WO2008064489A1 - Probiotics to inhibit inflammation - Google Patents

Abstract

The present invention provides compositions and methods for reducing inflammation. Certain probiotics have been shown to block an intermediate conductance calcium dependent potassium current. This blockade leads to a decrease in the production and/or effect of pro-inlfammatory compounds. Agents that inhibit this pathway are useful for the treatment of inflammation in the gut and airways.

Description

PROBIOTICS TO INHIBIT INFLAMMATION

FIELD OF INVENTION

[0001] The present invention relates to novel compositions and methods for preventing and/or treating inflammation, particularly inflammation in the gastrointestinal tract and in the respiratory tract.

BACKGROUND OF THE INVENTION

[0002] Inflammatory bowel disease (IBD) is a chronic relapsing, disease of the gut involving an immune reaction to elements of the intestinal tract. The 2 major classes of IBD are ulcerative colitis and Crohn's disease. These are diseases with complex etiologies that present with diarrhea and dysmotility, pain and systemic symptoms. Activation of the intestinal immune system against commensal bacteria appears to be responsible for the characteristic relapsing course of these diseases [4]. Activation of T lymphocytes producing IL-6, IL-13, IL-17, and the release of tumor necrosis factor (TNF) appear to have key roles for these inflammatory processes [5].

[0003] An increasing number of clinical studies have revealed therapeutic effects of feeding different strains of probiotics on IBD, including ulcerative colitis, Crohn's disease, and pouchitis. Administration of either Lactobacillus reuteri [7] or L. salivarius [8] decreases colitis seen in interleukin-10 deficient mice. Colitis in mice induced by 2,4,6-Trinitrobenzenesulfonic acid (TNBS) [9] or dextran sulfate sodium (DSS) [10] is attenuated by Lactobacilli species. Overall, the evidence from clinical and animal studies is that some probiotics have a beneficial effect in gut inflammation including IBD [11-13]. Because of this, efforts are being made to identify the mechanisms by which probiotics may decrease gut inflammation and diminish its effects [10, 14]. Optimal probiotic species and their combinations are also being sought [9]. In addition, insight into how these organisms may act can contribute to understanding the underlying pathologies of IBD. [0004] In addition to effects in the gut, there is evidence that probiotics, given perinatally, may be protective against manifestations of atopic disease. Clinical trials have demonstrated that Lactobacillus rhamnosus GG may be effective in treatment and prevention of early atopic disease in children. Lactobacillus fermentum was shown to be beneficial in improving the extent and severity of atopic dermatitis in young children. Such findings have lead to an increased interest in the role of commensal organisms in the regulation of systemic immune responses.

[0005] There is evidence indicating that oral administration of certain microbial organisms can modulate immune responses in the lung. Rats orally immunized with killed P. aeruginosa exhibited enhanced bacterial clearance from the airways compared to non-immunized donors following intra-tracheal challenge with live P. aeruginosa. This response was associated with increases in bronchoalveolar neutrophils and in recruitment and phagocytic activity of alveolar macrophages. Furthermore, infection with the gut-restricted bacterium, Citrobacter rodentium attenuated the airway eosinophilia that result from pulmonary Cryptococcus neoformans infection. A double blind randomized trial found that feeding with the probiotic L. rhamnosus GG reduced the rate and severity of respiratory virus infection in children. Oral treatment of mice with L. casei increased pulmonary NK cell activity and enhanced IFNγ and TNF production by nasal lymphocytes. The probiotic treated animals also had reduced viral titers in nasal washings following influenza infection.

[0006] Despite indications that probiotic treatment can modulate some immune responses in the lung there have been no previous reports of the effects of oral treatment with a probiotic organism on major characteristics of asthma, including allergic airway inflammation and bronchial hyper-responsiveness. SUMMARY OF THE INVENTION

[0007] A method to increase the release of anti-inflammatory transmitters from the ISNs that are decimated in chronic inflammation would be of considerable therapeutic benefit. The present invention addresses that need.

[0008] In addition, the ability of a probiotic organism L.reuteriXo attenuate antigen - induced eosinophil influx to the airway as well as local cytokine responses and hyper-responsiveness to methacholine in an OVA-sensitized mouse model of allergic airway disease is disclosed.

[0009] Feeding with the probiotic organism Lactobacillus rhamnosus (LB) leads to activation of enteric sensory neurons by inhibition of a specific calcium dependent potassium ion channel conductance. The consequence of this is a decrease in gut motility and increased release of anti-inflammatory neuropeptides.

[0010] According to an aspect of the present invention there is provided a method of reducing inflammation, said method comprising administering an effective amount of a probiotic. The probiotic is preferably Lactobacillus, bifidobacterium or a combination thereof.

[0011] According to another aspect of the invention a method of reducing inflammation is provided. The method comprises administering an agent that inhibits an intermediate conductance calcium dependent potassium current(IKCa). The agent is preferably formulated for mucosal delivery. The agent may be administered orally or via an aerosol. The method is useful for the treatment of inflammation in the gastrointestinal tract or in the respiratory tract.

