Variation in essential oil components and anti-inflammatory activity of Allophylus edulis leaves collected in central-western Brazil.

ETHNOPHARMACOLOGICAL RELEVANCE: An infusion obtained from the leaves of "chal-chal" (Allophylus edulis Radlk.) is used for popular treatment of intestinal disorders and as an anti-inflammatory throat treatment. Because of the anti-inflammatory medicinal folk use, a previous work reported scientific research confirming the anti-inflammatory activity of A. edulis essential oil collected in Dourados, MS, Brazil, in March 2015. AIM OF THE STUDY: The aim of this study was to evaluate the variation in the chemical profile of the essential oil of A. edulis plants collected in Dourados (EOAE-D) and Bonito (EOAE-B), two cities in Mato Grosso do Sul State, Brazil. Additionally, we evaluated the anti-inflammatory effects of the essential oil, as well as that of the major compounds (caryophyllene oxide and α-zingiberene), in experimental in vivo models of inflammation in mice. MATERIALS AND METHODS: Leaves were collected from plants at both sites in July 2018. The composition of the essential oil (EOAE-D and EOAE-B) was determined by GC/MS, and major compounds (caryophyllene oxide and α-zingiberene) were isolated and identified by chromatographic methods and NMR spectroscopy. Anti-inflammatory capacities were assessed using two classical models of inflammatory models, carrageenan- and CFA-induced paw inflammation (mechanical and thermal hyperalgesia). RESULTS: Both EOAE-D and EOAE-B showed sesquiterpenes as a major constituent, namely, caryophyllene oxide (29.5%) and α-zingiberene (45.0%), respectively. In tests, EOAE, caryophyllene oxide and α-zingiberene-induced antiedematogenic and antihyperalgesic effects were found in the different utilized models. CONCLUSIONS: The results indicate that samples from the two cities differed in chemical composition but not in their anti-inflammatory and antihyperalgesic effects. This finding corroborates the use of A. edulis as a medicinal plant and indicates its potential in the therapy of inflammatory conditions.

A hyperalgesic ulcer on return from Senegal.

Diphtheria is an infectious disease transmitted by air or by contact. There are three main species of corynebacterium: diphtheriae, ulcerans, and pseudotuberculosis. They may or may not secrete a toxin responsible for serious cardiac or neurological complications. The frequent clinical presentations are diphtheria angina and cutaneous diphtheria. When it is suspected, it is urgent to send cutaneous or pharyngeal samples to the National Reference Center. If toxin is present, serotherapy should be instituted and the case should be reported. Antibiotic therapy with amoxicillin or azithromycin is the standard treatment. Vaccine prevention begun in the middle of the 20th century has helped to slow epidemics.

Contribution of spinal cord glial cells to L. amazonensis experimental infection-induced pain in BALB/c mice.

