An opioid receptor-independent mechanism underlies motility dysfunction and visceral hyperalgesia in opioid-induced bowel dysfunction.

Constipation and abdominal pain are commonly encountered in opioid-induced bowel dysfunction (OBD). The underlying mechanisms are incompletely understood, and treatments are not satisfactory. As patients with OBD often have fecal retention, we aimed to determine whether fecal retention plays a pathogenic role in the development of constipation and abdominal pain in OBD, and if so to investigate the mechanisms. A rodent model of OBD was established by daily morphine treatment at 10 mg/kg for 7 days. Bowel movements, colonic muscle contractility, visceromotor response to colorectal distention, and cell excitability of colon-projecting dorsal root ganglion neurons were determined in rats fed with normal pellet food, or with clear liquid diet. Morphine treatment (Mor) reduced fecal outputs starting on day 1, and caused fecal retention afterward. Compared with controls, Mor rats demonstrated suppressed muscle contractility, increased neuronal excitability, and visceral hypersensitivity. Expression of cyclooxygenase-2 (COX-2) and nerve growth factor (NGF) was upregulated in the smooth muscle of the distended colon in Mor rats. However, prevention of fecal retention by feeding rats with clear liquid diet blocked upregulation of COX-2 and NGF, restored muscle contractility, and attenuated visceral hypersensitivity in Mor rats. Moreover, inhibition of COX-2 improved smooth muscle function and fecal outputs, whereas anti-NGF antibody administration attenuated visceral hypersensitivity in Mor rats. Morphine-induced fecal retention is an independent pathogenic factor for motility dysfunction and visceral hypersensitivity in rats with OBD. Liquid diet may have therapeutic potential for OBD by preventing fecal retention-induced mechanotranscription of COX-2 and NGF.NEW & NOTEWORTHY Our preclinical study shows that fecal retention is a pathogenic factor in opioid-induced bowel dysfunction, as prevention of fecal retention with liquid diet improved motility and attenuated visceral hyperalgesia in morphine-treated animals by blocking expression of cyclooxygenase-2 and nerve growth factor in the colon.

Novel role of Vpmas as major adhesins of Mycoplasma agalactiae mediating differential cell adhesion and invasion of Vpma expression variants.

Mycoplasma agalactiae exhibits antigenic variation by switching the expression of multiple surface lipoproteins called Vpmas. Although implicated to have a significant influence on the pathogenicity, their exact role in pathogen-host interactions has not been investigated so far. Initial attachment to host cells is regarded as one of the most important steps for colonization but this pathogen lacks the typical mycoplasma attachment organelle. The aim of this study was to determine the role of Vpmas in adhesion of M. agalactiae to host cells. 'Phase-Locked' Mutants (PLMs) steadily expressing single well-characterized Vpma lipoproteins served as ideal tools to evaluate the role of each of the six Vpmas in cytadhesion, which was otherwise not possible due to the high-frequency switching of Vpmas in the wildtype strain PG2. Using in vitro adhesion assays with HeLa and sheep mammary epithelial (MECs) and stromal (MSCs) cells, we could demonstrate differences in the adhesion capabilities of each of the six PLMs compared to the wildtype strain. The PLMV mutant expressing VpmaV exhibited the highest adhesion rate, whereas PLMU, which expresses VpmaU showed the lowest adhesion values explaining the reduced in vivo fitness of PLMU in sheep during experimental intramammary and conjunctival infections. Furthermore, adhesion inhibition assays using Vpma-specific polyclonal antisera were performed to confirm the role of Vpmas in M. agalactiae cytadhesion. This led to a significant decrease (p<0.05) in the adhesion percentage of each PLM. Immunofluorescence staining of TX-114 phase proteins extracted from each PLM showed binding of the respective Vpma to HeLa cells and MECs proving the direct role of Vpmas in cytadhesion. Furthermore, as adhesion is a prerequisite for cell invasion, the ability of the six PLMs to invade HeLa cells was also evaluated using the gentamicin protection assay. The results showed a strong correlation between the adhesion rates and invasion frequencies of the individual PLMs. This is the first report that describes a novel function of Vpma proteins in cell adhesion and invasion. Besides the variability of these proteins causing surface antigenic variation, the newly identified phenotypes are likely to play critical roles in the pathogenicity potential of this ruminant pathogen.

Simultaneous Identification of Potential Pathogenicity Factors of Mycoplasma agalactiae in the Natural Ovine Host by Negative Selection.