[0012] In a further aspect of the invention an agent for the treatment of inflammation is provided. The agent comprises an IKCa blocker. In one embodiment, the agent is derived from a probiotic, preferably lactobacillus or bifidobacterium. The agent may comprise a probiotic cell wall component or a mimetic of a probiotic component. [0013] In yet another embodiment a method of screening probiotics for antiinflammatory effects is provided. The method comprises the steps of: administering a probiotic to an animal; and measuring IKCa currents wherein a decrease in IKCa currents is indicative of an anti-inflammatory effect.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] These and other features of the invention can become more apparent from the following description in which reference is made to the appended drawings wherein:

[0015] Figure 1 shows a conceptual model whereby Lactobacilli (LB) decrease IKca activity in CGRP-containing intrinsic sensory neurons in the myenteric plexus;

[0016] Figure 2 shows a recording from intrinsic sensory neuron in longitudinal muscle myenteric plexus preparation;

[0017] Figure 3 demonstrates IKCa activity recorded after action potential volley;

[0018] Figure 4 shows the effect of Lactobacillus rhamnosus on slow AHP and excitability;

[0019] Figure 5 shows the effect of oral treatment with live (LR) heat killed (HK) and γ-irradiated (IR) Lreυteh (1x10⁹ organisms daily for 9 consecutive days) on total (A), differential (S) cell counts (macrophages, eosinophils, neutrophils, and lymphocytes) and (C) absolute numbers of macrophages and eosinophils in BAL fluid from OVA- sensitized male mice 24 hours after challenge with intranasal OVA or saline;

[0020] Figure 6 shows representative sections of lung tissue from Lreuteri treated (A) and untreated (B) OVA-sensitized mice following antigen challenge:

[0021] Figure 7 shows the effect of oral treatment with live (LR) heat killed (HK) and γ-irradiated (IR) Lreuteri (1x10⁹ organisms daily for 9 consecutive days) on cytokine levels in BAL fluid from OVA-sensitized male mice 24 hours after challenge with intranasal OVA or saline;

[0022] .Figure 8 shows the effect of oral treatment with live (LR) Lreuterϊ (A) and heat killed (HK) and γ-irradiated (IR) organisms (B) on airway reactivity (slope R_RS) and maximum resistance (Max R_RS) in OVA-sensitized male mice 24 hours after challenge with intranasal OVA or saline.

[0023] Figure 9 shows the effect of oral treatment with live (LR) heat killed (HK) and γ-irradiated (IR) L.reuteri (1x10⁹ organisms daily for 9 consecutive days) on activity of indoleamine 2,3-dioxygenase in plasma and lung tissue as assessed by kynurenine (Kyn) to tryptophan (Tryp) ratio and maximum IDO activity per mg of protein respectively.

[0024] Figure 10 shows the effect of oral treatment with live (LR) L.reuteri (1x10⁹ organisms daily for 9 consecutive days) on eosinophil numbers in BAL fluid (A) and airway responsiveness to methacholine (B) in OVA sensitized TLR-9 deficient mice.

DETAILED DESCRIPTION

[0025] Probiotics, such as Lactobacillus rhamnosus (LB), can decrease symptoms of inflammation and animal models [1 , 2]. When certain probiotics are applied to the gut they evoke relaxation and decreased motility [3]. The enteric nervous system (ENS) is thus a potential target for probiotic action as peptidergic and cholinergic enteric nerves can influence the immune system. The present inventors have discovered that L. rhamnosus excite gut intrinsic sensory neurons (ISNs). The ENS exerts a constitutive inhibitory effect on motility and mucosal inflammation, thus the increased excitability would counterbalance the decrease in enteric neuron number that occurs in chronic IBD.

[0026] Referring now to the Figures, Figure 1 shows a conceptual model whereby Lactobacilli (LB) decrease IK_Ca activity in CGRP-containing intrinsic sensory neurons in the myenteric plexus. The effect is to decrease the post-action potential relative refractory period (slow afterhyperpolarizatiorΛ thus making the neuron more excitable. Increased excitability leads to increased CGRP release in the mucosa because neurotransmitter release is facilitated by action potential invasion of terminal fibers. In IBD or animal models, increased CGRP release compensates for the decrease in enteric neurons associated with inflammation.

[0027] Figure 2 shows a recording from intrinsic sensory neuron in longitudinal muscle myenteric plexus preparation. CGRP immunoreactivity, neurobiotin fill and action potential (AP) parameters measured. A. Left panel, CGRP immunoreactive cell bodies with large oval Dogiel type 2 shape in myenteric ganglion. Right panel, Double labeling with Texas red-avidan reveals that one of the immunoreactive neurons was filled with neurobiotin after being recorded from. B. Upper, Action potential from cell recorded from in A. Action potential amplitude, upstroke speed, width at half height and amplitude and duration of fast afterhyperpolarization are measured. Lower, same as upper but slow after hyperpolarization is shown.

[0028] Figure 3 demonstrates IKCa activity recorded after action potential volley. A. Cell attached recording of IKCa opening after action potentials with symmetrical 140 mM K+. All points histograms constructed for recordings made a two different patch electrode potentials give unitary ion channel currents. Slope of I-V relation gives unitary conductance (g) of about 80 pS, in the upper intermediated conductance range. B. After breaking though patch, action potentials elicit a robust slow afterhyperpolarization. Maximal slow afterhyperpolarization at double arrows corresponds to period when single channel recording was taken. Image from cell yielding recordings for this figure is a digital capture of Texas red fluorescence showing typical multipolar Dogiel type 2 shape of ISN in rat colon myenteric plexus.

[0029] Figure 4 shows the effect of Lactobacillus rhamnosus on slow AHP and excitability A, 3 action potentials (truncated for display) evoke prolonged slow afterhyperpolarization in ISN of control rat. B, slow AHP from neuron in Lactobacillus rhamnosus fed rat: these were typically shorter than for controls. The slow AHP is caused by opening of IKca channels. C, a 500 ms duration. 2 x threshold, test current pulse evoked a single action potential in ISN of control rat. D₁ shows representative recording from Lactobacillus fed rat. ISNs from lactobacillus fed rats fired more action potentials (were more excitable) than those from control rats when stimulated a 2 x threshold intensity.