BACKGROUND: The cellular and molecular pathophysiological mecha\nisms of pain processing in neglected parasitic infections such as leishmaniasis remain unknown. The present study evaluated the participation of spinal cord glial cells in the pathophysiology of pain induced by Leishmania amazonensis infection in BALB/c mice. METHODS: Mice received intra-plantar (i.pl.) injection of L. amazonensis (1 × 105) and hyperalgesia, and paw edema were evaluated bilaterally for 40 days. The levels of TNF-α and IL-1ß, MPO activity, and histopathology were assessed on the 40th day. ATF3 mRNA expression was assessed in DRG cells at the 30th day post-infection. Blood TNF-α and IL-1ß levels and systemic parasite burden were evaluated 5-40 days after the infection. At the 30th day post-infection L. amazonensis, the effects of intrathecal (i.t.) treatments with neutralizing antibody anti-CX3CL1, etanercept (soluble TNFR2 receptor), and interleukin-1 receptor antagonist (IL-1ra) on infection-induced hyperalgesia and paw edema were assessed. In another set of experiments, we performed a time course analysis of spinal cord GFAP and Iba-1 (astrocytes and microglia markers, respectively) and used confocal immunofluorescence and Western blot to confirm the expression at the protein level. Selective astrocyte (α-aminoadipate) and microglia (minocycline) inhibitors were injected i.t. to determine the contribution of these cells to hyperalgesia and paw edema. The effects of i.t. treatments with glial and NFκB (PDTC) inhibitors on spinal glial activation, TNF-α, IL-1ß, CX3CR1 and CX3CL1 mRNA expression, and NFκB activation were also evaluated. Finally, the contribution of TNF-α and IL-1ß to CX3CL1 mRNA expression was investigated. RESULTS: L. amazonensis infection induced chronic mechanical and thermal hyperalgesia and paw edema in the infected paw. Mechanical hyperalgesia was also observed in the contralateral paw. TNF-α, IL-1ß, MPO activity, and epidermal/dermal thickness increased in the infected paw, which confirmed the peripheral inflammation at the primary foci of this infection. ATF3 mRNA expression at the ipsilateral DRG of the infected paw was unaltered 30 days post-infection. TNF-α and IL-1ß blood levels were not changed over the time course of disease, and parasitism increased in a time-dependent manner in the ipsilateral draining lymph node. Treatments targeting CX3CL1, TNF-α, and IL-1ß inhibited L. amazonensis-induced ongoing mechanical and thermal hyperalgesia, but not paw edema. A time course of GFAP, Iba-1, and CX3CR1 mRNA expression indicated spinal activation of astrocytes and microglia, which was confirmed at the GFAP and Iba-1 protein level at the peak of mRNA expression (30th day). Selective astrocyte and microglia inhibition diminished infection-induced ipsilateral mechanical hyperalgesia and thermal hyperalgesia, and contralateral mechanical hyperalgesia, but not ipsilateral paw edema. Targeting astrocytes, microglia and NFκB diminished L. amazonensis-induced GFAP, Iba-1, TNF-α, IL-1ß, CX3CR1 and CX3CL1 mRNA expression, and NFκB activation in the spinal cord at the peak of spinal cord glial cells activation. CX3CL1 mRNA expression was also detected in the ipsilateral DRG of infected mice at the 30th day post-infection, and the i.t. injection of TNF-α or IL-1ß in naïve animals induced CX3CL1 mRNA expression in the spinal cord and ipsilateral DRG. CONCLUSIONS: L. amazonensis skin infection produces chronic pain by central mechanisms involving spinal cord astrocytes and microglia-related production of cytokines and chemokines, and NFκB activation contributes to L. amazonensis infection-induced hyperalgesia and neuroinflammation.

Role of gram-positive bacteria in chronic pelvic pain syndrome (CPPS).

INTRODUCTION: Chronic pelvic pain syndrome (CPPS) is a complex disorder that affects a large proportion of all men. A limited understanding of its etiology and pathogenesis is reflected by the absence of effective therapies. Although CPPS is deemed clinically non-infectious with no well-defined etiological role for microbes, bacteria is readily isolated from both healthy and patient prostate secretion and urine samples. Our laboratory has previously demonstrated that a specific gram-negative bacterial isolate can induce CPPS-like symptoms in mice. Here we aimed to expand on these findings examining the role of gram-positive patient-derived bacteria in CPPS. METHODS: A retrospective analysis of bacterial cultures from CPPS patients from a single center was performed. Gram-positive bacteria were isolated from the expressed prostatic secretion (EPS) of three CPPS-patients (pain inducers, PI) and one from a healthy volunteer (non-pain inducer, NPI). These bacteria were inoculated intra-urethrally in two mouse backgrounds and analyzed for their ability to induce tactile allodynia, voiding dysfunction, and colonize the murine prostate. Host immune responses to bacterial instillation were analyzed by flow cytometry. RESULTS: PI strains (Staphylococcus haemolyticus 2551, Enterococcus faecalis 427, and Staphylococcus epidermidis 7244) induced and maintained tactile allodynia responses (200% increase above baseline) for 28 days in NOD/ShiLtJ mice. Conversely the healthy subject derived strain (Staphylococcus epidermidis NPI) demonstrated no significant pelvic allodynia induction. Intra-urethral inoculation of the four bacterial strains into C57BL/6 mice did not induce significant increases in pain responses. Infected NOD/ShiLtJ displayed significant voiding dysfunction compared to their control counterparts. Colony counts of prostate tissues from both NOD/ShiLtJ and C57BL/6 mice at day 28 demonstrated that bacterial strains colonized equally well, including NPI. We also determined that mechanistically, the patient-isolates induced prostate inflammation specifically involving T-cells and monocytes. CONCLUSIONS: Gram-positive isolates from CPPS patients showed enhanced ability to induce tactile allodynia compared to a single taxonomically similar gram-positive strain isolated from a healthy control. Responses were shown to be dependent on host genetic background and not on colonization differences between strains.