Mycoplasmas possess complex pathogenicity determinants that are largely unknown at the molecular level. Mycoplasma agalactiae serves as a useful model to study the molecular basis of mycoplasma pathogenicity. The generation and in vivo screening of a transposon mutant library of M. agalactiae were employed to unravel its host colonization factors. Tn4001mod mutants were sequenced using a novel sequencing method, and functionally heterogeneous pools containing 15 to 19 selected mutants were screened simultaneously through two successive cycles of sheep intramammary infections. A PCR-based negative selection method was employed to identify mutants that failed to colonize the udders and draining lymph nodes in the animals. A total of 14 different mutants found to be absent from ≥ 95% of samples were identified and subsequently verified via a second round of stringent confirmatory screening where 100% absence was considered attenuation. Using this criterion, seven mutants with insertions in genes MAG1050, MAG2540, MAG3390, uhpT, eutD, adhT, and MAG4460 were not recovered from any of the infected animals. Among the attenuated mutants, many contain disruptions in hypothetical genes, implying their previously unknown role in M. agalactiae pathogenicity. These data indicate the putative role of functionally different genes, including hypothetical ones, in the pathogenesis of M. agalactiae. Defining the precise functions of the identified genes is anticipated to increase our understanding of M. agalactiae infections and to develop successful intervention strategies against it.

In vitro and in vivo cell invasion and systemic spreading of Mycoplasma agalactiae in the sheep infection model.

Generally regarded as extracellular pathogens, molecular mechanisms of mycoplasma persistence, chronicity and disease spread are largely unknown. Mycoplasma agalactiae, an economically important pathogen of small ruminants, causes chronic infections that are difficult to eradicate. Animals continue to shed the agent for several months and even years after the initial infection, in spite of long antibiotic treatment. However, little is known about the strategies that M. agalactiae employs to survive and spread within an immunocompetent host to cause chronic disease. Here, we demonstrate for the first time its ability to invade cultured human (HeLa) and ruminant (BEND and BLF) host cells. Presence of intracellular mycoplasmas is clearly substantiated using differential immunofluorescence technique and quantitative gentamicin invasion assays. Internalized M. agalactiae could survive and exit the cells in a viable state to repopulate the extracellular environment after complete removal of extracellular bacteria with gentamicin. Furthermore, an experimental sheep intramammary infection was carried out to evaluate its systemic spread to organs and host niches distant from the site of initial infection. Positive results obtained via PCR, culture and immunohistochemistry, especially the latter depicting the presence of M. agalactiae in the cytoplasm of mammary duct epithelium and macrophages, clearly provide the first formal proof of M. agalactiae's capability to translocate across the mammary epithelium and systemically disseminate to distant inner organs. Altogether, the findings of these in vitro and in vivo studies indicate that M. agalactiae is capable of entering host cells and this might be the strategy that it employs at a population level to ward off the host immune response and antibiotic action, and to disseminate to new and safer niches to later egress and once again proliferate upon the return of favorable conditions to cause persistent chronic infections.

Combinations of the azaquinazoline anti-Wolbachia agent, AWZ1066S, with benzimidazole anthelmintics synergise to mediate sub-seven-day sterilising and curative efficacies in experimental models of filariasis.

Lymphatic filariasis and onchocerciasis are two major neglected tropical diseases that are responsible for causing severe disability in 50 million people worldwide, whilst veterinary filariasis (heartworm) is a potentially lethal parasitic infection of companion animals. There is an urgent need for safe, short-course curative (macrofilaricidal) drugs to eliminate these debilitating parasite infections. We investigated combination treatments of the novel anti-Wolbachia azaquinazoline small molecule, AWZ1066S, with benzimidazole drugs (albendazole or oxfendazole) in up to four different rodent filariasis infection models: Brugia malayi-CB.17 SCID mice, B. malayi-Mongolian gerbils, B. pahangi-Mongolian gerbils, and Litomosoides sigmodontis-Mongolian gerbils. Combination treatments synergised to elicit threshold (>90%) Wolbachia depletion from female worms in 5 days of treatment, using 2-fold lower dose-exposures of AWZ1066S than monotherapy. Short-course lowered dose AWZ1066S-albendazole combination treatments also delivered partial adulticidal activities and/or long-lasting inhibition of embryogenesis, resulting in complete transmission blockade in B. pahangi and L. sigmodontis gerbil models. We determined that short-course AWZ1066S-albendazole co-treatment significantly augmented the depletion of Wolbachia populations within both germline and hypodermal tissues of B. malayi female worms and in hypodermal tissues in male worms, indicating that anti-Wolbachia synergy is not limited to targeting female embryonic tissues. Our data provides pre-clinical proof-of-concept that sub-seven-day combinations of rapid-acting novel anti-Wolbachia agents with benzimidazole anthelmintics are a promising curative and transmission-blocking drug treatment strategy for filarial diseases of medical and veterinary importance.