[0030] The data indicate that L. rhamnosus excite ISNs by blocking a K⁺ ion channel whose opening inhibits the neurons. Since blockade of the same channel in T lymphocytes prevents their activation, this channel may represent a common pathway through which LB can modulate inflammation and motility via both immune and neural systems. CGRP and acetylcholine have anti-inflammatory functions in the mucosa and the great majority of CGRP and cholinergic fibers in the mucosa derive mainly from the 100 million ISNs present in the gut wall.

[0031] Direct sensory responses have been recorded in mouse AH/Dogiel type Il neurons proving that, as for guinea pig , they are also sensory. An in situ patch clamp technique was used to directly record from ISNs in the terminal rat colonand were identified electrophysiologically and by shape.14/14 AH cells, that were filled with marker, were Dogiel type Il in shape. Of these 6/6 ISNs were immunoreactive for CGRP. The results also show that rat ISNs express an intermediate conductance potassium channel (IKCa) channel whose opening coincides with the evolution of the slow afterhyperpolarization (AHP). This conductance has the biophysical characteristics of the IKCa channel whose opening underlies the slow AHP .

[0032] Experiments were performed to test the effect on ISNs of feeding 10⁹ Lactobacillus rhamnosus (LB) organisms daily for 9 days. LB was chosen because of its demonstrated ability to reduce disease severity in a rat model of TNBS induced colitis. LB reduced the duration of the slow AHP for a standard 3 action potential pulse protocol [48] by about 50 % (Table 1 & Figure 4).

TABLE 1.

[0033] No. of APs per current density (pA/100 μm² cell membrane, at 2 x threshold). *P = 0.05, ^**P = 0.04

[0034] The correlation between IK_Ca opening and the slow AHP provides strong evidence that IKca conductance has been inhibited by LB. The slow AHP represents the relative refractory period of the action potential and thus LB ought to make ISNs more excitable when they are depolarized by current. Experiments with current injection via the patch pipette confirmed that this was the case with a measurable increase in the number of action potentials discharged (Table 1 & Figure 3).

[0035] The results provide evidence that probiotics, especially (LB), increases excitability of sensory neurons in the ENS. Therefore, action by LB on the ENS may be a significant route by which they achieve their beneficial effects in inflammation.

[0036] Figure 5 shows the effect of oral treatment with live (LR) heat killed (HK) and γ-irradiated (IR) L.reuteri (1x10⁹ organisms daily for 9 consecutive days) on total (A), differential (S) cell counts (macrophages, eosinophils, neutrophils, and lymphocytes) and (C) absolute numbers of macrophages and eosinophils in BAL fluid from OVA- sensitized male mice 24 hours after challenge with intranasal OVA or saline. The effect of live L salivarius treatment (1 x10⁹ organisms daily for 9 consecutive days) on eosinophil numbers in BAL fluid is also shown (D). Each column represents the mean ± SEM (n =10). *p < 0.05; **p < 0.01 compared with MRS broth treated control.

[0037] Figure 6 shows representative sections of lung tissue from L.reuteri treated (A) and untreated (B) OVA-sensitized mice following antigen challenge. A section from a saline challenged control animal is shown for comparison (C). [0038] Figure 7 shows the effect of oral treatment with live (LR) heat killed (HK) and γ-irradiated (IR) Lreuteri (1x10⁹ organisms daily for 9 consecutive days) on cytokine levels in BAL fluid from OVA-sensitized male mice 24 hours after challenge with intranasal OVA or saline. Each column represents the mean ± SEM (n =10). *p < 0.05 compared to OVA challenged, MRS broth treated control.

[0039] .Figure 8 shows the effect of oral treatment with live (LR) L.reuteri (A) and heat killed (HK) and γ-irradiated (IR) organisms (B) on airway reactivity (slope RRS) and maximum resistance (Max R_RS) in OVA-sensitized male mice 24 hours after challenge with intranasal OVA or saline. Airway responsiveness was measured in response to increasing doses of intravenous methacholine. Using the resulting dose response curve, indices of airway reactivity and maximal degree of bronchoconstriction (Max R_Rs) were determined. Each column represents the mean ± SEM (n =10). ^*p < 0.05; compared OVA challenged, MRS broth treated.

[0040] Figure 9 shows the effect of oral treatment with live (LR) heat killed (HK) and γ-irradiated (IR) L.reuteri (1x10⁹ organisms daily for 9 consecutive days) on activity of indoleamine 2,3-dioxygenase in plasma and lung tissue as assessed by kynurenine (Kyn) to tryptophan (Tryp) ratio and maximum IDO activity per mg of protein respectively. IDO activity was assessed in OVA sensitized mice 24 hours after challenge with intranasal OVA or saline. Each column represents the mean ± SEM (n =10). *p < 0.05 compared to OVA challenged, MRS broth treated control.

[0041] Figure 10 shows the effect of oral treatment with live (LR) Lreuteri (1x10⁹ organisms daily for 9 consecutive days) on eosinophil numbers in BAL fluid (A) and airway responsiveness to methacholine (B) in OVA sensitized TLR-9 deficient mice. Cell counts and airway responsiveness were assessed 24 hours after challenge with intranasal OVA or saline. Airway responsiveness was measured in response to increasing doses of intravenous methacholine. Using the resulting dose response curve, indices of airway reactivity (slope RRS) and maximal degree of bronchoconstriction (Max R_RS) were determined. Each column represents the mean ± SEM (n =6) *p < 0.05 compared to OVA challenged, MRS broth treated control.

[0042] The present invention also demonstrates that oral administration of live L. reuteri resulted in significant attenuation of the allergic airway response. This effect appears to depend on both viable organisms and TLR-9 and may be associated with systemic evidence reflecting IDO activation. The results presented herein indicate that the immunomodulatory effects of oral treatment with L reuteri are not limited to the gastrointestinal tract and demonstrate that oral treatment with a probiotic organism can attenuate major characteristics of an asthmatic response, including airway eosinophilia, local cytokine responses and hyper-responsiveness to methacholine. These beneficial effects may be effected by various types of regulators of allergic airway disease that are based on the mechanisms behind the quantitative and qualitative differences in immune regulation that exist among probiotic organisms.