Beneficial effect of butyrate-producing Lachnospiraceae on stress-induced visceral hypersensitivity in rats.

BACKGROUND AND AIM: Emerging evidence indicates that psychological stress is involved in the pathogenesis of irritable bowel syndrome, which is characterized by visceral hypersensitivity and may be accompanied by gut dysbiosis. However, how such stress contributes to the development of visceral hypersensitivity is incompletely understood. Here, we aimed to investigate the influence that stress-induced microbial changes exert on visceral sensitivity, as well as the possible underlying mechanisms associated with this effect. METHODS: Male Sprague-Dawley rats underwent chronic water avoidance stress (WAS) to induce visceral hypersensitivity. Visceral sensitivity, colonic tight junction protein expression, and short-chain fatty acids of cecal contents were measured. Fecal samples were collected to characterize microbiota profiles. In a separate study, oral gavage of Roseburia in WAS rats was conducted to verify its potential role in the effectiveness on visceral hypersensitivity. RESULTS: Repeated WAS caused visceral hypersensitivity, altered fecal microbiota composition and function, and decreased occludin expression in the colon. Stressed rats exhibited reduced representation of pathways involved in the metabolism of butyrate and reduced abundance of several operational taxonomic units associated with butyrate-producing bacteria, such as Lachnospiraceae. Consistently, supplementation with Roseburia hominis, a species belonging to Lachnospiraceae, significantly increased cecal butyrate content. Moreover, Roseburia supplementation alleviated visceral hypersensitivity and prevented the decreased expression of occludin. CONCLUSIONS: Reduction in the abundance of butyrate-producing Lachnospiraceae, which is beneficial for the intestinal barrier, was involved in the formation of visceral hypersensitivity. R. hominis is a potential probiotic for treating stress-induced visceral hypersensitivity.

A prostate derived commensal Staphylococcus epidermidis strain prevents and ameliorates induction of chronic prostatitis by UPEC infection.

Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS) is a common syndrome with limited therapies and an unknown etiology. Previously, our laboratory has defined a potential role for pathogenic infection in disease onset. Intra-urethral infection with a uropathogenic Escherichia coli strain isolated from a CP/CPPS patient, CP1, induces prostatic inflammation and tactile allodynia in mice. We have also demonstrated that a prostate specific Staphylococcus epidermidis bacterial isolate, NPI (non-pain inducing), from a healthy subject reduces pain and inflammation in an experimental autoimmune prostatitis (EAP) murine model. Here we focus on the interplay between these human isolates in the context of prostatitis development and resolution. NOD/ShiLtJ mice were inoculated with either NP1 or CP1, or combinations of both. Infection with CP1 induced pelvic tactile allodynia after 7 days, while NPI instillation alone induced no such response. Instillation with NPI 7 days following CP1 infection resolved pelvic tactile allodynia and prophylactic instillation 7 days prior to CPI infection prevented its onset. Prophylactic NPI instillation also prevented CP1 colonization of both prostate and bladder tissues. In vitro analyses revealed that CP1 and NPI do not directly inhibit the growth or invasive potential of one another. Immunological analyses revealed that specific markers associated with CP1-induced pelvic allodynia were decreased upon NPI treatment or repressed by prophylactic colonization. This study demonstrates that a commensal bacterial isolate can inhibit the colonization, pain responses, and immunological activation to uropathogenic bacteria, emphasizing the power of a healthy prostatic microflora in controlling health and disease.

Allatostatin C modulates nociception and immunity in Drosophila.

