancer. MicroRNAs are small non-coding RNA molecules involved in transcriptional and post-translational regulation of gene expression by inhibiting gene translation. Using
Probiotic bacteria represent specific bacterial species that are a common part of human microbiota. It is widely known that the interaction between the normal gut microflora and the human mucosa is an essential phenomenon for proper intestinal function. Nutrigenomic studies have shown that gut micrflora influence the efficiency of energy extracted from diet and fat storage to the degree that microflora have essential therapeutic relevance, and could counter a disease such as obesity
Cytokine secretion by intestinal epithelial cells (IEC) and macrophages is up or down regulated by probiotics through modulations of key signaling pathways such as NF-kB and mitogen-activated protein kinases (MAPKs)
The small non-coding miRNA molecules have been correlated with different inflammatory activities, but few miRNAs have been validated with regard to endothelial cell function regulation. We will study the role of these miRNAs for different anti inflammation activities, and will explore the mechanism by which probiotics affect the signaling pathways of NFκB (in response to different inducing agents such as TNF-a or LPS, MAPKs, and transcriptional regulators such as heat shock transcription factor 1 and PPARγ), using total RNA extracted from LCM colon tissue or from stool, perform RT reaction, then carry out oligonucleotide microarray studies, focused microarray expressions studies, followed by modified real-time TaqMan® quantitative RT-qPCR reactions to quantify miRNAs.
The Food and Agricultural Organization of the United Nations and the World Health Organization (FAO/WHO) considers probiotics as “Live microorganisms, which when administered in adequate amounts confer a health benefit on the host”
We outline five objectives and detail methodologies for carrying out a nutrigenomic molecular research study. Time-line for achieving the outlined objectives is detailed in
Method-Aim/Months | Objective 1: Select three potent probiotic strains with anti-inflammatory effect of conditional media & heat killed probiotic strains isolated from fermented foods ( |
Objective 2: Employ |
Objective 3: Employ |
Objective 4: Study the role of statistics in realizing aims 1 through 3 | Objective 5: Provide & carry out alternate standardized methods to achieve study goals, if necessary |
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1. Select the three most potent probiotic strains having an anti-inflammatory activity by employing an in vitro assessment of the anti-inflammatory effect of the conditional media (probiotics) and the heat killed bacteria (secreted probiotics factors). Three probiotics strains isolated from fermented foods and belong to the genera (Lactobacillus, Bifidobacterium and Propionibacterium) will be selected.
2. 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.
3.Employ in vivo mouse model to evaluate the effect of three different nutrients and dietary components on developed biomarkers in health states.
4.Study the role of statistics in realizing aims 1 through 3.
5.Provide and carry out alternate standardized technical methods for achieving the above aims in the unlikely event that the proposed approach, or the outlined methods fail to achieve study goals.
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
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
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
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
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
MicroRNAs (miRNAs) are a small non-coding RNA molecules
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
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.
Select the three most potent probiotic strains having an anti-inflammatory activity by employing an in vitro assessment of the anti-inflammatory effect of the conditional media (probiotics) and the heat killed bacteria (secreted probiotics factors) of three different probiotic genera (Lactobacillus, Bifidobacterium and Propionibacterium) isolated from fermented foods.
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
2.The resistance to bile acids’ antibacterial effects
3.The adherence (binding) to human gut epithelial tissues in order to colonize the gastrointestinal tract
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
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
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
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
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
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
. IL-1α | . IL-10 | . IFN-γ |
. IL-1β | . IL-12 (p40) | . KC |
. IL-2 | . IL-12 (p70) | . MCP-1 (MCAF) |
. IL-3 | . IL-13 | . MIP-1α |
. IL-4 | . IL-17A | . MIP-1β |
. IL-5 | . eotaxin | . RANTES |
. IL-6 | . G-CSF | . TNF-α |
. IL-9 | . GM-CSF |
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
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
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).
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 1st 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
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
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
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
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
In literature where DSS-induced colitis model were used
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
Normal saline (vehicle) in the colitis control group or the tested bacterial strain (3 ml; 3x 10
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
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
Histological changes | Score | |||
0 | 1 | 2 | 3 | |
Severity of inflammation | None | Mild | Average | High severity |
Extent of inflammation | None | Mucosa | Mucosa and sub mucosa | Transmural (full-thickness) |
Crypt Damage percent | 33% | 66% | 100% with surface | 100% |
Epithelial erosion | None | Mild/focal | Evident/diffuse | Evident/diffuse |
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
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-Ldlrtm1Her/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.
As such, 40 mice/sex are used and divided to 5 groups of eight mice/sex according to power analysis, using as a signal factor a 80% power, assuming a 5% significance level and a two sided test after considering 10% loss of the animal during the course of the experiment due to death or any other reason.
Mutant strain, Ldlr−/− C57/BL6 mice (strain B6.129S7-LDLrtm1Her, Jackson Laboratory, Bar Harbor, ME), are used in this study. One group of mice are designated as the low fat negative control (LF-Con) group and will be fed an AIN-93M (diet containing 5.2% fat by weight). Conversely, the other four groups are fed high fat Western style diet (containing 21% by weight milk fat, 0.2% cholesterol), as shown in
Formula (percentage g/100g) | Low fat | Hi fat |
Casein | 19.5 | 20.4 |
DL-Methionine | 0.3 | 0.312 |
Sucrose | 12 | 35.721 |
Corn Starch | 43.3 | 15.691 |
Maltodextrin | 10 | - |
Anhydrous Milk fat | 3.72 | 16.529 |
Soybean Oil | 1.28 | 0.826 |
Cellulose | 5 | 5.23 |
Cholesterol | - | 0.157 |
Mineral Mix, AIN-76 (170915) | 3.5 | 3.661 |
Calcium Carbonate | 0.4 | 0.418 |
Vitamin Mix, Teklad (40060) | 1 | 1.046 |
Ethoxyquin, antioxidant | 0.001 | 0.004 |
Feces of each mouse is collected during the study and air dried. Blood is also collected. Adipose tissue, liver, muscle and heart tissue is harvested and divided to 3 parts. The first part is homogenized, weighed, snap frozen and stored at -80°C until use. The total amount of cholesterol, triglycerides, free fatty acids (FFA) is measured in the plasma samples. Total lipids, total triglycerides and cholesterol is measured in frozen tissues. Also, total lipids will be measured in dried feces for total fat excretion measurement. The other two parts of colon tissues will be used for cytokine measurements and miRNA analysis as described in the first model.