[0043] The above disclosure generally describes the present invention. It is believed that one of ordinary skill in the art can, using the preceding description, make and use the compositions and practice the methods of the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely to illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Other generic configurations can be apparent to one skilled in the art. Documents such as patents or patent applications referred to herein are hereby incorporated by reference.

EXAMPLES

[0044] Although specific terms have been used in these examples, such terms are intended in a descriptive sense and not for purposes of limitation. Methods referred to but not explicitly described in the disclosure and these examples are reported in the scientific literature and are well known to those skilled in the art.

Example 1. Animals and Methods.

[0045] Male Sprague-Dawley rats (Charles River Breeding Laboratories, Saint Constant, QC, Canada) weighing 200-250 g are used. Tissue is obtained from TNBS-treated and untreated animals that have been gavaged with either 10⁹ Lactobacillus rhamnosus or Bifidobacterium infantis in 0.2 mL MRS broth for 9 consecutive days, prior to induction of TNBS colitis and continuing for the duration for the experiment (13 days). Gavage with MRS broth alone is used as control. LB are prepared and administered as described for L. reuteri in Kamiya et al. [38]. Colon inflammation can be elicited by administration at day 9, per rectum, of a single dose of 0.5 mL of TNBS, (25 mg/mL in 25% ethanol) under deep surgical anesthesia, see Linden et al. 2003 [61] and Lomax et al. 2006 [62]. Per rectal administration of saline is used for control groups. The intensity of inflammatory damage is determined after overnight formaldehyde fixation of a colon segment (adjacent to the one taken for electrophysiology) 4 days following TNBS administration and assessment of macroscopic and histological changes using the scoring systems as described in McCafferty et al. [63]. Electrophysiology: Experiments is performed by making whole cell recordings from ISNs in the myenteric plexus of the rat large intestine. Electrophysiological recording is done 4 days after TNBS administration. Segments for patch clamp recording are opened and pinned in a plastic recording bath with mucosa uppermost and dissected to expose the myenteric plexus [64]. The recording dish is then mounted on an inverted microscope and the tissue continuously superfused (4 mL/min) with Krebs saline, gassed with 95% 02-5% CO2, and warmed to 35-37°C. A single ganglion is prepared for patch clamping as described in Kunze et al. (2000); briefly, the ganglion is exposed for 10-15 min to 3 ml of 0.01-0.02% protease type XIV (Sigma, http://www.siamapaldrich.com), then the upper surfaces of myenteric neurons are revealed by cleaning part of the ganglion with a fine hair until individual neuron soma became just visible. Whole cell patch clamp recording are made with pipettes filled with K⁺ rich intracellular solutions [64-66]. The standard pipette K⁺-rich solution contains (mM): 110 KMeSO4, 20 KCI, 9 NaCI, 0.09 CaCI2, 1 MgCI2, 10 HEPES, 0.2 BAPTA, and 3.0 ATP, 0.2 GTP, 5 neurobiotin™, pH 7.3 with addition of -2.8 ml_ 0.1 M KOH. Neurobiotin has been added to allow a posteriori verification by morphology that ISNs have been recorded [44, 67]. All recordings are made in the presence of hyoscine and nicardipine to minimize muscle contractions during recording [44, 68].

[0046] Active and passive neuronal properties including excitability are assessed in the current clamp (voltage recording) mode. Membrane potential (V_m) and whole cell background conductance (g_BG) are measured immediately after going whole cell. Action potential amplitude, duration, polarization velocities, and fast and slow afterhyperpolarizations are measured after injection of short depolarizing current pulses. Firing threshold and accommodation at 2x threshold are measured by injection of a 2 s duration current pulses. A standard presynaptic stimulus protocol for evoking sustained postsynaptic excitation [44, 69] is used. Electrical stimulation of presynaptic axons at high frequency (10 pulses at 20 Hz) is used to activate SP (NK3 receptor) dependent excitation while prolonged low frequency stimulation is used to evoke non-tachykinergic, CGRP excitation [70, 71]. Changes in V_m, g_BG, and active membrane properties are monitored before and during synaptic stimulation. If LB modulates metabotropic neurotransmission, NK3 and NK1 or CGRP1 receptor blockade can be used to identify the mode of neurotransmission that may have been modulated.

[0047] Results can be analyzed using nonparametric analysis of variance with post- hoc tests for statistical significance. Pilot experiments suggest that it is preferable to record from 40 ISNs in each treatment group of which 10-15 can have been tested for sustained synaptic input. Results have been from 24 cells in uninflamed colon.

[0048] Standard immunohistochemistry can be used in guinea pig and mouse to test myenteric neurons recorded from for immunoreactivity for calretinin or calbindin (partial makers for Dogiel type Il cells in rat) [37, 57, 68, 72], CGRPalpha and SP. All cells are filled with neurobiotin by ionophoresis and visualized using streptavidin- Texas red fluorescence [44, 57, 73].

[0049] Ion channel currents (single channel and whole cell) can be directly recorded in voltage clamp mode using appropriate extra- and intracellular solutions and specific ion channel blockers to isolate IK_Ca [44, 47]. We can record IK_Ca from ISNs and T-lymphocytes harvested from animals in separate experiments.

