John Bienenstock

The probiotic Bifidobacteria infantis: An assessment of potential antidepressant properties in the rat

It is becoming increasingly apparent that probiotics are important to the health of the host. The absence of probiotic bacteria in the gut can have adverse effects not only locally in the gut, but has also been shown to affect central HPA and monoaminergic activity, features that have been implicated in the aetiology of depression. To evaluate the potential antidepressant properties of probiotics, we tested rats chronically treated with Bifidobacteria infantis in the forced swim test, and also assessed the effects on immune, neuroendocrine and central monoaminergic activity. Sprague-Dawley rats were treated for 14 days with B. infantis. Probiotic administration in naive rats had no effect on swim behaviours on day 3 or day 14 following the commencement of treatment. However, there was a significant attenuation of IFN-gamma, TNF-alpha and IL-6 cytokines following mitogen stimulation (p<0.05) in probiotic-treated rats relative to controls. Furthermore, there was a marked increase in plasma concentrations of tryptophan (p<0.005) and kynurenic acid (p<0.05) in the bifidobacteria-treated rats when compared to controls. Bifidobacteria treatment also resulted in a reduced 5-HIAA concentration in the frontal cortex and a decrease in DOPAC in the amygdaloid cortex. The attenuation of pro-inflammatory immune responses, and the elevation of the serotonergic precursor, tryptophan by bifidobacteria treatment, provides encouraging evidence in support of the proposition that this probiotic may possess antidepressant properties. However, these findings are preliminary and further investigation into the precise mechanisms involved, is warranted.

Lactobacillus reuteri-induced regulatory T cells protect against an allergic airway response in mice.

RATIONALEnWe have previously demonstrated that oral treatment with live Lactobacillus reuteri can attenuate major characteristics of the asthmatic response in a mouse model of allergic airway inflammation. However, the mechanisms underlying these effects remain to be determined.nnnOBJECTIVESnWe tested the hypothesis that regulatory T cells play a major role in mediating L. reuteri-induced attenuation of the allergic airway response.nnnMETHODSnBALB/c mice were treated daily with L. reuteri by gavage. Flourescent-activated cell sorter analysis was used to determine CD4(+)CD25(+)Foxp3(+)T cell populations in spleens following treatment with L. reuteri or vehicle control. Cell proliferation assays were performed on immunomagnetic bead separated CD4(+)CD25(+) and CD4(+)CD25(-) T cells. CD4(+)CD25(+) T cells isolated from, ovalbumin naive, L. reuteri treated mice were transferred into ovalbumin-sensitized mice. Following antigen challenge the airway responsiveness, inflammatory cell influx and cytokine levels in bronchoalveolar lavage fluid of recipient mice were assessed.nnnMEASUREMENTS AND MAIN RESULTSnFollowing 9 days of oral L. reuteri treatment, the percentage and total number of CD4(+)CD25(+)Foxp3(+)T cells in spleens significantly increased. CD4(+)CD25(+) cells isolated from L. reuteri-fed animals also had greater capacity to suppress T-effector cell proliferation. Adoptive transfer of CD4(+)CD25(+) T cells from L. reuteri-treated mice to ovalbumin-sensitized animals attenuated airway hyper-responsiveness and inflammation in response to subsequent antigen challenge.nnnCONCLUSIONSnThese results strongly support a role for nonantigen-specific CD4(+)CD25(+)Foxp3(+) regulatory T cells in attenuating the allergic airway response following oral treatment with L. reuteri. This potent immuno-regulatory action may have therapeutic potential in controlling the Th2 bias observed in atopic individuals.

Live Lactobacillus reuteri Is Essential for the Inhibitory Effect on Tumor Necrosis Factor Alpha-Induced Interleukin-8 Expression