Bacterial induced inflammatory responses cause pain through direct activation of nociceptive neurons, and the ablation of these neurons leads to increased immune infiltration. In this study, we investigated nociceptive-immune interactions in Drosophila and the role these interactions play during pathogenic bacterial infection. After bacterial infection, we found robust upregulation of ligand-gated ion channels and allatostatin receptors involved in nociception, which potentially leads to hyperalgesia. We further found that Allatostatin-C Receptor 2 (AstC-R2) plays a crucial role in host survival during infection with the pathogenic bacterium Photorhabdus luminescens. Upon examination of immune signaling in AstC-R2 deficient mutants, we demonstrated that Allatostatin-C Receptor 2 specifically inhibits the Immune deficiency pathway, and knockdown of AstC-R2 leads to overproduction of antimicrobial peptides related to this pathway and decreased host survival. This study provides mechanistic insights into the importance of microbe-nociceptor interactions during bacterial challenge. We posit that Allatostatin C is an immunosuppressive substance released by nociceptors or Drosophila hemocytes that dampens IMD signaling in order to either prevent immunopathology or to reduce unnecessary metabolic cost after microbial stimulation. AstC-R2 also acts to dampen thermal nociception in the absence of infection, suggesting an intrinsic neuronal role in mediating these processes during homeostatic conditions. Further examination into the signaling mechanisms by which Allatostatin-C alters immunity and nociception in Drosophila may reveal conserved pathways which can be utilized towards therapeutically targeting inflammatory pain and chronic inflammation.

TRPV1 and the MCP-1/CCR2 Axis Modulate Post-UTI Chronic Pain.

The etiology of chronic pelvic pain syndromes remains unknown. In a murine urinary tract infection (UTI) model, lipopolysaccharide of uropathogenic E. coli and its receptor TLR4 are required for post-UTI chronic pain development. However, downstream mechanisms of post-UTI chronic pelvic pain remain unclear. Because the TRPV1 and MCP-1/CCR2 pathways are implicated in chronic neuropathic pain, we explored their role in post-UTI chronic pain. Mice were infected with the E. coli strain SΦ874, known to produce chronic allodynia, and treated with the TRPV1 antagonist capsazepine. Mice treated with capsazepine at the time of SΦ874 infection failed to develop chronic allodynia, whereas capsazepine treatment of mice at two weeks following SΦ874 infection did not reduce chronic allodynia. TRPV1-deficient mice did not develop chronic allodynia either. Similar results were found using novelty-suppressed feeding (NSF) to assess depressive behavior associated with neuropathic pain. Imaging of reporter mice also revealed induction of MCP-1 and CCR2 expression in sacral dorsal root ganglia following SΦ874 infection. Treatment with a CCR2 receptor antagonist at two weeks post-infection reduced chronic allodynia. Taken together, these results suggest that TRPV1 has a role in the establishment of post-UTI chronic pain, and CCR2 has a role in maintenance of post-UTI chronic pain.

Reversal of visceral hypersensitivity in rat by Menthacarin® , a proprietary combination of essential oils from peppermint and caraway, coincides with mycobiome modulation.

BACKGROUND: Irritable bowel syndrome (IBS) is a common gastrointestinal disorder associated with altered gastrointestinal microflora and increased nociception to colonic distension. This visceral hypersensitivity can be reversed in our rat maternal separation model by fungicides. Menthacarin® is a proprietary combination of essential oils from Mentha x piperita L. and Carum carvi. Because these oils exhibit antifungal and antibacterial properties, we investigated whether Menthacarin® can reverse existing visceral hypersensitivity in maternally separated rats. METHODS: In non-handled and maternally separated rats, we used the visceromotor responses to colorectal distension as measure for visceral sensitivity. We evaluated this response before and 24 hours after water-avoidance stress and after 7 days treatment with Menthacarin® or control. The pre- and post-treatment mycobiome and microbiome were characterized by sequencing of fungal internal transcribed spacer 1 (ITS-1) and bacterial 16s rDNA regions. In vitro antifungal and antimicrobial properties of Menthacarin® were studied with radial diffusion assay. KEY RESULTS: Menthacarin® inhibited in vitro growth of yeast and bacteria. Water-avoidance caused visceral hypersensitivity in maternally separated rats, and this was reversed by treatment. Multivariate analyses of ITS-1 and 16S high throughput data showed that maternal separation, induced changes in the myco- and microbiome. Menthacarin® treatment of non-handled and maternally separated rats shifted the mycobiomes to more similar compositions. CONCLUSIONS & INFERENCES: The development of visceral hypersensitivity in maternally separated rats and the Menthacarin® -mediated reversal of hypersensitivity is associated with changes in the mycobiome. Therefore, Menthacarin® may be a safe and effective treatment option that should be tested for IBS.

Paneth Cell Defects Induce Microbiota Dysbiosis in Mice and Promote Visceral Hypersensitivity.

