Scientific/Medical Studies

Here are the scientific and or medical studies that support the Alternative Protocol I have developed:

Slippery Elm Studies
VSL# 3 Probiotics Studies
Vitamin D, Krill Oil, Vitamin K Studies
Whey Protein supplementation Studies
Butyrate Enemas Studies
Glutamine Supplementation

1)Section 1 – Slippery Elm Studies

Slippery Elm Studies
As reported by the University of Maryland Medical Center:

Overview:
Slippery elm (Ulmus fulva) has been used as an herbal remedy in North America for centuries. Native Americans used slippery elm in healing salves for wounds, boils, ulcers, burns, and skin inflammation. It was also taken orally to relieve coughs, sore throats, diarrhea, and stomach problems.

Slippery elm contains mucilage, a substance that becomes a slick gel when mixed with water. It coats and soothes the mouth, throat, stomach, and intestines; it also contains antioxidants that help relieve inflammatory bowel conditions. Slippery elm also causes reflux stimulation of nerve endings in the gastrointestinal tract leading to increased mucus secretion. The increased mucus production may protect the gastrointestinal tract against ulcers and excess acidity.

There has been little scientific research on slippery elm, but it is often suggested for the following conditions:

Sore throat
Cough
Gastroesophogeal reflux disease (GERD)
Crohn’s disease and ulcerative colitis
Diarrhea
Wounds, burns, boils, psoriasis, and other skin conditions (external)

Plant Description:
Slippery elm is a medium-sized tree native to North America. It can reach well over 50 feet in height and is topped by spreading branches that form an open crown. The red-brown or orange branches grow downward, and the stalkless flowers are arranged in dense clusters. The plant’s leaves are long and green, and they darken in color during the fall. The bark has deep fissures, a gummy texture, and a slight but distinct odor.

Parts Used:
The inner bark is dried and powdered, and used for medicinal purposes.

Available Forms:
Available forms of slippery elm include the following:

Tablets and capsules
Lozenges
Finely powdered bark for making teas or extracts
Coarsely powdered bark for poultices

How to Take It:
Pediatric

Although there are no scientific studies examining the use of slippery elm in children, it is generally considered to be safe. Adjust the recommended adult dose to account for a child’s weight. Most herbal dosages for adults are calculated on the basis of a 150 lb (70 kg) adult. So if the child weighs 50 lb (20 – 25 kg), the appropriate dose of slippery elm would be 1/3 of the adult dose.

Adult

The following are recommended adult doses for slippery elm:

Tea: Pour 2 cups boiling water over 4 g (roughly 2 tablespoons) of powdered bark and then steep for 3 – 5 minutes. Drink 3 times per day.
Tincture: 5 mL 3 times per day. Note: Contains alcohol.
Capsules: 400 – 500 mg 3 – 4 times daily for 4 – 8 weeks. Take with a full glass of water.
Lozenges: follow dosing instructions on label.
External application: Mix coarse powdered bark with boiling water to make a poultice; cool and apply to affected area. Never apply slippery elm to an open wound.

Precautions:
The use of herbs is a time-honored approach to strengthening the body and treating disease. Herbs, however, can trigger side effects and can interact with other herbs, supplements, or medications. For these reasons, you should take herbs with care, under the supervision of a health care provider.

Slippery elm has no serious side effects. Because it coats the digestive tract, it may slow down the absorption of other drugs or herbs. You should take slippery elm 2 hours before or after other herbs or medications you may be taking.

Scientists think slippery elm is safe in pregnancy and during breastfeeding, but no scientific studies have been done to confirm this. The outer bark of the elm tree, however, may contain substances that could increase the risk of miscarriage, so sometimes pregnant women are advised to avoid slippery elm.

Possible Interactions:
There are no scientific reports of slippery elm interacting with any other medications, although it may slow down the absorption of other drugs or herbs (see “Precautions” section).

Alternative Names:
Red elm; Sweet elm; Ulmus fulva; Ulmus rubra

Reviewed last on: 2/3/2009
Steven D. Ehrlich, NMD, private practice specializing in complementary and alternative medicine, Phoenix, AZ. Review provided by VeriMed Healthcare Network.

Supporting Research
Bock S. Integrative medical treatment of inflammatory bowel disease. Int J Integr Med. 2000;2(5):21-29.

Brown AC, Hairfield M, Richards DG, McMillin DL, Mein EA, Nelson CD. Medical nutrition therapy as a potential complementary treatment for psoriasis — five case reports. Altern Med Rev. 2004;9(3):297-307.

Kemper KJ. Slipper elm (Ulmus rubra or U. fulva). The Longwood Herbal Task Force and The Center for Holistic Pediatric Education and Research. Revised Sept. 29, 1999. Online at https://longwoodherbal.org/slipperyelm/slipperyelm.htm.

Langmead L, Dawson C, Hawkins C, Banna N, Loo S, Rampton DS. Antioxidant effects of herbal therapies used by patients with inflammatory bowel disease: an in vitro study. Aliment Pharmacol Ther. 2002;16(2):197-205.

Newall C, Anderson L, Phillipson J. Herbal Medicines: A Guide for Health-care Professionals. London: Pharmaceutical Press; 1996:248.

Rakel D. Rakel: Integrative Medicine, 2nd ed. Philadelphia, PA: Saunders Elsevier Inc.; 2007:43.

Rotblatt M, Ziment I. Evidence-based Herbal Medicine. Philadelphia, Penn: Hanley & Belfus, Inc.;2202:337-338.

Section 2 – VSL# 3 Probiotics Studies

VSL# 3 Probiotics Studies
A) The probiotic preparation, VSL#3 induces remission in patients with mild-to-moderately active ulcerative colitis.

Clin Gastroenterol Hepatol. 2009 Nov;7(11):1202-9, 1209.e1. Epub 2009 Jul 22.

Sood A, Midha V, Makharia GK, Ahuja V, Singal D, Goswami P, Tandon RK.

Department of Gastroenterology and Medicine, Dayanand Medical College and Hospital, Ludhiana, India.

Comment in:

Gastroenterology. 2010 Sep;139(3):1054-6; discussion 1056.

Abstract
BACKGROUND & AIMS: Probiotics can maintain ulcerative colitis (UC) in remission effectively, but little is known of their ability to induce remission. We conducted a multicenter, randomized, double-blind, placebo-controlled trial of a high-potency probiotic, VSL#3, for the treatment of mild-to-moderately active UC.

METHODS: Adult patients with mild-to-moderate UC were assigned randomly to groups that were given 3.6 x 10(12) CFU VSL#3 (n = 77) or placebo (n = 70), twice daily for 12 weeks. The primary end point was a 50% decrease in the Ulcerative Colitis Disease Activity Index (UCDAI) at 6 weeks. The secondary end points included remission by 12 weeks and reduction in total individual UCDAI parameters from baseline at 12 weeks. Intention-to-treat analysis was performed.

RESULTS: At week 6, the percentage of patients with an improvement in UCDAI score that was greater than 50% was significantly higher in the group given VSL#3 (25; 32.5%) than the group given placebo (7; 10%) (P = .001). At week 12, there were 33 patients given VSL#3 (42.9%) who achieved remission, compared with 11 patients given placebo (15.7%) (P < .001). Furthermore, significantly more patients given VSL#3 (40; 51.9%) achieved a decrease in their UCDAI that was greater than 3 points, compared with those given placebo (13; 18.6%) (P < .001). The VSL#3 group had significantly greater decreases in UCDAI scores and individual symptoms at weeks 6 and 12, compared with the placebo group.

CONCLUSIONS: VSL#3 is safe and effective in achieving clinical responses and remissions in patients with mild-to-moderately active UC.

PMID: 19631292 [PubMed – indexed for MEDLINE]

B) Immunosuppressive effects via human intestinal dendritic cells of probiotic bacteria and steroids in the treatment of acute ulcerative colitis.

Inflamm Bowel Dis. 2010 Aug;16(8):1286-98.

Ng SC, Plamondon S, Kamm MA, Hart AL, Al-Hassi HO, Guenther T, Stagg AJ, Knight SC.

Antigen Presentation Research Group, Faculty of Medicine, Imperial College London, Northwick Park and St Mark’s Campus, Watford Road, Harrow, UK.

Abstract
BACKGROUND: In ulcerative colitis (UC) gut bacteria drive inflammation. Bacterial recognition and T-cell responses are shaped by intestinal dendritic cells (DCs); therapeutic effects of probiotic bacteria may relate to modulation of intestinal DC. The probiotic mixture, VSL#3, increases interleukin (IL)-10 and downregulates IL-12p40 production by DC in vitro. We evaluated in vivo effects of oral VSL#3 and steroids on colonic DC in patients with acute UC.

METHODS: Rectal biopsies were obtained from patients with active UC before and after treatment with VSL#3, corticosteroids, or placebo, and from healthy controls. Myeloid colonic DC were studied from freshly isolated lamina propria cells using multicolor flow cytometry. Surface expression of activation markers, CD40, CD86, pattern recognition receptors, Toll-like receptor (TLR)-2 and TLR-4 were assessed. Changed function was measured from ongoing intracellular IL-10, IL-12p40, IL-6, and IL-13 production.