Many miRNAs have been reported to be related to inflammation such as miRNA-155 during the macrophage inflammatory response
A procedure that we used for extracting total RNA from laser capture microdissected (LCM) tissue or from stool is carried out using a guanidinium-based buffer, which comes with the RNeasy isolation Kit®, Qiagen, Valencia, CA, USA, as we have previously detailed
Systematic investigation of miRNA for a universal coverage using microarray expression profiling
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)
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)
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
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.
Number of animals used is 12 mice/sex per group according to power analysis using above mentioned parameters. A total of 72 mice/sex for this objective is divided into 6 groups, each of 12 mice/sex.
Mice used for testing this aim are JAX® Diet-Induced Obesity (DIO) model: http://jaxmice.jax.org/diomice/index.html, and not the LDL mutant strain used in Aim 2. Seven weeks old mice will be used in which the control group mice is fed on 10% kcals from fat diet, and same age matched intervention group mice fed on 60% kcals from fat diet (
60 kcal% fat diet Intervention group | 10 kcal% fat diet Control group | |||
gm% | kcal% | gm% | kcal% | |
Protein | 26 | 20 | 19.2 | 20 |
Carbohydrate | 26 | 20 | 67.3 | 70 |
Fat | 35 | 60 | 4.3 | 10 |
Total | 100 | 100 | ||
kcal/gm | 5.24 | 3.85 | ||
Ingredient | gm | kcal | gm | kcal |
Casein, 30 Mesh | 200 | 800 | 200 | 800 |
L-Cystine | 3 | 12 | 3 | 12 |
Corn Starch | 0 | 0 | 506.2 | 2024.8 |
Maltodextrin 10 | 125 | 500 | 125 | 500 |
Sucrose | 68.8 | 275 | 68.8 | 275.2 |
Cellulose, BW200 | 50 | 0 | 50 | 0 |
Soybean Oil | 25 | 225 | 25 | 225 |
Lard | 245 | 2205 | 20 | 180 |
Mineral Mix S10026 | 10 | 0 | 10 | 0 |
DiCalcium Phosphate | 13 | 0 | 13 | 0 |
Calcium Carbonate | 5.5 | 0 | 5.5 | 0 |
Potassium Citrate, 1 H2O | 16.5 | 0 | 16.5 | 0 |
Vitamin Mix V10001 | 10 | 40 | 10 | 40 |
Choline Bitartrate | 2 | 0 | 2 | 0 |
FD&C Yellow Dye #5 | 0 | 0 | 0.04 | 0 |
FD&C Blue Dye #1 | 0.05 | 0 | 0.01 | 0 |
Total | 773.85 | 4057 | 1055.05 | 4057 |
The statistical methodology for the analysis of study specific aims has been as follows:
Data consists of cytokine levels obtained for three different probiotics. For each probiotic there will be 4 replicates. Preliminary analysis will consist of numerical summaries and side-by-side boxplots for each of the roughly 23 response variables (cytokine levels). Additionally, scatter plots of pairs cytokine variables will be made to explore the relationship among these variables. One-way analysis of variance may be appropriate to formally compare the probiotics with contrasts being used to address specific questions concerning the relationship among these probiotics. Whether additional analyses are required or warranted will depend on the results of the preliminary analysis. For the issue of multiple comparisons among the 23 measured cytokines formal adjustments will be based on the particularly relevant TNF-α and IL-1β as explained in Aim 1.
There are two parts to this aim. The first part involves repeating the steps in Aim 1 for data collected from in vivo assessment. For this aim there will be 6 replicates (6 mice/group/sex). The second part considers whether there is a relationship between the cytokine levels and gene expression levels. Scatter plots are a natural tool for visualizing the relationship between two numeric variables. Since there will be 100 or more genes considered for this aim, this would involve more than 2000 scatter plots. Instead, correlations will be calculated and scatter plots will be assessed for only those with the most extreme numeric value (near -1 or near 1) for correlation coefficient. By specifying a cutoff value to indicate extreme correlations, e.g., above 0.9 or below -0.9 (depending on the experimental results), each cytokine will have associated with it a collection of genes that are strongly correlated with it. The composition of these highly correlated genes across the cytokines will be helpful in identifying the potential role of these genes. Genes whose expression levels are highly correlated with multiple cytokine levels will be studied in the next aim.
We anticipate using about 20 genes obtained from Aim 2. This aim will use 126 mice. The preliminary analysis will consist of treating each of the genes as a univariate response variable modeled on 3 factors: sex (2 levels), probiotic (3 levels), and nutrients (3 levels). Each treatment will have 7 replicates, while the 20 genes can be considered a multivariate response variable. Statistical analysis will be performed using the statistical software R
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
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
LNAsTM 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 Tm of nucleotide capture probes & improving mismatch discrimination of the target leading to better hybridization properties
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
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.
We express our thanks to Dr. Paul W. Vos of the Department of Biosatistics, Eact Carolina University, School of Allied Health Sciences, for his advice on statistical analysis.