BACKGROUND & AIMS: Separation of newborn rats from their mothers induces visceral hypersensitivity and impaired epithelial secretory cell lineages when they are adults. Little is known about the mechanisms by which maternal separation causes visceral hypersensitivity or its relationship with defects in epithelial secretory cell lineages. METHODS: We performed studies with C3H/HeN mice separated from their mothers as newborns and mice genetically engineered (Sox9flox/flox-vil-cre on C57BL/6 background) to have deficiencies in Paneth cells. Paneth cell deficiency was assessed by lysozyme staining of ileum tissues and lysozyme activity in fecal samples. When mice were 50 days old, their abdominal response to colorectal distension was assessed by electromyography. Fecal samples were collected and microbiota were analyzed using Gut Low-Density Array quantitative polymerase chain reaction. RESULTS: Mice with maternal separation developed visceral hypersensitivity and defects in Paneth cells, as reported from rats, compared with mice without maternal separation. Sox9flox/flox-vil-Cre mice also had increased visceral hypersensitivity compared with control littermate Sox9flox/flox mice. Fecal samples from mice with maternal separation and from Sox9flox/flox-vil-cre mice had evidence for intestinal dysbiosis of the microbiota, characterized by expansion of Escherichia coli. Daily gavage of conventional C3H/HeN adult mice with 109 commensal E coli induced visceral hypersensitivity. Conversely, daily oral administration of lysozyme prevented expansion of E coli during maternal separation and visceral hypersensitivity. CONCLUSIONS: Mice with defects in Paneth cells (induced by maternal separation or genetically engineered) have intestinal expansion of E coli leading to visceral hypersensitivity. These findings provide evidence that Paneth cell function and intestinal dysbiosis are involved in visceral sensitivity.

Gut microbiota is critical for the induction of chemotherapy-induced pain.

Chemotherapy-induced pain is a dose-limiting condition that affects 30% of patients undergoing chemotherapy. We found that gut microbiota promotes the development of chemotherapy-induced mechanical hyperalgesia. Oxaliplatin-induced mechanical hyperalgesia was reduced in germ-free mice and in mice pretreated with antibiotics. Restoring the microbiota of germ-free mice abrogated this protection. These effects appear to be mediated, in part, by TLR4 expressed on hematopoietic cells, including macrophages.

Intestinal Fungal Dysbiosis Is Associated With Visceral Hypersensitivity in Patients With Irritable Bowel Syndrome and Rats.

BACKGROUND & AIMS: Visceral hypersensitivity is one feature of irritable bowel syndrome (IBS). Bacterial dysbiosis might be involved in the activation of nociceptive sensory pathways, but there have been few studies of the role of the mycobiome (the fungal microbiome) in the development of IBS. We analyzed intestinal mycobiomes of patients with IBS and a rat model of visceral hypersensitivity. METHODS: We used internal transcribed spacer 1-based metabarcoding to compare fecal mycobiomes of 18 healthy volunteers with those of 39 patients with IBS (with visceral hypersensitivity or normal levels of sensitivity). We also compared the mycobiomes of Long-Evans rats separated from their mothers (hypersensitive) with non-handled (normally sensitive) rats. We investigated whether fungi can cause visceral hypersensitivity using rats exposed to fungicide (fluconazole and nystatin). The functional relevance of the gut mycobiome was confirmed in fecal transplantation experiments: adult maternally separated rats were subjected to water avoidance stress (to induce visceral hypersensitivity), then given fungicide and donor cecum content via oral gavage. Other rats subjected to water avoidance stress were given soluble ß-glucans, which antagonize C-type lectin domain family 7 member A (CLEC7A or DECTIN1) signaling via spleen-associated tyrosine kinase (SYK), a SYK inhibitor to reduce visceral hypersensitivity, or vehicle (control). The sensitivity of mast cells to fungi was tested with mesenteric windows (ex vivo) and the human mast cell line HMC-1. RESULTS: α diversity (Shannon index) and mycobiome signature (stability selection) of both groups of IBS patients differed from healthy volunteers, and the mycobiome signature of hypersensitive patients differed from that of normally sensitive patients. We observed mycobiome dysbiosis in rats that had been separated from their mothers compared with non-handled rats. Administration of fungicide to hypersensitive rats reduced their visceral hypersensitivity to normal levels of sensitivity. Administration of cecal mycobiomes from rats that had been separated from their mothers (but not non-handled mycobiome) restored hypersensitivity to distension. Administration of soluble ß-glucans or a SYK inhibitor reduced visceral hypersensitivity, compared with controls. Particulate ß-glucan (a DECTIN-1 agonist) induced mast cell degranulation in mesenteric windows and HMC-1 cells responded to fungal antigens by release of histamine. CONCLUSIONS: In an analysis of patients with IBS and controls, we associated fungal dysbiosis with IBS. In studies of rats, we found fungi to promote visceral hypersensitivity, which could be reduced by administration of fungicides, soluble ß-glucans, or a SYK inhibitor. The intestinal fungi might therefore be manipulated for treatment of IBS-related visceral hypersensitivity.