RESULTS: Acute UC colonic myeloid DC were producing more IL-10 and IL-12p40 than control DC (P = 0.01). In VSL#3-treated patients DC TLR-2 expression decreased (P < 0.05), IL-10 production increased and IL-12p40 production decreased (P < 0.005); 10/14 patients on VSL#3 showed a clinical response. Corticosteroids also resulted in increased IL-10 and reduced IL-12p40 production by DC. Conversely, in patients on placebo, TLR-2 expression and intensity of staining for IL-12p40 and IL-6 increased (all P < 0.05); 5/14 patients on placebo showed a clinical response (P = NS).

CONCLUSIONS: Despite small numbers of human colonic DC available, we showed that treatment of UC patients with probiotic VSL#3 and corticosteroids induced “favorable” intestinal DC function in vivo, increasing regulatory cytokines and lowering proinflammatory cytokines and TLR expression. These effects may contribute to therapeutic benefit.

PMID: 20155842 [PubMed – indexed for MEDLINE]

C) Gut microbial diversity is reduced by the probiotic VSL#3 and correlates with decreased TNBS-induced colitis.

Inflamm Bowel Dis. 2010 Jun 17. [Epub ahead of print]

Uronis JM, Arthur JC, Keku T, Fodor A, Carroll IM, Cruz ML, Appleyard CB, Jobin C.

Department of Medicine, Division of Gastroenterology and Hepatology and Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

Abstract
BACKGROUND: Compositional changes within the normal intestinal microbiota have been associated with the development of various intestinal inflammatory disorders such as pouchitis and inflammatory bowel diseases (IBD). Therefore, it has been speculated that manipulation of a dysbiotic intestinal microbiota has the potential to restore microbial homeostasis and attenuate inflammation.

METHODS: We performed community composition analyses by terminal restriction fragment length polymorphism (T-RFLP) of the bacterial 16S ribosomal RNA gene to investigate the impact of the probiotic VSL#3 on colonic microbial community composition and development of trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats.

RESULTS: TNBS-induced chronic colitis was significantly reduced in VSL#3-fed rats compared to controls (P < 0.05). T-RFLP analysis revealed distinct microbial communities at luminal versus mucosal sites. Within the luminal microbiota, chronic colitis was associated with an overall decrease in bacterial richness and diversity (Margalef’s richness, P < 0.01; Shannon diversity, P < 0.01). This decrease in luminal microbial diversity was enhanced in TNBS-treated rats fed VSL#3 (Margalef’s richness, P < 0.001; Shannon diversity, P < 0.001) and significantly correlated with reduced clinical colitis scores (Pearson correlation P < 0.05).

CONCLUSIONS: Our data demonstrate that the probiotic VSL#3 alters the composition of the intestinal microbiota and these changes correlate with VSL#3-induced disease protection. (Inflamm Bowel Dis 2010;).

PMID: 20564535 [PubMed – as supplied by publisher]PMCID: PMC2953593 [Available on 2011/12/1]

Scientific/Medical Studies

D) Probiotics and Gastrointestinal Disease: Clinical Evidence and Basic Science

Antiinflamm Antiallergy Agents Med Chem. Author manuscript; available in PMC 2010 September 29.
Published in final edited form as:
Antiinflamm Antiallergy Agents Med Chem. 2009 September 1; 8(3): 260–269.
doi: 10.2174/187152309789151977. PMCID: PMC2947383
NIHMSID: NIHMS233283

Elaine O. Petrof*
Elaine O. Petrof, Department of Medicine, GIDRU and Division of Infectious Diseases, Queen’s University, Kingston, ON Canada;
*Address correspondence to this author at the Queen’s University, Dept. of Medicine, GIDRU wing, 76 Stuart St., Kingston, ON K7L 2V7, Canada; Email: eop@queensu.ca

Abstract
Introduction
Examples of Probiotics VSL#3: A Mixture of Different Gram-positive Bacteria
E. Coli Nissle 1917: A Gram-negative Probiotic
LGG and L. Reuteri: Gram-positive Probiotics
Lactobacillus Rhamnosus GG
Lactobacillus Reuteri
Saccharomyces Boulardii: A Probiotic Yeast
Conclusions
References Abstract
Our intestinal microbiota serve many roles vital to the normal daily function of the human gastrointestinal tract. Many probiotics are derived from our intestinal bacteria, and have been shown to provide clinical benefit in a variety of gastrointestinal conditions. Current evidence indicates that probiotic effects are strain-specific, they do not act through the same mechanisms, and nor are all probiotics indicated for the same health conditions. However, they do share several common features in that they exert anti-inflammatory effects, they employ different strategies to antagonize competing microorganisms, and they induce cytoprotective changes in the host either through enhancement of barrier function, or through the upregulation of cytoprotective host proteins. In this review we focus on a few selected probiotics – a bacterial mixture (VSL#3), a Gram-negative probiotic (E. coli Nissle 1917), two Gram-positive probiotic bacteria (LGG, L. reuteri), and a yeast probiotic (S. boulardii) – for which sound clinical and mechanistic data is available. Safety of probiotic formulations is also discussed.Keywords: Probiotics, intestinal microbiota, inflammation, colitis

Introduction
Bacteria form an integral component of the normal daily function of the human body. Our bacteria outnumber our human cells by 10 to 1[1], and the number of bacteria in our gastrointestinal tract measures in the range of 1012 in magnitude. These microorganisms play a key role in human metabolism and nutrition. They synthesize compounds such as vitamin K and B vitamins, they break down cholesterol, they produce short chain fatty acids such as butyrate, and digest dietary polysaccharides that would not otherwise be salvageable for energy use [2, 3]. They also contribute to host defense by priming the dendritic cells of the immune system [4], and they inhibit the colonization of pathogenic bacteria through competition for binding sites along the intestinal epithelial cell surface, a phenomenon known as “colonization resistance” [5]. In addition, they produce bactericidal products, such as small molecular weight peptides called bacteriocins, that kill other pathogenic bacteria [6]. Bacteriocins produced by lactobacilli, for example, are able to kill common food-borne pathogens such as Listeria monocytogenes [7], Bacillus cereus, Clostridium botulinum and Staphylococcus aureus [6]. Our commensal bacteria also provide defense by competing with pathogenic bacteria for nutrients. For the most part, we have developed a harmonious, symbiotic relationship with our commensal microbes: we provide them with food and a habitat, and in turn they play a key role in human metabolism and nutrition, and protect us from harmful pathogens (Fig. (1)). Hence, it comes as no surprise that many probiotic bacteria used today were originally isolated from our expansive repertoire of human commensal bacteria.

Beneficial effects of commensal and probiotic bacteria

Beneficial effects of commensal and probiotic bacteria
Schematic of the many vital roles of bacteria in the intestine. Commensal/probiotic bacteria are purple, epithelial cells are in light blue, apical and basolateral sides of epithelial cells are also indicated. Our intestinal bacteria serve an important role by 1) Inhibiting pathogen growth through production of antimicrobials; 2) Enhancing tight junction and barrier function; 3) Priming dendritic cells (drawn in black) and the immune system; 4) Assisting in digestion and breakdown of micro-nutrients from otherwise undigestable material (indicated in light green), synthesis of short chain fatty acids (SCFAs); 5) Synthesizing key vitamins such as vitamin K and B vitamins.

The United Nations and World Health Organization define probiotics as “live microorganisms which, when administrated in adequate amounts, confer a health benefit on the host”. To the average person, the quantity of probiotic bacteria now available on the market is daunting in number, and this number only continues to rise. Regrettably, many of the bacteria advertised as probiotics have never been evaluated for clinical efficacy. Since they are considered as food and food supplements instead of pharmacologic agents, probiotics are not regulated with the same standards as drugs, even though they are often marketed with the same health claims as many drugs. Several studies of marketed probiotics have found that the viability of organisms and the composition of the probiotic formulation are often not as advertised. For example, a study in Britain tested several probiotic supplements and found that the numbers of viable bacteria, and even the identity of the actual strains of bacteria, were not correctly indicated by the product labels [8]. Lack of consistency in terms of dose, species and strain used, origin of strain, delivery vehicle (pill, liquid, food, etc.), makes interpretation of the available data from clinical trials even more difficult. Indeed, because probiotics are not inert chemical compounds but are live organisms which (like all bacteria) possess the capacity to mutate/change their phenotypes, they present their own unique challenges for regulatory agencies and for researchers performing clinical trials on probiotics [9, 10].Recognizing this fact, in 2002 the United Nations FAO/WHO Working Group generated new guidelines for the development and evaluation of probiotics found in foods [11]. These guidelines included the following recommendations: (1) appropriate methods to identify genus and species of the probiotic strain, (2) in vitro tests to screen potential probiotic organisms as well as target-specific in vitro tests to correlate with in vivo results, (3) standards to ensure that a probiotic strain is safe and free of contamination, (4) In vivo studies using animals and humans (clinical trials) to test specific health claims of the probiotic in question, and (5) guidelines on how the probiotic should be labeled, including proper storage conditions and minimum viable numbers of organism at the end of the indicated shelf-life.This review is not meant to be a comprehensive treatise on all probiotics, but rather will attempt to address some basic concepts of probiotics and to illustrate the fact that “not all probiotics are created equal”. Current evidence indicates that probiotic effects are strain-specific, they do not act through the same mechanisms, and nor are all probiotics indicated for the same health conditions. We will focus on a few selected probiotics, of different species, for which clinical and mechanistic data is available. Other probiotic reviews may provide the reader with more information on the subject [12-14].