[0050] T cell isolation: MLN and Peyer's patches can be removed from animals and forced through a stainless steel wire mesh in Hanks balanced salt solution. After discontinuous Ficoll gradient centrifugation, mononuclear cells can be pooled and suspended in Hanks balanced salt solution containing 5% fetal calf serum. CD4⁺ and CD8⁺ T cells can be purified from mononuclear cells by negative selection. In brief, cells can be incubated for 30 min on ice with a mixture of the following mouse monoclonal antibodies: anti-rat CD45RA, specific for B cells and anti-rat CD8 (OX-8; Serotec) for CD4⁺ T-cell selection or anti-rat CD45RA and anti-rat CD4 for CD8⁺ T- cell selection. After being washed, cells can be incubated with a suspension of magnetic beads coated with goat anti-mouse IgG antibody. The cells bound to beads and the free magnetic beads can then be removed using a magnetic field. The remaining cells can be passed through glass bead columns coated with polyclonal anti-rat IgG (heavy plus light chains) and anti-mouse IgG (heavy plus light chains) antibodies. The purity of CD4⁺ and CD8⁺ T-cell populations thus obtained can be determined by flow cytometric analysis and is expected to be greater than 98%. T- cells can be patched after allowing adhesion to polylysine coated bottom of a plastic Petri dish.

[0051] Single channel recording: For ISNs neurons, preparation for patch clamp recordings can be made as described in detail above [44, 65, 87].

[0052] For both neurons and T-cells, single ion channel recordings can initially be made in cell-attached mode. Using an intracellular solution of (mM): 130 KMeSO4, 9 NaCI, 0.09 CaCI2, 1 MgCI2, 10 HEPES, 0.2 BAPTA, and 3.0 ATP, 0.2 GTP, 5 neurobiotin, pH 7.3 with addition of -2.8 mL 0.1 M KOH. Extracellular bathing solution can be normal Krebs with or without the addition of TRAM-34 a highly specific blocker of IKc_a. IKca can be identified (as we have previously done) on the basis of its unitary conductance and differentiated from background (leak) K⁺ channels by its negligible open probability at rest and by its activation by burst of action potentials and dependence on intracellular Ca²⁺ [44, 46] [47, 88]. The density of active ion channels can be estimated by counting the number of patches that contain active IKca channel activity. Density is calculated from the area (capacitance) of the patch compared to that for the whole cell. For example, using this method [89], the number of IK_Ca channels per ISN has been computed as 3210 for cells with an average body surface area of 3400 urn². TNBS inflammation and LB may alter IK_Ca functionally rather than altering channel density. We can also determine if total IKca current is altered due to changes in opening or closing kinetics rather than in total number of channels. Therefore, we can analyze open and closed dwell times with respect to the reaction state scheme that best describes the channel's gating behavior [90]. This gives information about the nature of changes in IKca open probability, which together with channel no. determines total current flow.

[0053] Whole cell recording: Measurements of IKca currents can be made to complement the single channel data. In neurons, activation of IKca current, which is equal to the slow AHP K+ current in ISNs can activated by 6 brief (50 ms) depolarizing prepulses to +50 mV from a holding potential of -60 mV [91]. Pilot experiments have shown that this robust protocol elicits a measurable IKca current in ISN, even when they are taken from inflamed intestine. Sensitivity to IK_Ca current blockers clotrimazole and TRAM-34, and insensitivity to the SK_Ca and BK_Ca channel blockers apamin and iberiotoxin make up the standard pharmacological sieve that is used to confirm the biophysical identification of IK_Ca [44]. For T-cells IK_Ca, currents are evoked by incrementing, depolarizing voltage steps or quasi-steady state ramp voltage commands [65]. Then, as for neurons, the current is isolated using ion substitution and differential sensitivity to TRAM-34, clotrimazole, apamine and iberiotoxin.

[0054] Example 2. The effect of probiotic ingestion on intrinsic sensory neuron action potentials and excitability in normal and TNBS treated rat colon.

[0055] Patch clamp recording is used to investigate ISN excitation, which is the "gold standard" for measuring the activity of cells and their ion channels in real time [49, 50], Patch clamping is a mature and evolving nanotechnology tool [49, 50], which like the polymerase chain reaction shows no sign of leveling off in potential. It is able to provide a kinetic fingerprint of an ion channel in the microsecond domain and can discern if there have been changes in protein function even if there has been no alteration in mRNA, protein expression, translocation or folding. This provides a great advantage over post hoc methods such as visualization of Fos protein that only indicate that there may have, in the past, been a alteration in gene expression. An exemplar of its power with regard to other methods is provided by the hyperpolarization activated nucleotide gate channel in mouse ENS. Message (mRNA) for this channel is present in >90 % of enteric neurons and Western analysis and immunohistochemistry confirm that the protein is present and expressed in the plasmalemma [51]; yet this channel is only functional in 20 % of enteric ISNs [44, 51]. A novel method was devised for patch clamping guinea pig myenteric neurons in the intact longitudinal muscle - myenteric plexus (LMMP) preparation obviating the need for cell dispersion for patch clamp recording. This method has led to new discoveries of mechano-transducing ion channels, novel membrane currents and intracellular transduction. Preliminary data show that ISN excitability is increased by feeding LB. At present, there is no other available data on effects of lactobacilli on neurons.

