Stephanie Blum

Inflammatory Disease Processes and Interactions with Nutrition

Inflammation is a stereotypical physiological response to infections and tissue injury; it initiates pathogen killing as well as tissue repair processes and helps to restore homeostasis at infected or damaged sites. Acute inflammatory reactions are usually self-limiting and resolve rapidly, due to the involvement of negative feedback mechanisms. Thus, regulated inflammatory responses are essential to remain healthy and maintain homeostasis. However, inflammatory responses that fail to regulate themselves can become chronic and contribute to the perpetuation and progression of disease. Characteristics typical of chronic inflammatory responses underlying the pathophysiology of several disorders include loss of barrier function, responsiveness to a normally benign stimulus, infiltration of inflammatory cells into compartments where they are not normally found in such high numbers, and overproduction of oxidants, cytokines, chemokines, eicosanoids and matrix metalloproteinases. The levels of these mediators amplify the inflammatory response, are destructive and contribute to the clinical symptoms. Various dietary components including long chain omega-3 fatty acids, antioxidant vitamins, plant flavonoids, prebiotics and probiotics have the potential to modulate predisposition to chronic inflammatory conditions and may have a role in their therapy. These components act through a variety of mechanisms including decreasing inflammatory mediator production through effects on cell signaling and gene expression (omega-3 fatty acids, vitamin E, plant flavonoids), reducing the production of damaging oxidants (vitamin E and other antioxidants), and promoting gut barrier function and anti-inflammatory responses (prebiotics and probiotics). However, in general really strong evidence of benefit to human health through anti-inflammatory actions is lacking for most of these dietary components. Thus, further studies addressing efficacy in humans linked to studies providing greater understanding of the mechanisms of action involved are required.

Divergent patterns of colonization and immune response elicited from two intestinal Lactobacillus strains that display similar properties in vitro

ABSTRACT Lactobacilli derived from the endogenous flora of normal donors are being increasingly used as probiotics in functional foods and as vaccine carriers. However, a variety of studies done with distinct strains of lactobacilli has suggested heterogeneous and strain-specific effects. To dissect this heterogeneity at the immunological level, we selected two strains of lactobacilli that displayed similar properties in vitro and studied their impact on mucosal and systemic B-cell responses in monoxenic mice. Germfree mice were colonized with Lactobacillus johnsonii (NCC 533) or Lactobacillus paracasei (NCC 2461). Bacterial loads were monitored for 30 days in intestinal tissues, and mucosal and systemic B-cell responses were measured. Although both Lactobacillus strains displayed similar growth, survival, and adherence properties in vitro, they colonized the intestinal lumen and translocated into mucosal lymphoid organs at different densities. L. johnsonii colonized the intestine very efficiently at high levels, whereas the number of L. paracasei decreased rapidly and it colonized at low levels. We determined whether this difference in colonization correlated with an induction of different types of immune responses. We observed that colonization with either strain induced similar germinal center formation and immunoglobulin A-bearing lymphocytes in the mucosa, suggesting that both strains were able to activate mucosal B-cell responses. However, clear differences in patterns of immunoglobulins were observed between the two strains in the mucosa and in the periphery. Therefore, despite similar in vitro probiotic properties, distinct Lactobacillus strains may colonize the gut differently and generate divergent immune responses.

Effects of parenteral lipid emulsions with different fatty acid composition on immune cell functions in vitro

BACKGROUND Numerous studies suggest that immune function may be compromised by lipid emulsions rich in polyunsaturated fatty acids (PUFAs). In our study, we compared the effect of a new olive oil-based lipid emulsion (ClinOleic) containing a moderate level of PUFAs, with emulsions based on soybean oil (Intralipid or Ivelip), on immune functions of human cell in vitro. METHODS Peripheral white blood cells were collected from healthy volunteers. Lymphocyte proliferation was evaluated by [3H]-thymidine incorporation after stimulation with either phytohemagglutinin (PHA) or antibodies against T-cell specific antigens. Lymphocytes subsets and T-cell activation markers (CD25 and HLA-DR) were measured by flow cytometry. The release of cytokines (interleukin [IL]-2, IL-1beta, and tumor necrosis factor-alpha [TNF-alpha]) was measured by enzyme-linked immunosorbent assay (ELISA), after lymphocytes or monocytes/macrophages stimulation with PHA or lipopolysaccharide (LPS). RESULTS A significant dose-dependent inhibition of thymidine incorporation was observed with Intralipid and Ivelip (incorporation down to 39.9% of control, p < .001) whereas ClinOleic showed no inhibitory effect. Activation antigen expression on both CD4+ and CD8+ T-cells tended to decrease with Intralipid (CD25: -53.4% on CD4+ and -57.4% on CD8+; HLA-DR: -61.5% on CD4+ and -58.5% on CD8+) but not with ClinOleic (from -2.9% for CD25 on CD4+ to 16.7% for HLA-DR on CD4+). Intralipid decreased significantly IL-2 production (-39.0%, p < .05) whereas ClinOleic had little effect (-13.0%, NS). Intralipid and ClinOleic tended to inhibit to a similar extent the release of pro-inflammatory cytokines (TNF-alpha: -21.5% and -34.8%, IL-1beta: -45.1% and -40.3%; respectively). CONCLUSIONS Our results suggest that an olive oil-based lipid emulsion could modulate immune response selectively, maintaining protective immunity and reducing inflammatory response. Olive oil may offer an immunologically neutral alternative to soybean oil for use in parenteral lipid emulsions.