Conclusions
ReferencesExamples of Probiotics VSL#3: A Mixture of Different Gram-positive Bacteria

The probiotic VSL#3 is a mixture of 8 different species of bacteria, namely Streptococcus salivarius subsp. thermophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacteria longum, Bifidobacteria infantis, and Bifidobacteria breve. These bacteria are all Gram-positive bacteria and they were originally isolated from the stool of a healthy human volunteer [15]. The bacteria in VSL#3 survive well in the human gastrointestinal tract, being recoverable in stool as viable bacteria after ingestion [15].

Clinical Evidence
VSL#3 has been shown to improve the clinical outcome of chronic intestinal inflammation in clinical trials. In a randomized, double-blinded, placebo-controlled trial of 40 patients suffering from at least 3 relapses per year of recurrent pouchitis, relapse occurred in only 15% (3/20) of the patients assigned to receive twice daily dosing of VSL#3 whereas those patients assigned to receive twice daily placebo all relapsed within four months [16]. Similar results were obtained in a subsequent study of once daily dosing of VSL#3 in patients with recurrent pouchitis. Out of 36 patients enrolled, remission was maintained after 12 months in 17 patients (85%) on VSL#3 and in one patient (6%) on placebo [17]. A standardized questionnaire was used as a scoring tool to determine quality of life assessment, and scores were much higher in the VSL#3 group as compared to placebo [17]. Another study examining the onset of acute pouchitis in the year following ileal pouch-anal anastomosis after colectomy in patients with ulcerative colitis suggested that VSL#3 may be helpful as prophylactic treatment in patients with pouchitis, in addition to its utility in maintenance therapy [18].
In an open label trial to determine whether patients with active UC would benefit from VSL#3, patients with mild to moderate UC (n=34) were given the probiotic mixture for 6 weeks and then reassessed. Using intention to treat analysis, a remission rate of 53% was noted in the VSL#3 group, no response was seen in 9% and worsening of symptoms noted in 9%, with 5% failing to complete the final assessment [19]. An interesting component of this study was the determination of biopsy-associated bacteria using nucleic acid-based sequencing of 16S rRNA to test for the presence of VSL#3 species. The investigators were able to demonstrate that two of the bacteria found in VSL#3, S. salivarius subspecies thermophilus and B. infantis, were detectable in the biopsies of 3 of the patients in remission. Of interest, none of the other VSL#3 bacteria were detected, which led the authors to postulate that these two components of VSL#3 may therefore be the primary active ingredients of the bacterial mixture in vivo and led them to suggest that these strains may be of interest in future studies of probiotic treatment of UC.

Basic Science

In the laboratory, VSL#3 displays many interesting properties both in vitro and in vivo which may account for its clinical activities. VSL#3 attenuates intestinal inflammation in the IL-10 deficient mouse model of enterocolitis, resulting in a decrease in TNF-alpha secretion and an improvement in histologic scores. In addition, less epithelial hyperplasia, less mucosal ulceration, and decreased neutrophilic infiltration was observed in the VSL#3 group as compared to controls [20]. Barrier function was also assessed using mannitol flux assays and after 4 weeks of VSL#3 treatment, barrier function normalized in these mice. Using the T84 human intestinal epithelial cell line it was further shown that VSL#3 and products secreted by these bacteria enhanced intestinal epithelial barrier function in vitro, and pre-exposure of T84 monolayers to VSL#3 provided a dose-dependent decrease in cellular invasion by the pathogenic bacteria Salmonella enterica serovar Dublin. A subsequent study corroborated these findings and further found that VSL#3 upregulated the expression of several mucins which are postulated to play an important cytoprotective role in host defense against pathogens [21].

Further work on VSL#3 showed this probiotic secreted products which inhibited the key pro-inflammatory transcription factor NF-kappaB, and inhibited degradation of IkappaB (an NF-kappaB inhibitor), by blocking proteasome activity in intestinal epithelial cells [22]. In addition, products produced by VSL#3 induced the expression of heat shock proteins which protected cells against oxidant injury. Heat shock proteins were induced through activation of the transcription factor HSF-1 (Heat Shock Factor-1). Both the live VSL#3 bacteria and secreted bacterial factors were able to elicit heat shock protein induction in intestinal epithelial cells, in a dose-dependent manner [22].

Two studies tested DNA isolated from VSL#3 bacteria for anti-inflammatory activity in two different models of experimental colitis [23, 24]. In the first study, Il-10 deficient mice were fed DNA from VSL#3 for 2 weeks and then their colons removed for analysis. The animals receiving VSL#3 DNA showed less histologic disease and a decrease in TNF-alpha as compared to controls [23]. In the second study, a DSS animal model of experimental colitis was used. Animals were pretreated with DNAse-treated, methylated or unmethylated DNA from VSL#3, or E. coli DNA for 10 days prior to DSS exposure and then colitis severity was assessed after 7 days of DSS treatment. It was found that the DSS-treated groups who received VSL#3 probiotic DNA – and the E. coli DNA – displayed less severe colitis than the groups that received DNAase-treated probiotic and methylated probiotic DNA. Further studies using TLR9-deficient mice led the authors to conclude that TLR9 signaling played an essential role in mediating these anti-inflammatory effects [24].

Taken together, the data from all of these studies suggest that VSL#3 may act through several different mechanisms to protect the intestine against inflammatory injury and colitis. Given that VSL#3 is a complex bacterial mixture of multiple different types of bacteria, it is likely that different species within the mixture account for so many of these different mechanisms, and one would expect that many mechanisms of action for VSL#3 have yet to be elucidated.

Conclusions
ReferencesE. Coli Nissle 1917: A Gram-negative Probiotic
E. coli Nissle 1917 is unusual as a probiotic because it is a Gram-negative bacillus, whereas most bacterial probiotics are Gram positive bacteria. It also has a very long and interesting history. First discovered in 1917, the microbiologist Alfred Nissle isolated this strain from the stool of a soldier in World War I who was relatively unaffected by an outbreak of Shigellosis [25]. Nissle characterized the strain of E. coli, and then marketed it for the treatment of infectious diarrhea long before the availability of conventional antibiotics. This strain has now been on the market for nearly 100 years for the treatment of diarrhea, and in Germany it is used as an acceptable alternative to mesalazine for maintaining remission of ulcerative colitis [26].

Clinical Evidence
Several studies comparing E. coli Nissle to the gold standard mesalazine have shown equivalent efficacy of E. coli Nissle in maintaining remission of ulcerative colitis [26-29]. In 1997, a double-blinded study of 120 patients with inactive ulcerative colitis were given either mesalazine or the probiotic E. coli Nissle for 12 weeks and then relapse rates, relapse-free times and global assessments were compared. Relapse rates of 11.3% were reported for mesalazine and 16.0% for E. coli Nissle, with a relapse-free time of 103 +/- 4 days for mesalazine and 106 +/- 5 days for E. coli Nissle 1917. Global assessments and tolerability were similar for both groups [28]. Another clinical trial in the U.K. showed similar results, with E. coli Nissle being as effective as mesalazine in maintaining remission of ulcerative colitis [29]. A larger double-blind clinical trial followed, with 327 UC patients in disease remission randomized to receive either mesalazine or E. coli Nissle for 12 months, and then assessed using endoscopic and histological activity indices. The relapse rate by “per protocol” analysis was 40/110 (36.4%) in the E. coli Nissle group and 38/112 (33.9%) in the mesalazine group (significant equivalence p = 0.003). Using “Intention to Treat” analysis, including all patients who did not strictly follow protocol but who took at least one dose of the study medication, a relapse rate of 45.1% was calculated for the Nissle group and 37.0% for the mesalazine group (significant equivalence p = 0.013). No serious adverse events were reported [27].

Basic Science
Unlike the harmful strains of E. coli that cause human disease, the probiotic E. coli Nissle 1917 lacks many of the virulence factors normally found in its more pathogenic relatives. In addition, it possesses several “fitness factors” which confer a survival advantage over both pathogenic and non-pathogenic strains of E. coli [26]. Based on clinical trial data, clearly, E. coli Nissle must confer some anti-inflammatory and cytoprotective effects on the bowel mucosa, but the mechanisms by which this probiotic exerts it anti-inflammatory effects remain unclear. Several groups have shown that Nissle confers a protective effect in animal models of inflammatory colitis [30-32]. In an attempt to determine whether the protective effects of Nissle in inflammatory colitis were TLR-mediated, a DSS model of experimental colitis was used. Wild-type, TLR2-deficient and TLR4-deficient mice were fed Nissle or saline control, and then the animals were assessed for disease activity, mucosal damage, and cytokine secretion. E. coli Nissle improved colitis scores and decreased TNF-alpha and MCP-1 secretion in the wild-type mice but there was no improvement in DAI score, microscopic inflammation or in neutrophil recruitment (MPO activity) observed with the TLR-2 or TLR-4 knockout mice [31], suggesting that the anti-inflammatory mechanism of E. coli Nissle 1917 may occur through TLR-2- and TLR-4-dependent pathways, presumably mediated through NF-kappaB. However, another study performed in the human intestinal epithelial cell line HCT15 showed that E. coli Nissle 1917 inhibited TNF-alpha-induced IL-8 production but did not affect NF-kappaB activation, nuclear translocation, or DNA binding [33]. Thus, the mechanism(s) by which Nissle exerts its anti-inflammatory effects still continues to be a subject of intense investigation.