Isomalto-oligosaccharides ameliorate visceral hyperalgesia with repair damage of ileal epithelial ultrastructure in rats.

BACKGROUND: Treatment of irritable bowel syndrome (IBS) with probiotics has achieved effectiveness to a certain extent. Whether prebiotics will work is still unclear. This study aimed to investigate the therapeutic effects of the prebiotic isomalto-oligosaccharides (IMO) on visceral hypersensitivity (VHS) in rats and to explore potential mechanism. METHODS: Water avoidance stress (WAS) was used to induce VHS in rats. The score for the abdominal withdrawal reflex (AWR) was determined while colorectal distension and compared between VHS group and control group in order to validate VHS preparation. Rats with VHS were then divided into an IMO-treated group (intragastric 5% IMO, 2 mL/d, 14 days) and a water-control group (intragastric water). After treatment, AWR score and intestinal transit rate (ITR) were determined, stool culture was performed, the ultrastructure of the ileum epithelium was observed with scanning electron microscopy (SEM), and serum cytokines were measured. RESULTS: WAS significantly increased AWR score responding to colorectal distension, and lowered the pain threshold. IMO treatment improved VHS with a reduction in AWR score on graded colorectal distension and an increase in pain threshold. SEM showed damages on the ileal epithelial ultrastructure in VHS rats, which was attenuated by IMO treatment. ITR, fecal microbiota and serum cytokine levels were comparable among control group, water-control group, and IMO-treated rats. CONCLUSION: In this randomized placebo-controlled study, the results showed that IMO ameliorated WAS-induced visceral hyperalgesia in rats, this effect may be attributed to the repair of damages on intestinal epithelial ultrastructure.

Irritable bowel syndrome: a gut microbiota-related disorder?

Irritable bowel syndrome (IBS) is one of the most common gastrointestinal (GI) disorders. Despite its prevalence, the pathophysiology of IBS is not well understood although multiple peripheral and central factors are implicated. Recent studies suggest a role for alterations in gut microbiota in IBS. Significant advances in next-generation sequencing technology and bioinformatics and the declining cost have now allowed us to better investigate the role of gut microbiota in IBS. In the following review, we propose gut microbiota as a unifying factor in the pathophysiology of IBS. We first describe how gut microbiota can be influenced by factors predisposing individuals to IBS such as host genetics, stress, diet, antibiotics, and early life experiences. We then highlight the known effects of gut microbiota on mechanisms implicated in the pathophysiology of IBS including disrupted gut brain axis (GBA), visceral hypersensitivity (VH), altered GI motility, epithelial barrier dysfunction, and immune activation. While there are several gaps in the field that preclude us from connecting the dots to establish causation, we hope this overview will allow us to identify and fill in the voids.

Visceral hypersensitive rats share common dysbiosis features with irritable bowel syndrome patients.

AIM: To evaluate gut microbial dysbiosis in two visceral hypersensitive models in comparison with irritable bowel syndrome (IBS) patients and to explore the extent to which these models capture the dysbiosis of IBS patients. METHODS: Visceral hypersensitivity was developed using the maternal separation (MS) rat model and post-inflammatory rat model. The visceral sensitivity of the model groups and control group was evaluated using the abdominal withdraw reflex score and electromyography in response to graded colorectal distention. The 16S ribosomal RNA gene from fecal samples was pyrosequenced and analyzed. The correlation between dysbiosis in the microbiota and visceral hypersensitivity was calculated. Positive findings were compared to sequencing data from a published human IBS cohort. RESULTS: Dysbiosis triggered by neonatal maternal separation was lasting but not static. Both MS and post-inflammatory rat fecal microbiota deviated from that of the control rats to an extent that was larger than the co-housing effect. Two short chain fatty acid producing genera, Fusobacterium and Clostridium XI, were shared by the human IBS cohort and by the maternal separation rats and post-inflammatory rats, respectively, to different extents. Fusobacterium was significantly increased in the MS group, and its abundance positively correlated with the degree of visceral hypersensitivity. Porphyromonadaceae was a protective biomarker for both the rat control group and healthy human controls. CONCLUSION: The dysbiosis MS rat model and the post-inflammatory rat model captured some of the dysbiosis features of IBS patients. Fusobacterium, Clostridium XI and Porphyromonadaceae were identified as targets for future mechanistic research.