Lactobacillus johnsonii La1 Antagonizes Giardia intestinalis In Vivo

ABSTRACT This study describes the in vivo activity of Lactobacillus johnsonii La1 (NCC533) in Giardia intestinalis-infected gerbils (Meriones unguiculatus). Daily administration of lactobacilli in the drinking water from 7 days before inoculation with Giardia trophozoites efficiently prevented G. intestinalis strain WB clone C6 from infecting gerbils. More specifically, shedding of fecal Giardia antigens (GSA65 protein) was diminished in the La1-treated group, and resolution of infection was observed by 21 days postinoculation. Histology and analysis of enzymatic markers of microvillus membrane integrity revealed that probiotic administration also protected against parasite-induced mucosal damage. In addition, a cellular response to Giardia antigens was stimulated in spleen cells from La1-treated gerbils. Results show for the first time the antigiardial effect of probiotic lactobacilli in vivo and provide further insight into the antagonistic properties of lactic acid bacteria against protozoa involved in intestinal infections.

Probiotics as a Treatment Strategy for Gastrointestinal Diseases

Current interest in probiotics is motivated not only by the clinical data showing efficacy of some probiotic bacteria but also by the increasing antibiotic resistance of pathogenic bacteria (particularly in hospitals) and the rise of consumers’ demand for natural substitutes of drugs. Only few randomized, double-blind placebo-controlled human trials are available, and some involved only small numbers of patients. They are difficult to compare because of differences in probiotic strains employed, doses and formulation. Among probiotic applications, reduction of diarrhea is probably the best-documented effect confirmed by recent meta-analyses. Literature on Helicobacter pylori indicates that probiotics are unable to eradicate the infection but could be useful in decreasing infection levels and as adjuvants of therapy-associated side effects. Studies performed in inflammatory bowel disease suggest that high doses of probiotics and most likely a combination of different lactobacilli and bifidobacteria are more effective in decreasing inflammatory score and maintaining patients in remission than a single probiotic strain. Probiotic studies evaluating amelioration of symptoms in irritable bowel syndrome would require more sustained patient numbers. However, accumulated data is encouraging and suggests that efficacy is strain-dependent. Finally, too few probiotic intervention trials have been reported on colon cancer to allow any firm conclusion.

Selection of Bifidobacteria Based on Adhesion and Anti-Inflammatory Capacity In Vitro for Amelioration of Murine Colitis

ABSTRACT Adhesion and anti-inflammatory properties of eight strains of bifidobacteria were tested using the intestinal epithelial cell lines Caco-2, T84, and HT29. Two strains were selected for further assessment of their anti-inflammatory capacity in two murine models of colitis. In vivo results confirmed the high anti-inflammatory capacity of a Bifidobacterium bifidum strain.

Metabolic assessment of gradual development of moderate experimental colitis in IL-10 deficient mice.

Evidence has linked genetic predisposition and environmental exposures to the worldwide pandemic of inflammatory bowel diseases (IBD), but underlying biochemical events remain largely undefined. Here, we studied the gradual development of colitis in Interleukin 10 deficient mice using a combination of (i) histopathological analysis of intestinal sections, (ii) metabolic profiling of blood plasma, and (iii) measurement of plasma inflammatory biomarkers. Data integration using chemometric tools, including Independent Component Analysis, provided a new strategy for measuring and mapping the metabolic effects associated with the development of intestinal inflammation at the age of 1, 8, 16, and 24 weeks. Chronic inflammation appeared at 8 weeks and onward, and was associated with altered cecum and colon morphologies and increased inflammatory cell infiltration into the mucosa and the submucosa. Blood plasma profiles provided additional evidence of loss of energy homeostasis, impaired metabolism of lipoproteins and glycosylated proteins. In particular, IL-10-/-mice were characterized by decreased levels of VLDL and increased concentrations of LDL and polyunsaturated fatty acids, which are related to the etiology of IBD. Moreover, higher levels of lactate, pyruvate, citrate and lowered glucose suggested increased fatty acid oxidation and glycolysis, while higher levels of free amino acids reflected muscle atrophy, breakdown of proteins and interconversions of amino acids to produce energy. These integrated system investigations demonstrate the potential of metabonomics for investigating the mechanistic basis of IBD, and it will provide novel avenues for management of IBD.