Two other properties also likely contribute to E. coli Nissle’s effectiveness as a probiotic organism. Like VSL#3, E. coli Nissle may enhance intestinal barrier function. In experiments measuring transepithelial resistance of infected T84 cells, E. coli Nissle protected T84 monolayers against barrier disruption by enteropathogenic E. coli (EPEC) infection [34]. Based on DNA microarray analysis, the authors concluded that the mechanism involved downregulation of PKCzeta activity (one of the protein kinase C isoforms important in epithelial barrier disruption), and modulation of tight junction protein expression, such as zonula occludin-2 (ZO-2) [34]. However, unlike VSL#3, this protective barrier effect was not seen with E. coli Nissle when monolayers were infected with the invasive pathogen Salmonella enterica serovar Dublin [21], indicating that probiotics may not all possess the same protective capabilities against all intestinal pathogens.
Another group of investigators showed that E. coli Nissle upregulates human beta-defensin expression [35, 36]. Defensins are antimicrobial peptides produced by the gut which, as the name implies, protect and defend the host against bacterial pathogens. There is an association between IBD and low defensin levels [37], therefore it is possible that Nissle provides protection through a defensin mechanism, for example by limiting the adherence of certain harmful populations of bacteria to the intestinal epithelial cell lining of the gut.
LGG and L. Reuteri: Gram-positive Probiotics

The next two probiotics that will be discussed (LGG, L. reuteri) are both Gram- positive bacilli of the Lactobacillus genus. Of all bacteria, Lactobacillus species are probably the most commonly used as probiotics. They are generally regarded as safe because of their long history of use in the food and dairy industry, which probably accounts for their popularity as probiotic choices. Interestingly, however, overall they comprise only a small portion of the natural intestinal microbiota [38]. As the name implies, they all produce lactate and lactic acid.

Conclusions
ReferencesLactobacillus Rhamnosus GG

Lactobacillus rhamnosus GG, or LGG was first discovered by Sherwood Gorbach and Barry Goldwin. In a search for good candidate probiotic organisms, bacteria were isolated from the stool specimens of healthy human volunteers and screened for their ability to survive bile and acid exposure; their adherent properties to epithelial cells were also tested [39]. Out of this screening process, LGG was first discovered. Subsequent studies were performed in which 76 volunteers were given LGG either in the form of a frozen concentrate of bacteria or a fermented milk preparation and then feces analyzed for the presence of viable LGG [40]. LGG was recovered from 87% of volunteers after four days of stopping the LGG and in 33% of volunteers one week after terminating the treatment, confirming the ability of LGG to survive in the human GI tract. Further tests on the viability of LGG, ingested in the form of gelatin capsules, have confirmed that Lactobacillus GG also survives well as a capsular formulation and can be recovered from the feces of humans after 3 days of daily ingestion of capsules containing 1.2 × 1010 CFU of bacteria [41].

Clinical Evidence
Several clinical trials have been performed using LGG for the treatment of diarrhea, and the organism has been used successfully in the treatment of acute diarrhea in pediatric populations [42-45]. LGG appears most effective against rotavirus diarrhea, resulting in a decrease both in duration and frequency of diarrhea in this group of patients [44, 46]. It is also effective in the treatment of nosocomial and antibiotic-associated diarrhea [47, 48]. A meta-analysis examining the use of probiotics for antibiotic-associated diarrhea included a total of 6 trials using LGG, which yielded a total combined number of 817 patients. The calculated weighted event rates were 8% for the LGG group and 27% for the control, resulting in an average NNT (number to treat) of six [49, 50]. In other words, to prevent one patient from developing antibiotic-associated diarrhea, one would need to treat 6 patients receiving antibiotics with LGG. Results from another meta-analysis yielded similar results [51]. Therefore, most experts agree that LGG is one of the probiotics for which there is the most evidence of clinical efficacy, particularly for antibiotic-associated diarrhea and for rotavirus diarrhea in children [50, 52-54].

Basic Science
LGG has been shown to produce antimicrobial substances which inhibit other intestinal bacteria such as Clostridium and other anaerobes, Pseudomonas, Salmonella and E. coli, as well as Staphylococcus and Streptococcus species [55]. However, based on the small molecular size and other properties, the authors concluded that rather than resembling a bacteriocin, the antimicrobial substance was more likely to be similar to the microcins produced by E. coli [55]. The antimicrobial from LGG showed no inhibitory effect on other lactobacilli.

LGG also enhances barrier function. In a study of children with mild to moderately active Crohn’s disease, daily administration of LGG resulted in improved intestinal permeability as measured by a cellobiosemannitol sugar permeability test, with a maximal effect being seen after 12 weeks of probiotic treatment [56]. In animals, LGG also improves intestinal barrier function [57]. Two week old rats were gavaged with either cow’s milk, milk plus LGG, or water and Ussing chambers were then used to assess intestinal permeability. LGG was found to protect against the increased gut permeability induced by cow’s milk in suckling rats. In vitro, LGG pre-treatment of epithelial cells results in protection of barrier function against the intestinal pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 [58]. In this study, cells were pretreated with LGG, infected with E. coli O157:H7 and then examined using electron microscopy. Although not able to prevent cytoplasmic vacuolization, treatment with LGG protected cellular architecture, particularly tight junction disruption, caused by E. coli O157:H7. It was further noted that LGG pretreatment partially protected against E. coli O157:H7-induced loss of barrier function in human intestinal T84 cells grown in monolayers, as measured by transepithelial resistance (TER). Collectively, these human, in vivo, and in vitro studies provide evidence that LGG protects intestinal barrier function.

Other potential mechanisms that may contribute to the probiotic effects of LGG include the induction of cytoprotective heat shock proteins [59]. Small molecular weight compounds synthesized and released by LGG induce a time and concentration-dependent induction of cytoprotective heat shock proteins Hsp25 and Hsp72 in intestinal epithelial cells. In this study, pretreatment of cells with LGG factors (LGGCM) caused activation of the transcription factor Heat Shock Factor – 1, upregulated Hsp72 expression and Hsp25 expression on microarray analysis and real-time PCR, and protected intestinal epithelial cells against oxidant injury. When siRNA was used to silence the expression of Hsp25 and Hsp72, it was found that Hsp72 (and not Hsp25) played the major role in protecting the intestinal epithelial cells against oxidant injury. This finding is in keeping with other published data, showing that Hsp72 stabilizes and prevents denaturation of cellular proteins, and protects intestinal epithelial cells against oxidant-induced damage [60]. Two proteins secreted by LGG have been described, p40 and p75, which also likely contribute to protecting intestinal epithelial cells against oxidant injury. These LGG-derived proteins were found to attenuate hydrogen-peroxide-induced oxidant injury of intestinal monolayers in vitro [61]. In addition, they play a role in inhibiting cytokine-induced apoptosis in human and mouse epithelial cells [62].

Conclusions
ReferencesLactobacillus Reuteri
Lactobacillus reuteri was named after the German microbiologist Gerhard Reuter and was first recognized as a distinct species of the Lactobacillus genus in 1980. Studies indicate that each animal has a species-specific strain of L. reuteri which has presumably evolved to adapt to its particular host and L. reuteri is a ubiquitous colonizer of the intestine, having been isolated from the gastrointestinal tract of humans and of many animal species including pigs, rats, and even poultry [63-65].

Clinical Evidence
Clinical trials indicate that, like LGG, L. reuteri may be useful for the treatment of acute diarrhea, particularly in children [52]. In one study, 40 children aged 6 to 36 months hospitalized with acute diarrhea were randomized to receive either human-origin L. reuteri or placebo for the length of hospitalization or up to 5 days. Analysis of the stool samples showed an increase of roughly 5 log of L. reuteri after 48 h in the L. reuteri group. The total amount of measurable lactobacilli also increased by 2 logs in the L. reuteri group after 48 h. Levels of total lactobacilli in feces were low in the placebo group, and L. reuteri was not detectable. L. reuteri decreased the duration of watery diarrhea as compared to the placebo-treated group, with watery diarrhea persisting in 26% of the L. reuteri group as compared to 81% in the control group by the second day [66]. Results from another study focused primarily on pediatric rotavirus gastroenteritis were similar, with a decrease in the duration of diarrhea noted in the L. reuteri group as compared to placebo [67].