Multispecies probiotic protects gut barrier function in experimental models.

AIM: To investigate the effect of the probiotic combination Lactibiane Tolerance(®) (LT) on epithelial barrier function in vitro and in vivo. METHODS: The effect of the multispecies probiotic LT was assessed on several models of epithelial barrier function both in vitro (in basal and inflammatory conditions) and in vivo [visceral hypersensitivity induced by chronic stress or by colonic perfusion of a fecal supernatant (FSN) from patients with irritable bowel syndrome (IBS)]. In vitro, we measured the permeability of confluent T84 cell monolayers incubated with or without LT by evaluating the paracellular flux of macromolecules, in basal conditions and after stimulation with lipopolysaccharide (LPS) or with conditioned medium of colonic biopsies from IBS patients (IBS-CM). In vivo, male C57/Bl6 mice received orally NaCl or LT for 15 d and were submitted to water avoidance stress (WAS) before evaluating visceral sensitivity by measuring the myoelectrical activity of the abdominal muscle and the paracellular permeability with (51)Cr-EDTA. Permeability and sensitivity were also measured after colonic instillation of FSN. Tight-junctions were assessed by immunoblotting and TLR-4 expression was evaluated by immunohistochemistry RESULTS: Incubation of T84 cell monolayers with LT in basal conditions had no significant effect on permeability (P > 0.05 vs culture medium). By contrast, addition of LT bacterial bodies (LT) completely prevented the LPS-induced increase in paracellular permeability (P < 0.01 vs LPS 10 ng/mL (LPS 10); P < 0.01 vs LPS 100 ng/mL (LPS 100), P > 0.05 vs culture medium). The effect was dose dependent as addition of 10(9) LT bacterial bodies induced a stronger decrease in absorbance than 10(6) LT (10(9) LT + LPS 10: -20.1% ± 13.4, P < 0.01 vs LPS 10; 10(6) LT + LPS 10: -11.6% ± 6.2, P < 0.01 vs LPS 10; 10(9) LT + LPS 100: -14.4% ± 5.5, P < 0.01 vs LPS 100; 10(6) LT + LPS 100: -11.6% ± 7.3, P < 0.05 vs LPS 100). Moreover, the increase in paracellular permeability induced by culturing T84 cells with conditioned medium of colonic biopsies from IBS patients (IBS-CM) was completely inhibited in the presence of 10(9) LT (P < 0.01 vs IBS-CM). LT also significantly prevented the epithelial disruption induced by intracolonic infusion of fecal supernatant from IBS patients (P < 0.01 vs IBS FSN) or water avoidance stress P < 0.01 vs WAS) in C57/Bl6 mice and increased the expression of occludin in vitro and in vivo, as assessed by immnunoblotting. The WAS-induced effect on visceral sensitivity was prevented by LT treatment since values obtained for all steps of colorectal distension were significantly (P < 0.01) different from the WAS group. Finally, LT down-regulated the response mediated through TLR-4 in vitro (decrease in tumor necrosis factor α secretion in response to LPS: -65.8% for 10(9) LT and -52.5% for 10(6) LT, P < 0.01 vs LPS) and in vivo (inhibition of WAS induced an increase in TLR-4 expression in the LT treated mice colon, P < 0.01 vs WAS). CONCLUSION: The probiotic LT mix prevented the disruption to the epithelial barrier induced by LPS, stress or colonic soluble factors from IBS patients and prevented visceral hypersensitivity.

Rifaximin alters intestinal bacteria and prevents stress-induced gut inflammation and visceral hyperalgesia in rats.