Improvement of the human intestinal flora by ingestion of the probiotic strain Lactobacillus johnsonii La1.

To exert beneficial effects for the host, for example, improving the intestinal microflora, a probiotic must reach the intestine as a viable strain. These properties must be demonstrated by in vitro as well as in vivo methods. However, only a few well-designed human clinical studies have shown these properties. Lactobacillus johnsonii La1 has been shown to give many beneficial effects for the host, but it is unclear whether a viable strain of L. johnsonii La1 has the effect of improving host intestinal microflora. In the present study, a randomised double-blind placebo-controlled cross-over trial was conducted to elucidate the effect of L. johnsonii La1 on human intestinal microflora. Twenty-two young healthy Japanese women were randomly divided into two groups, and either received fermented milk with L. johnsonii La1 or a fermented milk without L. johnsonii La1 (placebo) daily for 21 d. Consumption of the fermented milk: (a) increased total Bifidobacterium and Lactobacillus, and decreased lecithinase-positive Clostridium in the faeces; (b) increased the faecal lactic acid concentrations; (c) decreased the faecal pH; (d) increased the defecation frequency. These changes were stronger than those observed with the placebo. L. johnsonii La1 was identified in all subjects only after the consumption of the fermented milk. These results suggest that L. johnsonii La1 can contribute to improve intestinal microflora with probiotic properties.

Patchy distribution of mucosal lesions in ileal Crohn's disease is not linked to differences in the dominant mucosa‐associated bacteria: A study using fluorescence in situ hybridization and temporal temperature gradient gel electrophoresis

Background The mucosa‐associated bacteria (MAB) are suspected of being involved in the pathogenesis of Crohns disease. We analyzed and compared the MAB in noninflamed and inflamed ileal mucosa of Crohns disease patients (n = 22). Methods Tissue samples from the inflamed ileal mucosa and from the adjacent noninflamed ileal mucosa were taken from surgical resection specimens. The MAB were investigated using fluorescence in situ hybridization with 7 group‐specific probes and temporal temperature gradient gel electrophoresis (TTGE). Results Samples from both noninflamed and inflamed mucosa were obtained from 15 patients. The distribution of the bacterial populations was not different between noninflamed and inflamed mucosa. The Bacteroidetes phylum was dominant and accounted for 29% of MAB (0%–74%) in noninflamed tissues and 32% (0%–70%) in inflamed areas. The &ggr; Proteobacteria represented 12% (0%–70%) of MAB both in noninflamed and inflamed areas. The Clostridium coccoides group (Firmicutes phylum) represented 15% of MAB in noninflamed tissues versus 7% in inflamed areas. For most of the patients the similarity index between TTGE paired profiles was very high. Conclusion The dominant MAB do not differ between noninflamed and inflamed ileal mucosa in Crohns disease. This argues against a localized dysbiosis to explain the patchy distribution of mucosal lesions. (Inflamm Bowel Dis 2007)

IL-10 Producing CD14low Monocytes Inhibit Lymphocyte-Dependent Activation of Intestinal Epithelial Cells by Commensal Bacteria

Intestinal epithelial cell (IEC) activation by non‐pathogenic, commensal bacteria was demonstrated to require the presence of immunocompetent cells. In this study, HT‐29 and CaCO‐2 transwell cultures, reconstituted with CD4+ and CD8+ T cells, CD19+ B cells and CD14high monocytes, were challenged with nonpathogenic Gram negative Escherichia coli and Gram positive lactobacilli. Cytokine expression was analysed by reverse transcription‐polymerase chain reaction (RT‐PCR) and enzyme linked immunosorbent assays (ELISA). Expression of tumour necrosis factor alpha (TNF‐α) and interleukin (IL)‐8 mRNA in E. coli or L. sakei challenged IEC was promoted by lymphocyte populations predominantly CD4+ T cells, while monocytes failed to mediate an inflammatory cytokine response. The monocyte phenotype and function were further characterised by flow cytometry and mixed lymphocyte reaction (MLR). During the co‐culture with IEC and bacterial stimulated IEC, CD14high peripheral blood monocytes acquired a CD14low CD16low phenotype with reduced expression co‐stimulatory (CD80, CD86, CD58) cell surface molecules. Immunosuppressive functions of IEC conditioned CD14low monocytes were demonstrated by the predominant secretion of IL‐10 and IL‐1Ra and their reduced potential to trigger an allogeneic lymphocyte response. In conclusion, IEC contribute to the development of CD14low CD16low monocytes with immunosuppressive function and antagonised a lymphocyte‐mediated activation of the intestinal epithelium in response to intestinal and food derived bacteria. These results strengthen the hypothesis that the gut epithelium constitutes an important functional element in the regulation of mucosal immune homeostasis towards commensal bacteria.