Basic Science
In an IL-10 deficient animal model of experimental colitis, L. reuteri colonization was shown to attenuate the development of colitis [68]. Its anti-inflammatory properties are likely not limited to effects on the intestinal mucosa, as demonstrated by a study using monocyte-derived macrophages from children with Crohn’s disease (both with active disease and in remission). It was found that L. reuteri suppressed TNF-alpha and MCP-1 release from activated cells, leading the authors to suggest that L. reuteri may decrease inflammation by suppressing both monocyte and macrophage chemotaxis and cellular activation. Further mechanistic investigations revealed that, unlike other lactobacilli [23, 69], L. reuteri achieved suppression of TNF-alpha release through the suppression of c-Jun phosphorylation and AP-1 activation rather than through inhibition of NF-kappaB – in fact, NF-kappaB activity appeared to be unaffected [70]. It is interesting to note that, in contrast, the same group did report suppression of TNF-alpha-induced NF-kappaB activation by L. reuteri in a different type of cell (human myeloid cells), which in this case led to increased cellular apoptosis; these effects were mediated at least in part through inhibition of ubiquitination of the inhibitor of NF-kappaB (IkappaB-alpha) and through MAPK signaling [71].

Under anaerobic growth conditions, Lactobacillus reuteri produces a potent antimicrobial compound called reuterin, which is a β-hydroxypropionaldehyde derivative of glycerol. This compound exerts broad-spectrum antimicrobial activity against multiple pathogenic intestinal bacteria. Several human-derived strains of L. reuteri tested for their ability to inhibit growth of enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), Salmonella enterica, Shigella sonnei and Vibrio cholerae were all able to inhibit growth of the intestinal pathogens but to varying degrees, indicating some strains are more effective than others [72]. In addition, the antimicrobial capacity of the L. reuteri strains did not always correlate with their level of reuterin production, indicating that other as-yet-unidentified antimicrobial factors may be synthesized by these bacteria.

Conclusions
ReferencesSaccharomyces Boulardii: A Probiotic Yeast
A yeast distantly related to Saccharomyces cerevisiae (Brewer’s yeast), Saccharomyces boulardii is one of the few microorganisms commonly used as a probiotic which is not of bacterial origin. It was first described in the 1920’s by the microbiologist Henri Boulard, who isolated the yeast from the skins of lychee nuts from his travels in Indochina [73]. The yeast, which now bears his name, is a substrain of Saccharomyces cerevisiae, has an optimal growth temperature of 37° C and survives passage through all levels of the GI tract. It does not permanently colonize the colon, however, and kinetic studies indicate that it disappears within 5 days of discontinuing administration [74].

Clinical Evidence
Several clinical trials of S. boulardii have demonstrated its efficacy in the prevention of antibiotic-associated diarrhea and in treating recurrent Clostridium difficile-associated disease [73, 75, 77-82]. Clostridium difficile-associated disease, or CDAD, is caused by Clostridium difficile, a toxin-producing bacteria which usually results in a diarrheal illness. When severe, CDAD can cause colitis which can be life-threatening and even fatal. A risk factor for acquiring CDAD is antimicrobial use, and CDAD accounts for 15-25% of antibiotic-associated diarrhea [83]. Many people receive treatment for CDAD, experience multiple relapses and never completely clear their infection. S. boulardii has been shown in several studies to provide clinical benefit in ameliorating antibiotic-associated diarrhea as well as to improve the eradication of recurrent C. difficile colitis [78-81]. S. boulardii has been shown to decrease recurrent C. difficile disease, especially when administered in combination with high-dose vancomycin [82]. Some suggest that S. boulardii may decrease CDAD recurrences by up to 50% [84].
Although a promising probiotic candidate for the treatment of AAD and recurrent CDAD, experts recommend that caution be used with this probiotic. There have been several reports of fungemia associated with S. boulardii use, and there is even a report of a hospital outbreak of Saccharomyces boulardii bloodstream infections occurring in three patients who did not actually receive the probiotic, but who shared the same hospital ward as other patients who had received S. boulardii [85]. Therefore, concerns have been raised about the safety of administering this live yeast as a probiotic, especially in the elderly, the very ill and in immunocompromised patient populations [86, 87].

Basic Science
Saccharomyces boulardii has some interesting properties: it adheres well to epithelial cells throughout all levels of the gastrointestinal tract, and it produces a protease which can cleave C. difficile toxins A and B [88, 89]. In addition, S. boulardii administration in rodent models stimulates secretory IgA and induces a specific IgA immune response to C. difficile toxin A [90, 91]. This has particularly important implications for the treatment of CDAD, because evidence suggests that IgA titers are protective against the toxin [92].

It has been demonstrated that S. boulardii protects and preserves epithelial barrier function in the setting of EPEC infection and decreases the extent of bacterial invasion and translocation [76]. Our own studies have shown that heat-inactivated media taken from cultures of S. boulardii protects epithelial cells in vitro against the injurious effects of C. difficile toxin A on barrier function. Intestinal epithelial cells were grown on transwell permeable supports and pretreated overnight with heat-inactivated S. boulardii CM prior to addition of 100ng/ml of C. difficile toxin A to the apical compartment. TER measurements were then taken using an EVOM chopstick voltohmmeter one hour prior to addition of toxin and then at intervals after toxin addition as indicated (Fig. (2)). The cells pretreated with S. boulardii CM displayed less loss of barrier function than controls, suggesting that S. boulardii may synthesize and secrete additional, yet-to-be-identified factors which protect intestinal epithelial cells against injury.

Heat-treated media from S. boulardii protects epithelial barrier function against C. difficile toxin A.

Heat-treated media from S. boulardii protects epithelial barrier function against C. difficile toxin A.
Human intestinal Caco2 Bbe cells were grown to confluence in monolayers on transwell supports. S. boulardii conditioned media (SB-CM) was boiled for 10 minutes and then added to the apical (AP) and basolateral (BL) sides (1:10) and allowed to incubate overnight (boiled media was used as control). C. difficile toxin A (kind gift of Dr. C. Pothoulakis) was then added to the apical side of the cells and the transepithelial resistance of the monolayers was measured every 30 minutes for 120 minutes with a chopstick voltohmmeter. Schematic of the experimental design is shown on the left. SB-CM protected the intestinal epithelial barrier function, with only 17% loss of barrier function recorded after 120 minutes as compared to 69% loss of barrier function noted in the C. difficile toxin A-treated group that received control media only (n=3, error bars calculated as S.E.M.)

Conclusions
Although more clinical trials are needed, what we know to date indicates that probiotics are useful for certain clinical indications (e.g. VSL#3 for pouchitis, E. coli Nissle for some subsets of UC patients, LGG and L. reuteri for rotavirus diarrhea, S. boulardii for certain subsets of C. difficile disease, LGG and S. boulardii for antibiotic-associated diarrhea, etc). It can also be seen that, in addition to differences in clinical use and composition, probiotics vary in their proposed mechanisms of action. Nonetheless, many of them seem to have cytoprotective (induction of heat shock protein, mucin expression) as well as anti-inflammatory effects (often by affecting the same inflammatory pathways but at different steps – see Fig. (3)). There is an urgent need to better define their appropriate clinical use, especially as probiotics are not always benign [87, 93]. There are many reports of probiotics causing infections, and in particular there is an increased risk of invasive infection in patients with indwelling intravenous catheters [86, 94]. Probiotic use can even turn deadly: in one clinical trial examining probiotics for pancreatitis, the trial had to be stopped early because the probiotic group actually fared much worse [95]. There were 24 deaths in the probiotic group and 9 in the control group, and 9 cases of bowel ischemia reported in the probiotic group, whereas none were seen in the control group [95]. The results of this trial provide a good illustration of how we still do not entirely understand the complex mechanisms of action of probiotics, and of the urgent need to better determine the scientific basis for their function. This clinical trial used rigorously-tested probiotics, so it is not difficult to imagine the additional potential dangers lurking in probiotics of inadequately controlled quality [8, 96]. Fortunately, probiotic use is generally safe and it shows much promise for clinical efficacy in many gastrointestinal disorders, but we still have a long way to go. Ongoing research in this area, and a better understanding of host-microbial interactions through ongoing research on the human microbiome [1, 97, 98], will undoubtedly lead to further advances in this important field of Gl research.

Different probiotics act at different steps along the NF-κB activation pathway.

Different probiotics act at different steps along the NF-κB activation pathway

NF-κB activation involves several steps, including: 1) Phosphorylation of the inhibitor IκB molecule; 2) Ubiquitination of the inhibitor IκB molecule, which targets it for degradation (L. reuteri acts here); 3) Degradation of the inhibitor IκB molecule by the proteasome (VSL#3 acts here); 4) Translocation of NF-κB to the nucleus, now in active form; 5) Binding of NF-κB to pro-inflammatory gene targets in the nucleus; 6) Production of the gene products of inflammatory gene targets (e.g., MCP-1, IL-8) (E. coli Nissle, L. reuteri act here)

Acknowledgments
I would like to acknowledge Dr. J. Sun for her suggestions and help with the manuscript, Dr. E. B. Chang and NIH grant DK42086 for support with the S. boulardii experiments and Dr. C. Pothoulakis for providing the toxin A used in Fig. (2). Probiotic research in our laboratory is supported by the National Institutes of Health (AT004044-01A2) and by the Crohn’s and Colitis Foundation of Canada.