[0056]TNBS inflammation has a similar immune profile to IBD [53] and the inflammation is reported to be reliably decreased by prior feeding with LB [54]. Thus, it is possible to compare changes in neuron behaviour and directly correlate this with the anti-inflammatory action of LB. ISNs connect with each other to form an excitatory network which determines the overall excitability set-point of the ENS [36, 55, 56]. Transmission between ISNs is almost exclusively metabotropic [56] with CGRP and SP being the main transmitters [34, 36]. Short (500 ms), high frequency (20 Hz) presynaptic stimuli elicit tachykininergic slow EPSPs that are blocked by neurokinin 3 (and 1) receptor blockade [57]. Whereas, ongoing, low frequency (1-2 Hz) presynaptic stimulation elicits sustained excitation which is not diminished by tachykinin or acetylcholine receptor blockers. Thus, it remains that CGRP like LHRH [58] elsewhere is likely to be preferentially released using continuous repetitive low frequency stimulation. Given the anti-inflammatory action attributed to CGRP release in the mucosa as well as its role as a neurotransmitter, LB could modulate ISN to ISN metabotropic transmission and thus neuron excitability. Studies suggest that candidate probiotics induce both general immunostimulatory and immunoregulatory responses. Much of this disparity in immune response appears to be due to differing inherent characteristics of specific probiotic organisms that include degree of colonization, adhesion and intrinsic immunogenicity. Given the clear distinctions in the immunomodulatory ability of different probiotic strains it is probable that the potential for neuromodulation can also vary between organisms. For this reason, the efficacy of an additional probiotic species, Bifidobacterium infantis, to modulate ENS excitability is tested. B. infantis has been demonstrated to decrease disease severity in a rat model of colitis [59] and to reduce symptomatology in human irritable bowel syndrome [60].

Example 3 Suppression of IKCa represents a significant component of the antiinflammatory and relaxation response evoked bv probiotic treatment.

[0057] TNBS colitis is a well-established model of inflammation that is characterized by colonic ulceration, infiltration of inflammatory cells, disruption of gut motility and a decrease in enteric neuron number similar to that observed in tissue taken from patients with IBD [53]. LB has also been clearly demonstrated to reduce disease severity in the TNBS model. [0058] The TNBS model of intestinal inflammation can be used to determine the effect of the IKCa receptor antagonist TRAM34 (120 mg/kg s.c.) and the channel activator 1-EBIO (40 mg/kg s.c [101]) both alone and in conjunction with probiotic feeding. Both of these channel modulators have previously been used successfully in vivo [96, 101]. Parameters assessed include histological score, determination of MPO activity, serum and gut tissue cytokine profile and ex vivo measures of gut motility. The effects of TRAM-34 and 1-EBIO on function of cells isolated from Peyer's patches and mesenteric lymph nodes (MLN) can be determined.

[0059] For all studies there were at least 6 animals in each of 8 treatment groups (colitis/control+broth, colitis/control+ probiotc, colitis/control+ IKCa opener/antagonist, colitis/control+ IK_Ca opener/ antagonist+probiotc). At least two independent experiments are performed making a total of 12 animals per treatment group.

[0060] Male Sprague Dawley rats 200-250 g are given 10⁹ live, LB in 200 μl of broth via a gavaging needle (20 gauge, 3.8 cm long,) for 9 consecutive days, prior to induction of TNBS colitis and continuing for the duration for the experiment (13 days in total). TRAM-34 or 1-EBIO can be administered over the same period through daily subcutaneous injection at doses of 120 mg/kg per day [96] and 35 mg/kg [101] respectively. MRS broth can serve as control for probiotic treatment while vehicle (peanut oil) can serve as the control for antagonist/opener treatment.

[0061] TNBS model: TNBS colitis can be induced as described in Objective 1. Animals can be sacrificed 4 days after TNBS administration. Following sacrifice the ileum, caecum, proximal colon and distal colon can be fixed in 10 % formalin, paraffin embedded and assessed histologically in a blinded fashion. Haematoxylin- eosin stained tissue sections of intestinal specimens can be graded using a histological index, as previously published [63]. This index is based on the degree of epithelial cell erosion, goblet cell depletion and inflammatory cell infiltrate. Serum samples can be taken from rats and TNF, IL-4 IL-6 and IL-10 and IFNγ levels measured using a cytometric bead assay (BD™ Flex Set system). Tissue levels can be measured in homogenized colon tissue. Tissues for assessment of myeloperoxidase content can be snap-frozen in liquid nitrogen and stored at -70 ⁰C and MPO can be measured as previously described (51 ). Additional tissue can be collected for RT-PCR analysis, immunohistochemistry and in situ hybridization

[0062] Cell stimulation assays: The effect of probiotic and/or TRAM34/I-EBIO treatment on stimulated cytokine release from cells isolated from the Peyer's patches and MLN of rats from each treatment group can be assessed. The response of total mononuclear cell populations and isolated CD4+ and CD8+ cells can be investigated. Following isolation, cells (10⁶/ml) suspended in the RPM1 1640 medium can be stimulated with plate-bound anti-rat CD3 and soluble anti-rat CD28 (anti- CD3/CD28) mouse monoclonal antibodys (G4.18 [10 μg/ml] and JJ319 [1 μg/ml], respectively; Pharmingen, San Diego, Calif) in 96-well flat-bottomed culture plates at 37⁰C in a 5% CO₂ atmosphere. Culture supematants can be harvested 72 h after initiation and stored at -8O⁰C until use. Cytokine (TNF, IFNγ, IL-4, IL-6, IL-10) levels in supematants can be measured using the BD™ Cytometric Bead Array (CBA) Flex Set system.

[0063] Gut motility: Gut motility recordings can be used as a direct index of ENS function and output. Migrating motor complexes (MMCs) in the colon as well as peristalsis [102, 103] [104] are dependent on the ENS whose constitutive activity controls MMC frequency [103]. MMCs, spontaneous contractions and distension- evoked peristalsis can be measured as pressure waves [105] in a modified Trendelenburg organ bath preparation [105, 106] using colon segments. A 6 cm segment of distal colon is removed under deep surgical anesthesia, placed in a 40 ml_ plexiglass chamber, and superfused with normal Krebs saline saturated with 95 % O2-5 % CO2 and heated to 37 ⁰C. Intraluminal pressure can be recorded using an custom made pressure transducer [64] inserted into a T piece attached to a saline-filled glass tube inserted at the anal end. The segment is filled with fluid orally via a saline-filled tube inserted there with the pressure head driven by gravity. The signal is further amplified, displayed, and stored on computer. Motility is recorded from segments taken from normal and TNBS treated animals that have been fed either probiotic or broth. We can test bath application of IK_Ca blockers and activators; SKca and BKca blockers apamine and iberiotoxin can also be applied to further test the specificity of the response to the IKca modulators.