ABSTRACT The mechanism of the apparent anti-inflammatory action of probiotic organisms is unclear. Lactobacillus reuteri is effective in inhibiting colitis in interleukin-10 (IL-10)-deficient mice. Nerve growth factor (NGF), in addition to its activity on neuronal cell growth, has significant anti-inflammatory effects in several experimental systems in vitro and in vivo, including a model of colitis. Our experiments were designed to explore the mechanism of effect of L. reuteri in the human epithelial cell lines T84 and HT29 on cytokine and NGF synthesis and IL-8 response to tumor necrosis factor alpha (TNF-α). Epithelial cells were cultured for various times with live and killed L. reuteri and examined by reverse transcription-PCR for NGF, IL-10, and TNF-α-induced IL-8 expression. An enzyme-linked immunosorbent assay was used to quantitate intracellular IL-8 and secreted product. Western blotting and confocal microscopy were used to determine the effects on IκB and NF-κB, respectively. Live but not heat-killed or gamma-irradiated L. reuteri upregulated NGF and dose dependently inhibited constitutive synthesis by T84 and HT29 cells of IL-8 and that induced by TNF-α in terms of mRNA and intracellular and secreted protein. Similarly, L. reuteri inhibited IL-8 synthesis induced by Salmonella enterica serovar Typhimurium. L. reuteri required preincubation and adherence for effect, inhibited translocation of NF-κB to the nuclei of HeLa cells, and prevented degradation of IκB. Neither cellular lysates nor media supernatants had any effect on TNF-α-induced IL-8. The conclusion is that L. reuteri has potent direct anti-inflammatory activity on human epithelial cells, which is likely to be related to the activity of ingested probiotics. L. reuteri also upregulates an unusual anti-inflammatory molecule, NGF, and inhibits NF-κB translocation to the nucleus.

Mood and gut feelings

Evidence is accumulating to suggest that gut microbes (microbiota) may be involved in neural development and function, both peripherally in the enteric nervous system and centrally in the brain. There is an increasing and intense current interest in the role that gut bacteria play in maintaining the health of the host. Altogether the mass of intestinal bacteria represents a virtual inner organ with 100 times the total genetic material contained in all the cells in the human body. Surprisingly, the characterization of this extraordinarily diverse population is only just beginning, since some 60% of these microbes have never been cultured. Commensal organisms live in a state of harmonious symbiosis with each other and their host, however, a disordered balance amongst gut microbes is now thought to be an associated or even causal factor for chronic medical conditions as varied as obesity and inflammatory bowel diseases. While evidence is still limited in psychiatric illnesses, there are rapidly coalescing clusters of evidence which point to the possibility that variations in the composition of gut microbes may be associated with changes in the normal functioning of the nervous system. This review focuses on these data and suggests that the concept should be explored further to increase our understanding of mood disorders, and possibly even uncover missing links to a number of co-morbid medical diseases.

Lactobacillus reuteri enhances excitability of colonic AH neurons by inhibiting calcium-dependent potassium channel opening

Probiotics are live non‐pathogenic commensal organisms that exert therapeutic effects in travellers’ diarrhea, irritable bowel syndrome and inflammatory bowel disease. Little is known about mechanisms of action of commensal bacteria on intestinal motility and motility‐induced pain. It has been proposed that probiotics affect intestinal nerve function, but direct evidence for this has thus far been lacking. We hypothesized that probiotic effects might be mediated by actions on colonic intrinsic sensory neurons. We first determined whether sensory neurons were present in rat colon by their responses to chemical mucosal stimulation and identified them in terms of physiological phenotype and soma morphotype. Enteric neuron excitability and ion channel activity were measured using patch clamp recordings. We fed 109Lactobacillus reuteri (LR) or vehicle control to rats for 9 days. LR ingestion increased excitability (threshold for evoking action potentials) and number of action potentials per depolarizing pulse, decreased calcium‐dependent potassium channel (IKCa) opening and decreased the slow afterhyperpolarization (sAHP) in sensory AH neurons, similar to the IKCa antagonists Tram‐34 and clotrimazole. LR did not affect threshold for action potential generation in S neurons. Our results demonstrate that LR targets an ion channel in enteric sensory nerves through which LR may affect gut motility and pain perception.

What is the physiological function of mast cells

Abstract:u2002 Under physiological conditions, skin mast cells preferentially localize around nerves, blood vessels and hair follicles. This observation, which dates back to Paul Ehrlich, intuitively suggests that these enigmatic, multifacetted protagonists of natural immunity are functionally relevant to many more aspects of tissue physiology than just to the generation of inflammatory and vasodilatory responses to IgE‐dependent environmental antigens. And yet, for decades, mainstream‐mast cell research has been dominated by a focus on the – undisputedly prominent and important – mast cell functions in type I immune responses and in the pathogenesis and management of allergic diseases. Certainly, it is hard to believe that the very large and rather selectively distributed number of mast cells in normal, uninflamed, non‐infected, non‐traumatized mammalian skin or mucosal tissue is simply hanging around there lazily day and night, just to wait for the odd allergen or parasite‐associated antigen to come by so the mast cell can finally swing into action. Indeed, the past decade has witnessed a renaissance of mast cell research ‘beyond allergy’, along with a more systematic exploration of the surprisingly wide range of physiological functions that mast cells may be involved in. The current debate sketches many of the exciting new horizons that have recently come into our vision during this intriguing, ongoing search.