Section 3 – Vitamin D, Krill Oil, Vitamin K Studies

Vitamin D, Krill Oil, Vitamin K Studies

A) From Medscape Medical News –
Inflammatory Bowel Disease Associated With Bone Loss, Vitamin D Deficiency
Nancy A. Melville

October 22, 2010 (San Antonio, Texas) — Patients with inflammatory bowel disease (IBD) who are vitamin D deficient have a significantly increased risk for osteoporosis, osteopenia, and abnormal bone density levels, irrespective of other factors that could place them at a higher risk, according to research presented here at the American College of Gastroenterology (ACG) 2010 Annual Scientific Meeting and Postgraduate Course.

The study of 161 patients diagnosed with IBDs, such as ulcerative colitis and Crohn’s disease, found that 22% of patients had a reduction in bone density, and a diagnosis of osteopenia or osteoporosis. Of those with a bone density reduction, 50% were younger than 40 years.

“Bone loss is generally uncommon below the age of 40 in the normal population, so I was a little surprised to see a high number of my patients below that age with abnormal bone density. I do believe that it is further evidence of the effects of IBD,” said Bincy P. Abraham, MD, MS, lead author of the study.

Of patients with abnormal dual-energy x-ray absorptiometry (DXA) bone density exams, 40% had higher rates of vitamin D deficiency, defined as levels of 25-hydroxyvitamin D below 30 ng/mL, compared with 1% of those with normal scans (odds ratio [OR], 8.7; 95% confidence interval [CI], 2.4 – 19.8, P = .001).

The higher levels of vitamin D deficiency remained after patients were controlled for corticosteroid intake, age, and sex.

“If you were vitamin D deficient, you were nearly 9 times more likely to have abnormal bone deficiency” than if you weren’t, said Dr. Abraham, who is assistant professor of medicine in the Inflammatory Bowel Disease Program at Baylor College of Medicine in Houston, Texas.

“We looked at the other risk factors and still found that vitamin D was the major factor contributing to the abnormal bone density,” she said.

Previous studies have reported on the high prevalence of osteoporosis among patients with IBD, with the use of corticosteroid and excess of inflammatory cytokines potentially interfering with bone repair and remodeling. Vitamin D deficiencies have also been reported in such patients, but studies have disagreed about the association between the deficiency and bone density.

The prospective study evaluated patients between the ages of 10 and 70 years who were diagnosed with IBD on the basis of clinical, radiologic, endoscopic, and histologic data.

The results showed that patients with Crohn’s disease were much more likely to have abnormal bone density exams than those with ulcerative colitis (34% vs 13%; OR, 4.2; 95% CI, 1.8 – 11.7; P = .02). Those with osteoporosis plus Crohn’s disease or ulcerative colitis had significantly higher rates of vitamin D deficiency, regardless of prednisone intake.

The findings suggest that clinicians treating IBD patients need to consider the possibility of low vitamin D levels and be aware of the potential for bone loss among those patients,” Dr. Abraham said.

“The first step for clinicians treating IBD patients is to check their vitamin D levels, and if they find a deficiency, treat it,” she told Medscape Medical News.

“I prescribe 50,000 units of vitamin D weekly for 8 weeks and then recheck their levels. I’m usually able to get my patients back to good levels (between 30 to 50 ng/mL). . . . I then wait a year and recheck their DXA scans.”

IBD patients are known to be at increased risk for low bone density; however, the study’s findings are notable for showing a relation between vitamin D deficiencies and bone loss, said Jean Paul Achkar, MD, a gastroenterologist from the Cleveland Clinic, in Ohio.

“The ACG has guidelines regarding the need to monitor bone density in IBD patients,” he said. “It is also increasingly recognized that vitamin D levels need to be monitored and repleted if low in patients with IBD.”

“The interesting point of this abstract is the demonstration of a strong association between low vitamin D and abnormal DXA scan, and the fact that this association remained after adjustment for steroid intake and age.”

“The study highlights the importance of checking vitamin D levels in addition to routine DXA monitoring.”

The study did not receive funding. Dr. Abraham reports being on the speaker’s bureau for UCB, Abbott, Warner-Chilcott, Salix, and Prometheus, and the advisory committee for UCB. Dr. Achkar has disclosed no relevant financial relationships.

American College of Gastroenterology (ACG) 2010 Annual Scientific Meeting and Postgraduate Course: Poster P290. Presented October 17, 2010.

Journalist
Nancy A Melville
Nancy Melville is a freelance writer for Medscape.

B) Fish Oils Reduce Steroid Use by over 50% in Colitis Patients

St. Louis, Missouri – Eighteen (18) patients with active ulcerative colitis enrolled in a study looking at the possible health benefits of fish oils. All the patients’ disease was ‘active’ with diarrhea and rectal inflammation. Prescribed drug treatment continued throughout the study using Prednisone and Sulfasalazine.

Sulfasalazine is frequently used in ulcerative colitis treatment. It helps treat symptoms of bowel inflammation, diarrhea, rectal bleeding and abdominal pain.

Prednisone is a steroid commonly used to treat inflammation.

The National Institute of Health states both drugs come with a host of side effects including: extreme mood changes, slow healing of cuts and bruises, irregular or absent menstrual periods, seizures, depression, irregular heart beat, sudden weight gain and vomiting.

Omega 3 fatty acids have proven anti-inflammatory properties. Could they be an alternative to powerful drugs used in standard ulcerative colitis treatment? In this study, patients took either 18 Max-EPA capsules daily (equaling 3 grams eicosapentaenoic acid and 2 grams docosahexaenoic acid in total) or a placebo consisting of 18 capsules of vegetable oil. Study participants were evaluated at the start of the study and after each diet period.

Parameters used for monitoring included:

Flexible sigmoidoscopy (a small flexible scope used to visualize the surface of the colon), Tissue samples from the rectum, Blood tests to measure chemical levels of inflammatory markers.

And the results are:

Patients taking fish oils showed a major decrease in inflammatory markers.
Patients taking fish oils gained bodyweight (many patients suffering from ulcerative colitis experience weight loss).
Patients taking fish oils reduced their daily Prednisone requirements by more than 50%.
Patients taking the placebo didn’t do as well. They had to increase their Prednisone requirements by almost 24%.

Researchers concluded using fish oil supplements in ulcerative colitis:

Reduces the need for steroids by more than 50%
Decreases inflammatory markers in the body and helps patients gain bodyweight.

C) Fish Oils help Decrease Amount of Drugs used to treat Ulcerative Colitis

Martinez, California – A study from the American Journal of Gastroenterology shows fish oils improve symptoms of ulcerative colitis and reduced the amount of medication needed to treat the disease.

Eleven (11) patients with ulcerative colitis of mild to moderate severity were studied for an 8 month period. Patients took either a fish oil supplement providing about 4 grams of omega 3 fatty acids per day or a placebo.

Patients’ symptoms were monitored using a ‘Disease Activity Index’:

Sigmoidoscope was used to look at the actual tissue surface of the colon.

Levels of inflammatory chemicals measured using radio-immunoassay. This is a lab technique using radiation to measure specific substances in your body.
And the results are:

Patients taking fish oils had a 56% decrease in their symptoms according to the Disease Activity Index.

72% of the patients using fish oils were able to reduce their need for anti-inflammatory drugs. Some of these patients were able to stop taking anti-inflammatory drugs completely during the study.

There was no change in the inflammatory chemicals measured in both the fish oil and placebo patients. Nor was there any change in the actual tissue surface of the colon as viewed with the sigmoidoscope in both groups. Only 4% of the placebo patients had a decrease in their symptoms.

Researchers determined fish oil supplements help improve symptoms in mild to active ulcerative colitis and reduced the amount of anti-inflammatory drugs taken. They felt more studies are needed to determine the best dosage and duration when combining fish oils and ulcerative colitis treatment.

Comments:
What’s impressive about these fish oils and ulcerative colitis studies is the dramatic effect on inflammation. Omega 3 fish oils seem to help patients reduce the amount of corticosteroids needed to deal with their symptoms.

Corticosteroids form a common part of the treatment plan for patients with ulcerative colitis. Unfortunately, some people experience severe side effects from these powerful drugs.

While fish oils aren’t a cure for ulcerative colitis, studies show they may help reduce inflammation and improve the quality of life for many afflicted by this disease.

Research References:
Dietary Supplementation with Fish Oils and Ulcerative Colitis, Annals of Internal Medicine. 1992 Apr 15;116(8):609-14.

Fish Oil Fatty Acid Supplementation in Active Ulcerative Colitis: A Double-Blind, Placebo-Controlled, Crossover Study American Journal of Gastroenterology. 1992 Apr;87(4):432-7.

D) Vitamin K2 Studies

Japanese studies done in 2009, concluded that IBD patients have high prevalence of decreased Bone Mineral Density (BMD) and vitamin K and D deficiency probably caused by malabsorption of these vitamins. The study suggested that malabsorption is the basis for hypovitaminosis K and D and decreased BMD, which might be corrected or helped through Vitamin K and Vitamin D supplementation. Other studies done in Egypt in 2010 concurred that supplementation in IBD and UC patients would help in reducing risk factors in the lowering of Bone Mineral Desity directly associated in IBD and UC patients.

Section 4 – Whey Protein supplementation Studies

Whey Protein Supplementation Studies

Whey Protein contains high concentrations of Glutamine, which is one of the most important nutrients for your intestines. Glutamine is the primary energy source for your immune system. It has the ability to “repair Leaky Gut Syndrome” by maintaining the integrity of the bowels.