[0064] Example 4. Determination if the localization and/or release of CGRP and SP are altered through probiotic action on ENS and if such changes mediate the effects on inflammation and motility.

[0065] TNBS induced colonic inflammation in the rat is accompanied by a change in the distribution of CGRP and SP immunoreactive nerves [110]. Using this model we can investigate changes in expression and localization of SP and CGRP in normal and inflammed colon tissue. The ability of the CGRP receptor antagonist (CGRP8- 37) to inhibit the effects of LB on TNBS colitis can also be assess to determine the importance of enhanced CGRP release to the mechanism of action of the organism.

[0066] For all studies there are be 6 animals in each of 8 treatment groups (colitis / control + broth, colitis / control÷ probiotc, colitis / control + CGRP receptor antagonist, colitis / control+ CGRP receptor/ antagonist + probiotc) and at least two independent experiments can be performed making a total of 12 animals per treatment group.

[0067] CGRP receptor antagonist treatment: Male Sprague Dawley rats 200-250 g are treated with LB as described above. The CGRP receptor antagonist CGRP8-37 is administered (10 μg/kg s.c. 4 times/day) over the same period as LB administration. CGRP8-37 has been used successfully in vivo with this dose and route of administration in previous studies [111]. MRS broth can serve as control for probiotic treatment while vehicle can serve as the control for CGRP receptor antagonist treatment. TNBS colitis can then be induced and disease severity assessed. [0068] Tissue preparation: Colon tissue from CGRP antagonist treated can be fixed overnight in a mixture of 2% formaldehyde and 0.2% picric acid in phosphate buffer (pH 7.2) followed by washing in Tyrode's solution containing 10% sucrose. Fixed tissue can then be embedded in OCT, snap frozen and cut in a cryostat.

[0069] Immunohistochemistry: Cryostat-sections can be rehydrated in PBS containing 0.1% Triton X-100 (PBS-T) and incubated separately for 48 h at 4 ⁰C with the specific primary antisera directed against CGRP, SP₁ and/or the neuronal markers, neurone-specific enolase (NSE). Sections can then be washed with PBS-T and incubated for 1 h at room temperature with sheep anti-rabbit immunoglobulin G conjugated to fluorescent cyanine dye ^ΛCY3TM' (1:100; Sigma, St. Louis, MO, USA) alone or combined with goat anti-mouse immunoglobulin G conjugated to fluorescein isothiocyanate (FITC) (1 :50; Incstar, Stillwater, MN₁ USA). After further PBS-T washing sections can be mounted in bicarbonate-buffered glycerol (pH 8.6) and examined using a fluorescence microscope

/^"00707RT-PCR: RNA can be isolated from colonic tissue using the TRIzol protocol (Invitrogen, Carsbad, CA) and then treated with Amplification Grade Deoxyribonuclease I. Reverse transcription can be performed using Omniscript RT kit (Qiagen, Vanencia, CA) using random hexamers as primers. Real-time PCR can be carried out using the TaqMan assay. The PCR reactions can be carried out on an ABI Prism 7000 Sequence Detector.

[0071]\n situ hybridization: Radiolabeled probes for CGRP and β-preprotachykinin mRNA can be diluted in a hybridization buffer and applied to sections (approximately 500,000 CPM/section). Slides can be incubated overnight at 55⁰C in a humidified chamber. To reduce non-specific binding of the probe, slides can be washed in 20 μg/ml RNase solution for 30 min at room temperature, followed by 1 h each in 2XSSC at 5O⁰C, 0.2X SSC at 55°C and 60 ⁰C. Slides can be dehydrated and air- dried for autoradiography. Slides and 14C plastic standards containing known amounts of radioactivity can be placed in x-ray cassettes, apposed to film for 24-72h, and developed in an automatic film developer. To determine the cellular localization of hybridized probes, sections can be coated with nuclear track emulsion (NTB-2; Eastman Kodak) and exposed for 4-6 weeks, and counterstained with cresyl violet.

Example 5. Inflammatory cell influx to the airway

[0072] Total cell numbers in BAL fluids were significantly increased 24 h after the final antigen challenge in OVA sensitized mice compared with OVA sensitized saline- challenged mice (155.9 ±30.1 x10⁴vs 18.08 ±0.8 x10⁴ respectively n=12 pθ.001) (Figure 5a), thus confirming that challenge with OVA was effective . Although the cell population in BAL fluid from saline-challenge mice was almost exclusively alveolar macrophages, OVA challenge caused a dramatic increase in the proportion of eosinophils (Figure 5b). OVA-challenged mice fed with L. reuteri had a significant reduction in cells recovered in BAL fluid compared with MRS broth fed animals (59.2 ±11.8 x10⁴ vs 155.9 ±30.1 x10⁴ respectively, n=12 p = 0.004) (Figure 6a). This corresponded to a significant decrease in both eosinophil (38.2±2.7 x10⁴ vs 114.6 ±19.0 x10⁴) and macrophage numbers (19.1±3.6 x10⁴ vs 38.6 ±4.5 x10⁴) (Figure 5c). Histological analysis demonstrated that the increase in eosinophils in the lung parenchyma of OVA-sensitized and -challenged mice was also reduced by treatment with live L reuteri (Fig 6). Treatment of animals with either heat-killed or γ-irradiated organisms did not attenuate eosinophil influx to the airway (Figure 5). In contrast to L reuteri, live L salivarius did not modulate cellular influx to the airway (Figure 5d).