BACKGROUND & AIMS: Rifaximin is used to treat patients with functional gastrointestinal disorders, but little is known about its therapeutic mechanism. We propose that rifaximin modulates the ileal bacterial community, reduces subclinical inflammation of the intestinal mucosa, and improves gut barrier function to reduce visceral hypersensitivity. METHODS: We induced visceral hyperalgesia in rats, via chronic water avoidance or repeat restraint stressors, and investigated whether rifaximin altered the gut microbiota, prevented intestinal inflammation, and improved gut barrier function. Quantitative polymerase chain reaction (PCR) and 454 pyrosequencing were used to analyze bacterial 16S ribosomal RNA in ileal contents from the rats. Reverse transcription, immunoblot, and histologic analyses were used to evaluate levels of cytokines, the tight junction protein occludin, and mucosal inflammation, respectively. Intestinal permeability and rectal sensitivity were measured. RESULTS: Water avoidance and repeat restraint stress each led to visceral hyperalgesia, accompanied by mucosal inflammation and impaired mucosal barrier function. Oral rifaximin altered the composition of bacterial communities in the ileum (Lactobacillus species became the most abundant) and prevented mucosal inflammation, impairment to intestinal barrier function, and visceral hyperalgesia in response to chronic stress. Neomycin also changed the composition of the ileal bacterial community (Proteobacteria became the most abundant species). Neomycin did not prevent intestinal inflammation or induction of visceral hyperalgesia induced by water avoidance stress. CONCLUSIONS: Rifaximin alters the bacterial population in the ileum of rats, leading to a relative abundance of Lactobacillus. These changes prevent intestinal abnormalities and visceral hyperalgesia in response to chronic psychological stress.

Bacteria activate sensory neurons that modulate pain and inflammation.

Nociceptor sensory neurons are specialized to detect potentially damaging stimuli, protecting the organism by initiating the sensation of pain and eliciting defensive behaviours. Bacterial infections produce pain by unknown molecular mechanisms, although they are presumed to be secondary to immune activation. Here we demonstrate that bacteria directly activate nociceptors, and that the immune response mediated through TLR2, MyD88, T cells, B cells, and neutrophils and monocytes is not necessary for Staphylococcus aureus-induced pain in mice. Mechanical and thermal hyperalgesia in mice is correlated with live bacterial load rather than tissue swelling or immune activation. Bacteria induce calcium flux and action potentials in nociceptor neurons, in part via bacterial N-formylated peptides and the pore-forming toxin α-haemolysin, through distinct mechanisms. Specific ablation of Nav1.8-lineage neurons, which include nociceptors, abrogated pain during bacterial infection, but concurrently increased local immune infiltration and lymphadenopathy of the draining lymph node. Thus, bacterial pathogens produce pain by directly activating sensory neurons that modulate inflammation, an unsuspected role for the nervous system in host-pathogen interactions.

Measurement of tactile allodynia in a murine model of bacterial prostatitis.

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain, a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS). Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity. A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria. CP/CPPS is primarily characterized by the presence of tactile allodynia. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.

O-antigen modulates infection-induced pain states.

The molecular initiators of infection-associated pain are not understood. We recently found that uropathogenic E. coli (UPEC) elicited acute pelvic pain in murine urinary tract infection (UTI). UTI pain was due to E. coli lipopolysaccharide (LPS) and its receptor, TLR4, but pain was not correlated with inflammation. LPS is known to drive inflammation by interactions between the acylated lipid A component and TLR4, but the function of the O-antigen polysaccharide in host responses is unknown. Here, we examined the role of O-antigen in pain using cutaneous hypersensitivity (allodynia) to quantify pelvic pain behavior and using sacral spinal cord excitability to quantify central nervous system manifestations in murine UTI. A UPEC mutant defective for O-antigen biosynthesis induced chronic allodynia that persisted long after clearance of transient infections, but wild type UPEC evoked only acute pain. E. coli strains lacking O-antigen gene clusters had a chronic pain phenotype, and expressing cloned O-antigen gene clusters altered the pain phenotype in a predictable manner. Chronic allodynia was abrogated in TLR4-deficient mice, but inflammatory responses in wild type mice were similar among E. coli strains spanning a wide range of pain phenotypes, suggesting that O-antigen modulates pain independent of inflammation. Spinal cords of mice with chronic allodynia exhibited increased spontaneous firing and compromised short-term depression, consistent with centralized pain. Taken together, these findings suggest that O-antigen functions as a rheostat to modulate LPS-associated pain. These observations have implications for an infectious etiology of chronic pain and evolutionary modification of pathogens to alter host behaviors.