Although Glutamine is an amino acid that is frequently used as a sports and fitness supplement, it has been found to help modulate the immune system and protect the mucosal protective layer in the intestine. Studies have demonstrated that glutamine can help improve blood flow in inflamed segments of the colon in patients who have ulcerative colitis, although its benefits did not extend to the most seriously affected portion of the colon (Kruschewski M et al 1998). Glutamine is also able to reduce leakiness of the intestine, which may help to reduce symptoms of inflammatory bowel disease.

Glutathione, a small peptide found in the highest concentrations in fresh vegetables, fruits, and lean meats is also beneficial to the small intestine, since it can directly act as an antioxidant in the intestinal tract and help decrease damaging molecules that may be produced during inflammation. Vitamin C, from citrus fruits, and vitamin E, found in whole grain cereals and nut oils, are important antioxidants for the small intestine and work with glutathione to support intestinal healing.The cells that line the intestinal tract need fuel to continue their process of nutrient uptake. The preferred fuel for these cells is the amino acid glutamine, which can be obtained from proteins. Some studies have shown that short-chain fatty acids may also support the small intestinal tract barrier because they can serve as an alternate fuel for the cells that make up the intestinal lining. The small intestinal tract cells also require energy to maintain integrity of the cell wall, and production of energy requires healthy levels of vitamin B5. Mushrooms, cauliflower, sunflower seeds, corn, broccoli, and yogurt are concentrated sources of vitamin B5. The intestinal tract cells also require a number of vitamins, so adequate overall nutrition is necessary.

Section 5 – Butyrate Enemas Studies

Butyrate Enemas Studies
A) Butyrate enema therapy stimulates mucosal repair in experimental colitis in the rat.

Gut. 1996 April; 38(4): 568–573. PMCID: PMC1383116
J D Butzner, R Parmar, C J Bell, and V Dalal
Gastrointestinal Research Group, Faculty of Medicine, University of Calgary, Alberta, Canada.

Abstract
BACKGROUND–The short chain fatty acid (SCFA) butyrate provides energy for colonocytes, stimulates colonic fluid and electrolyte absorption and is recognised as an effective treatment for multiple types of colitis. AIM–To examine the impact of butyrate enema therapy on the clinical course, severity of inflammation, and SCFA stimulated Na+ absorption in a chronic experimental colitis. METHODS–Distal colitis was induced in rats with a trinitrobenzenesulphonic acid (TNBS) enema. Five days after induction, rats were divided into groups to receive: no treatment, saline enemas, or 100 mM Na-butyrate enemas daily. On day 24, colonic damage score and tissue myeloperoxidase (MPO) activity were evaluated. Colon was mounted in Ussing chambers and Na+ transport and electrical activities were measured during a basal period and after stimulation with 25 mM butyrate.

RESULTS–In the untreated and the saline enema treated TNBS groups, diarrhoea and extensive colonic damage were seen, associated with increased tissue MPO activities and absent butyrate stimulated Na+ absorption. In contrast, in the butyrate enema treated TNBS group, diarrhoea ceased, colonic damage score improved, and tissue MPO activity as well as butyrate stimulated Na+ absorption recovered to control values.

CONCLUSION—Butyrate enema therapy stimulated colonic repair, as evidenced by clinical recovery, decreased inflammation, and restoration of SCFA stimulated electrolyte absorption.

B) Effect of butyrate enemas on the colonic mucosa in distal ulcerative colitis.

Gastroenterology. 1992 Jul;103(1):51-6.

Scheppach W, Sommer H, Kirchner T, Paganelli GM, Bartram P, Christl S, Richter F, Dusel G, Kasper H.

Department of Medicine, University of Würzburg, Germany.

Comment in:

Gastroenterology. 1992 Nov;103(5):1709-10.
Gastroenterology. 1992 Jul;103(1):336-8.

Abstract
Short-chain fatty acid irrigation has been shown to ameliorate inflammation in diversion colitis. In this study the effect of butyrate enemas was tested in 10 patients with distal ulcerative colitis who had been unresponsive to or intolerant of standard therapy for 8 weeks. They were treated for 2 weeks with sodium butyrate (100 mmol/L) and 2 weeks with placebo in random order (single-blind trial). Before and after treatment, clinical symptoms were noted and the degree of inflammation was graded endoscopically and histologically. Rectal proliferation was assessed by autoradiography. After butyrate irrigation, stool frequency (n/day) decreased from 4.7 +/- 0.5 to 2.1 +/- 0.4 (P less than 0.01) and discharge of blood ceased in 9 of 10 patients. The endoscopic score fell from 6.5 +/- 0.4 to 3.8 +/- 0.8 (P less than 0.01). The histological degree of inflammation decreased from 2.4 +/- 0.3 to 1.5 +/- 0.3 (P less than 0.02). Overall crypt proliferation was unchanged, but the upper crypt-labeling index fell from 0.086 +/- 0.019 to 0.032 +/- 0.003 (P less than 0.03). On placebo, all of these parameters were unchanged.

These data support the view that butyrate deficiency may play a role in the pathogenesis of distal ulcerative colitis and that butyrate irrigation ameliorates this condition.

PMID: 1612357 [PubMed – indexed for MEDLINE]

Glutamine Supplementation

A)From the University of Maryland Medical Center

Glutamine
Overview:
Glutamine is the most abundant amino acid (building block of protein) in the body. The body can make enough glutamine for its regular needs, but extreme stress (the kind you would experience after very heavy exercise or an injury), your body may need more glutamine than it can make. Most glutamine is stored in muscles followed by the lungs, where much of the glutamine is made.

Glutamine is important for removing excess ammonia (a common waste product in the body). It also helps your immune system function and appears to be needed for normal brain function and digestion.

You can usually get enough glutamine without taking a supplement, because your body makes it and you get some in your diet. Certain medical conditions, including injuries, surgery, infections, and prolonged stress, can lower glutamine levels, however. In these cases, taking a glutamine supplement may be helpful.

Uses:

Woundhealing and recovery from illness
When the body is stressed (from injuries, infections, burns, trauma, or surgical procedures), it releases the hormone cortisol into the bloodstream. High levels of cortisol can lower your body’ s stores of glutamine. Several studies show that adding glutamine to enteral nutrition (tube feeding) helps reduce the rate of death in trauma and critically ill people. Clinical studies have found that glutamine supplements strengthen the immune system and reduce infections (particularly infections associated with surgery).
Glutamine supplements may also help in the recovery of severe burns.
Inflammatory bowel disease (IBD) – Glutamine helps to protect the lining of the gastrointestinal tract known as the mucosa. For that reason, some have suggested that people who have inflammatory bowel disease (ulcerative colitis and Crohn’ s disease) may not have enough glutamine. However, two clinical trials found that taking glutamine supplements did not improve symptoms of Crohn’ s disease. More research is needed. In the meantime, ask your doctor when deciding whether to use glutamine for IBD.
HIV/AIDS- People with HIV or AIDS often experience severe weight loss (particularly loss of muscle mass). A few studies of people with HIV and AIDS have found that taking glutamine supplements, along with other important nutrients including vitamins C and E, beta-carotene, selenium, and N-acetylcysteine, may increase weight gain and help the intestines better absorb nutrients.
Athletes – Athletes who train for endurance events (like marathons) may reduce the amount of glutamine in their bodies. It’ s common for them to catch a cold after an athletic event. Some experts think that may be because of the role glutamine plays in the immune system. For this select group of athletes, one study showed that taking glutamine supplements resulted in fewer infections. The same is not true, however, for most exercisers who work out at a much more moderate intensity.
Cancer – Many people with cancer have low levels of glutamine. For this reason, some researchers speculate that glutamine may be helpful when added to conventional cancer treatment for some people. Supplemental glutamine is often given to malnourished cancer patients undergoing chemotherapy or radiation treatments and sometimes used in patients undergoing bone marrow transplants. (See Interactions below.)
Glutamine seems to help reduce stomatitis (an inflammation of the mouth) caused by chemotherapy. Some studies, but not all, have suggested that taking glutamine orally may help reduce diarrhea associated with chemotherapy.

More clinical research is needed to know whether use of glutamine is safe or effective to use as part of the treatment regimen for cancer.

Dietary Sources:
Dietary sources of glutamine include plant and animal proteins such as beef, pork and poultry, milk, yogurt, ricotta cheese, cottage cheese, raw spinach, raw parsley, and cabbage.

Available Forms:
Glutamine, usually in the form of L-glutamine, is available by itself or as part of a protein supplement. These come in powder, capsule, tablet, or liquid form.

Standard preparations are typically available in 500 mg tablets or capsules.

How to Take It:
Take glutamine with cold or room temperature foods or liquids. It should not be added to hot beverages because heat destroys glutamine.

Pediatric

For children 10 years and younger: Do not give glutamine to a child unless your doctor recommends it as part of a complete amino acid supplement.

For children 10 – 18 years: Doses of 500 mg, 1 – 3 times daily, are generally considered safe.

Adult

For adults ages 18 and older: Doses of 500, 1 – 3 times daily, are generally considered safe. Doses as high as 5,000 – 15,000 mg daily (in divided doses) may be prescribed by a health care provider.