Inhibitory effects of Lactobacillus reuteri on visceral pain induced by colorectal distension in Sprague-Dawley rats

Background and aims: Probiotic bacteria are being investigated as possible treatments for many intestinal disorders. The present study aimed to explore the effects of live, heat killed, or gamma irradiated Lactobacillus reuteri on cardio-autonomic response and single fibre unit discharge in dorsal root ganglia to colorectal distension in healthy Sprague-Dawley rats housed under conventional conditions. The effects of this treatment on somatic pain were also examined. Methods: 1×109 bacteria were given by gavage for nine days. Colorectal distension occurred under anaesthesia. Heart rate was measured through continuous electrocardiography. Single fibre unit discharge was recorded from the 6th left lumbar dorsal root ganglion. Somatic pain was evaluated by the tail flick and paw pressure tests. Results: Colorectal distension caused a pressure dependent bradycardia in the control (native medium) group. Treatment with live, heat killed, or gamma irradiated bacteria as well as their products (conditioned medium) prevented the pain response even during the maximum distension pressure (80 mm Hg). Both viable and non-viable bacteria significantly decreased dorsal root ganglion single unit activity to distension. No effects on somatic pain were seen with any treatment. Conclusions: Oral administration of either live or killed probiotic bacteria or conditioned medium inhibited the constitutive cardio-autonomic response to colorectal distension in rats through effects on enteric nerves. These data may provide a novel explanation for beneficial probiotic effects on visceral pain.

Mast cell differentiation and heterogeneity

Major differences between mast cells from different tissues and species have been known for at least 20 years but have been rigorously studied only recently. A recent meeting focused on the ontogeny and differentiation of mast cells, their functional characteristics and the clinical and biological significance of their heterogeneity.

Communication between gastrointestinal bacteria and the nervous system

In the past few years, intestinal microbiota has emerged as a novel target for the treatment of gut-brain axis alterations. These include functional gastrointestinal disorders, such as irritable bowel syndrome (IBS), which can be comorbid with stress-related psychiatric conditions. Thus, modulation of the microbiota (e.g. with the use of probiotics) could be proposed as a novel strategy not only for the treatment of IBS but also as an adjuvant for psychiatric treatment of anxiety and depression.

The Spermatogenic Ig Superfamily/Synaptic Cell Adhesion Molecule Mast-Cell Adhesion Molecule Promotes Interaction with Nerves

Nerve-mast cell interaction is involved in both homeostatic and pathologic regulations. The molecules that sustain this association have not been identified. Because synaptic cell adhesion molecule (SynCAM), alternatively named spermatogenic Ig superfamily (SgIGSF), is expressed on both nerves and mast cells and because it binds homophilically, this molecule may be a candidate. To examine this possibility, mast cells with or without SgIGSF/SynCAM were cocultured with superior cervical ganglion neurons that express SgIGSF/SynCAM, and the number of mast cells attached to neurites was counted. The attachment of mast cells with SgIGSF/SynCAM, i.e., bone marrow-derived mast cells (BMMC) from wild-type mice, was inhibited dose-dependently by blocking Ab to SgIGSF/SynCAM. Mast cells without SgIGSF/SynCAM, i.e., BMMC from microphthalmia transcription factor-deficient mice and BMMC-derived cell line IC-2 cells, were defective in attachment to neurite, and transfection with SgIGSF/SynCAM normalized this. When the nerves were specifically activated by scorpion venom, one-quarter of the attached IC-2 cells mobilized Ca2+ after a few dozen seconds, and ectopic SgIGSF/SynCAM doubled this proportion. At points of contact between neurites and wild-type BMMC, SgIGSF/SynCAM was locally concentrated in both neurites and BMMC. SgIGSF/SynCAM on mast cells appeared to predominantly mediate attachment and promote communication with nerves.