Example 6. Blood eosinophils

[0073] To determine if the reduction in eosinophil influx to the airway was entirely due to a decrease in migration from the blood, or also a result of decreased production/release from bone marrow, we measured circulating eosinophils. In OVA challenged animals treatment with live L. reuteri resulted in a significant reduction in the population of circulating eosinophils compared to MRS broth fed animals (4.7 ±0.54 % vs 1.6 ±0.27 % of total white blood cells respectively, n=6 p<0.001 ) (data not shown). Example 7. Cytokine levels in BAL fluid

[0074] Following OVA challenge levels of MCP-1 , TNF, IL-5, IL-13 and eotaxin were significantly increased in BAL fluid compared to saline challenged animals (Figure 7). Levels of IFNγ, IL-12 and IL-10 were not significantly changed compared to saline controls. Treatment with L reuteri significantly attenuated the increase in MCP-1 , TNF, IL-5 and IL-13 but did not alter eotaxin levels. There was no effect of L. reuteri on IL-10 while IFNγ and IL-12 were below the limit of detection in all groups assessed. Treatment with either heat killed or irradiated L reuteri also significantly attenuated the increase in MCP-1 TNF and IL-5 following antigen challenge however in contrast to treatment with live bacteria, killed organisms did not significantly alter levels of IL-13 (Figure 8). While L salivarius had no effect on cytokine levels in BAL fluid, (data not shown).

Example 8. Airways response to methacholine

[0075] Challenge with OVA in sensitized animals produced an increase in airway responsiveness to methacholine as determined by an increase in the maximum resistance from 3.9 ± 0.9 to 7.0 ± 0.8 cmH20/ml/s (n=15 p<0.01 ) and slope of dose response curve from 1.9 ± 0.7 to 4.8 ± 0.7 (p<0.01 ). This hyper-responsiveness was significantly reduced by treatment with live L. reuteri (max resistance 4.5 + 0.7 cmH20/ml/s, slope 2.9 ± 0.6) but not with heat killed or irradiated organisms. (Figure 9).

[0076] L. salivarius did not modulate airway responsiveness to methacholine (data not shown).

[0077] lndoleamine 2, 3 Dioxygenase (IDO) activity.

[0078] The plasma ratio of kynurenine (Kyn) to tryptophan (Tryp) was significantly increased following treatment with live L reuteri vs. MRS broth (0.24 ± 0.05 vs 0.09 ± 0.03 respectively, n=10 p<0.05). Heat killed and irradiated organisms did not modulate the Kyn/Tryp ratio (Figure 1Oa). Maximal IDO in lung tissue was not changed by treatment with either live or killed organisms (Figure 10b).

Example 9. The role of TLR 9 in L reuteri treatment

[0079] As in wild type animals OVA sensitization and challenge of TLR9 -/- mice lead to eosinophil influx to the airway (76.2 ±12.4xlO⁴ vs 0.62 ± O.lxlO⁴ n=6, p<0.001) (Figure 7) and increased airway responsiveness compared to saline challenged animals. However treatment with live L. reuteri did not result in attenuation of eosinophil influx (99.2±32.7xl0⁴), BAL cytokine levels (data not shown) or airway responsiveness to methacholine (Figure 7).

Example 10. Effect of isolated L. reuteri DNA

[0080] Given the requirement for TLR-9 in the effects of L. reuteri we assessed the ability of DNA isolated from the organism to reduce allergic airway inflammation. As previously described, [24] i.p. administration of the synthetic TLR-9 ligand, ISS-ODN, significantly reduced eosinophil influx to the airway (Figure 8a), however neither treatment with L. reuteri nor calf thymus DNA had any effect on the allergic airway response when administered either orally or through i.p. injection (Figure 8b).

[0081] One or more currently preferred embodiments have been described by way of example. It can be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims. References

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Claims

WHAT IS CLAIMED IS:

1. A method of reducing inflammation, said method comprising administering an effective amount of a probiotic.

2. A method according to claim 1 wherein the probiotic is Lactobacillus, bifidobacterium or a combination thereof.

3. A method of reducing inflammation comprising administering an agent that inhibits an intermediate conductance calcium dependent potassium current(IKCa).

4. A method according to claim 3 wherein the agent is formulated for mucosal delivery.

5. A method according to claim 3 wherein the agent is administered orally.

6. A method according to claim 3 wherein the agent is administered via an aerosol.

7. A method according to claim 3 wherein the inflammation is in the gastrointestinal tract.

8. A method according to claim 3 wherein the inflammation is in the respiratory tract.

9. An agent for the treatment of inflammation, said agent comprising an IKCa inhibitor.

10. An agent according to claim 9 wherein the agent is derived from a probiotic.

11. An agent according to claim 10 wherein the probiotic is lactobacillus.

12. An agent according to claim 9 comprising a probiotic cell wall component.

13. An agent according to claim 9 comprising a mimetic of a probiotic component.

14. A method of screening probiotics or compounds for anti-inflammatory effects, said method comprising the steps of: i) administering a probiotic to an animal; and ii) measuring IKCa currents wherein a decrease in IKCa currents is indicative of an anti-inflammatory effect.

15. A kit for screening probiotics or compounds for anti-inflammatory effects, said kit comprising: i) an inflammation inducer; ii) a control selective IKCa antagonist; and iii) a control IKCa channel opener.

16. A kit according to claim 15 further comprising a control probiotic.

17. A kit according to claim 15 wherein the inflammation inducer is 2,4,6 - trinitrobenzenesulfonic acid (TNBS).

18. A kit according to claim 15 wherein the IKCa antagonist is TRAM 34.

19. A kit according to claim 15 wherein the IKCa channel opener is I-EBIO.