As an oral rinse for radiation therapy-induced mucositis and chemotherapy-induced stomatitis: Place 16 gm (one tablespoonful) of glutamine powder in 240 ml (8 ounces) normal saline or sterile water and mix. Then, swish 30 – 60 ml (1 – 2 ounces) and spit out, 4 times a day.

Precautions:
Because of the potential for side effects and interactions with medications, dietary supplements should be taken only under the supervision of a knowledgeable health care provider.

Glutamine appears to be safe in doses up to 14 g or higher per day.

Glutamine powder should not be added to hot beverages because heat destroys this amino acid. Glutamine supplements should also be kept in a dry location.

People with kidney disease, liver disease, or Reye syndrome (a rare, sometimes fatal disease of childhood that is generally associated with aspirin use) should not take glutamine.

Many elderly people have decreased kidney function and may need to reduce the dose of glutamine.

Glutamine is different from glutamate (glutamic acid), monosodium glutamate, and gluten. Glutamine should not cause symptoms (headaches, facial pressure, tingling, or burning sensation) associated with sensitivity to monosodium glutamate. People who are gluten sensitive can use glutamine without problems.

Possible Interactions:
If you are currently being treated with any of the following medications, you should not use glutamine supplements without first talking to your health care provider.

Cancer therapy — Glutamine may increase the effectiveness and reduce the side effects of chemotherapy treatments with doxorubicin, methotrexate, and 5-fluorouracil in people with colon cancer. Preliminary clinical studies suggest that glutamine supplements may prevent nerve damage associated with a medication called paclitaxel, used for breast and other types of cancers.

However, laboratory studies suggest that glutamine may actually stimulate growth of tumors. Much more research is needed before it is known whether it is safe to use glutamine if you have cancer.

Alternative Names:
L-glutamine

Reviewed last on: 6/20/2009
Steven D. Ehrlich, NMD, Solutions Acupuncture, a private practice specializing in complementary and alternative medicine, Phoenix, AZ. Review provided by VeriMed Healthcare Network.
Supporting Research
Abcouwer SF. The effects of glutamine on immune cells [editorial]. Nutrition. 2000;16(1):67-69.

Akobeng AK, Miller V, Stanton J, Elbadri AM, Thomas AG. Double-blind randomized controlled trial of glutamine-enriched polymeric diet in the treatment of active Crohn’s disease. J Pediatr Gastroenterol Nutr. 2000;30(1):78-84.

Antoon AY, Donovan DK. Burn Injuries. In: Behrman RE, Kliegman RM, Jenson HB, eds. Nelson Textbook of Pediatrics. Philadelphia, Pa: W.B. Saunders Company; 2000:287-294.

Avenell A. Symposium 4: Hot topics in parenteral nutrition Current evidence and ongoing trials on the use of glutamine in critically-ill patients and patients undergoing surgery. Proc Nutr Soc. 2009 Jun 3:1-8. [Epub ahead of print]

Buchman AL. Glutamine: commercially essential or conditionally essential? A critical appraisal of the human data. Am J Clin Nutr. 2001;74(1):25-32.

Clark RH, Feleke G, Din M, et al. Nutritional treatment for acquired immunodeficiency virus-associated wasting using beta-hydroxy-beta-methylbutyrate, glutamine, and arginine: a randomized, double-blind placebo-controlled study. JPEN: J Parenter Enteral Nutr. 2000;24(3):133-139.

Daniele B, Perrone F, Gallo C, et al. Oral glutamine in the prevention of fluorourcil induced intestinal toxicity: a double blind, placebo controlled, randomized trial. Gut. 2001;48:28-33.

Decker GM. Glutamine: indicated in cancer care? Clin J Oncol Nurs. 2002;6(2):112-115.

Fan YP, Yu JC, Kang WM, Zhang Q. Effects of glutamine supplementation on patients undergoing abdominal surgery. Chin Med Sci J. 2009 Mar;24(1):55-9.

Field CJ, Johnson IR, Schley PD. Nutrients and their role in host resistance to infection. J Leukoc Biol. 2002 Jan;71(1):16-32.

Furukawa S. Saito H, Inoue T, et al. Supplemental glutamine augments phagocytosis and reactive oxygen intermediate production by neutrophils and monocytes from postoperative patients in vitro. Nutrition. 2000;1695):323-329.

Garlick PJ. Assessment of the safety of glutamine and other amino acids.J Nutr. 2001 Sep;131(9 Suppl):2556S-61S. [Review].

Greenlee H, Hershman DL, Jacobson JS. Use of antioxidant supplements during breast cancer treatment: a comprehensive review. Breast Cancer Res Treat. 2009 Jun;115(3):437-52. Epub 2008 Oct 7.

Grimm H, Kraus A. Immunonutrition–supplementary amino acids and fatty acids ameliorate immune deficiency in critically ill patients. Langenbecks Arch Surg. 2001 Aug;386(5):369-376.

Lecleire S, Hassan A, Marion-Letellier R, Antonietti M, Savoye G, et al. Combined glutamine and arginine decrease proinflammatory cytokine production by biopsies from Crohn’s patients in association with changes in nuclear factor-kappaB and p38 mitogen-activated protein kinase pathways. J Nutr. 2008 Dec;138(12):2481-6.

Medina MA. Glutamine and cancer. J Nutr. 2001;131(9 Suppl):2539S-2542S; discussion 2550S-2551S.

Murray SM, Pindoria S. Nutrition support for bone marrow transplant patients. Cochrane Database Syst Rev. 2009 Jan 21;(1):CD002920. Review.

Neu J, DeMarco V, Li N. Glutamine: clinical applications and mechanism of action. Curr Opin Clin Nutr Metab Care. 2002;5(1):69-75

Reeds PJ, Burrin DG. Glutamine and the bowel. J Nutr. 2001;131(9 Suppl):2505S-8S.

Vahdat L, Papadopoulos K, Lange D, et al. Reduction of paclitaxel-induced peripheral neuropathy with glutamine. Clin Cancer Res. 2001;7(5):1192-1197.

van Stijn MF, Ligthart-Melis GC, Boelens PG, Scheffer PG, Teerlink T, et al. Antioxidant enriched enteral nutrition and oxidative stress after major gastrointestinal tract surgery. World J Gastroenterol. 2008 Dec 7;14(45):6960-9.

Wilmore DW. The effect of glutamine supplementation in patients following elective surgery and accidental injury. [Review]. J Nutr. 2001;131(9 Suppl):2543S-9S; discussion 2550S-1S.

Ziegler TR. Glutamine supplementation in cancer patients receiving bone marrow transplantation and high dose chemotherapy. [Review]. J Nutr. 2001;131(9 Suppl):2578S-84S; discussion 2590S.

B) Prophylactic effect of dietary glutamine supplementation on interleukin 8 and tumour necrosis factor α production in trinitrobenzene sulphonic acid induced colitis

AbstractGut 1997;41:487-493 doi:10.1136/gut.41.4.487
Inflammatory bowel disease – by C K Amehoa, A A Adjeib, E K Harrisonc, K Takeshitaa, T Moriokad, Y Arakakid, E Itod, I Suzukig, A D Kulkarnie, A Kawajirif, S Yamamotoh –
A) Department of Nutrition, University of the Ryukyus, Nishihara, Okinawa 903–01, Japan, B) Department of Bacteriology, C) Department of Adult Health, D) First Department of Pathology, University of the Ryukyus, E) Department of Surgery, St Louis Medical Center, St Louis, USA, F) Daidochuo Hospital, Naha, Okinawa, Japan, G) Department of Nutrition, Kumamoto Prefectural University, Kumamoto, Japan, H) Department of Nutrition, Facultyof Medicine, University of Tokushima, 3 Kuramoto, Tokushima 770, Japan
Professor S Yamamoto.
Accepted 25 March 1997

Background—It is well established that glutamine supplemented elemental diets result in less severe intestinal damage in experimental colitis. However, few studies have examined the mode of action of glutamine in reducing intestinal damage.

Aims—To examine the effects of glutamine supplemented elemental diets on the potent inflammatory cytokines interleukin 8 (IL-8) and tumour necrosis factor α (TNF-α) in trinitrobenzene sulphonic acid (TNBS) induced colitis which presents with both acute and chronic features of ulcerative colitis.

Methods—Sprague-Dawley rats were randomised into three dietary groups and fed 20% casein (controls), or 20% casein supplemented with either 2% glutamine (2% Gln) or 4% glutamine (4% Gln). After two weeks they received intracolonic TNBS to induce colitis.

Results—Both Gln groups of rats gained more weight than the control group (p<0.05) which had progressive weight loss. Colon weight, macroscopic, and microscopic damage scores for the Gln groups were lower than in the control group (p<0.05). IL-8 and TNF-α concentrations in inflamed colonic tissues were lower in the Gln groups than in the control group (p<0.05), and correlated well with disease severity. Bacterial translocation was lower both in incidence (p<0.05) and in the number of colony forming units (p<0.05) for the Gln groups, than in the control group. With respect to all indices studied, the 4% Gln group performed better than did the 2% Gln group.

Conclusion—Prophylactic glutamine supplementation modulates the inflammatory activities of IL-8 and TNF-α in TNBS induced colitis.

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