Mechanisms of lymphoid depletion in bowel obstruction.

Background and aims: Bowel obstruction (BO) causes not only gastrointestinal dysfunctions but also systemic responses such as sepsis, infections, and immune impairments. The mechanisms involved are not well understood. In this study, we tested the hypothesis that BO leads to lymphoid depletion in primary and peripheral lymphoid organs, which may contribute to systemic responses. We also sought to uncover mechanisms of lymphoid depletion in BO. Methods: Partial colon obstruction was induced with a band in the distal colon of Sprague-Dawley rats, and wild-type and osteopontin knockout (OPN-/-) mice. Obstruction was maintained for 7 days in rats and 4 days in mice. Thymus, bone marrow, spleen, and mesenteric lymph node (MLN) were taken for flow cytometry analysis. Results: The weight of thymus, spleen, and MLN was significantly decreased in BO rats, compared to sham. B and T lymphopoiesis in the bone marrow and thymus was suppressed, and numbers of lymphocytes, CD4+, and CD8+ T cells in the spleen and MLN were all decreased in BO. Depletion of gut microbiota blocked BO-associated lymphopenia in the MLN. Corticosterone antagonism partially attenuated BO-associated reduction of lymphocytes in the thymus and bone marrow. Plasma OPN levels and OPN expression in the distended colon were increased in BO. Deletion of the OPN gene did not affect splenic lymphopenia, but attenuated suppression of lymphopoiesis in the bone marrow and thymus in BO. Conclusions: BO suppresses lymphocyte generation and maintenance in lymphoid organs. Mechanical distention-induced OPN, corticosterone, and gut microbiota are involved in the immune phenotype in BO.

Sheep Infection Trials with 'Phase-Locked' Vpma Expression Variants of Mycoplasma agalactiae-Towards Elucidating the Role of a Multigene Family Encoding Variable Surface Lipoproteins in Infection and Disease.

The significance of large multigene families causing high-frequency surface variations in mycoplasmas is not well-understood. Previously, VpmaY and VpmaU clonal variants of the Vpma family of lipoproteins of M. agalactiae were compared via experimental sheep infections using the two corresponding 'Phase-Locked Mutants'. However, nothing is known about the infectivity of the remaining four Vpma expression variants VpmaX, VpmaW, VpmaZ and VpmaV as they were never evaluated in vivo. Here, in vivo infection and disease progression of all six Vpma expressers constituting the Vpma family of type strain PG2 were compared using the corresponding xer1-disrupted PLMs expressing single well-characterized Vpmas. Each of the six PLMs were separately evaluated using the intramammary sheep infection model along with the control phase-variable wildtype strain PG2. Thorough bacteriological, pathological and clinical examinations were performed, including assessment of milk quality, quantity and somatic cell counts. Altogether, the results indicated that the inability to vary the Vpma expression phase does not hamper the initiation of infection leading to mastitis for all six PLMs, except for PLMU, which showed a defect in host colonization and multiplication for the first 24 h p.i. and pathological/bacteriological analysis indicated a higher potential for systemic spread for PLMV and PLMX. This is the first study in which all isogenic expression variants of a large mycoplasma multigene family are tested in the natural host.

Mechanistic Study of Coffee Effects on Gut Microbiota and Motility in Rats.

Consumption of coffee has benefits in postoperative ileus. We tested the hypothesis that the benefits may be related to the effects of coffee on gut microbiota and motility and studied the mechanisms of action in rats. The in vitro and in vivo effects of regular and decaffeinated (decaf) coffee on gut microbiota of the ileum and colon were determined by bacterial culture and quantitative RT-PCR. Ileal and colonic smooth muscle contractility was determined in a muscle bath. In the in vivo studies, coffee solution (1 g/kg) was administered by oral gavage daily for 3 days. Compared to regular LB agar, the growth of microbiota in the colon and ileal contents was significantly suppressed in LB agar containing coffee or decaf (1.5% or 3%). Treatment with coffee or decaf in vivo for 3 days suppressed gut microbiota but did not significantly affect gut motility or smooth muscle contractility. However, coffee or decaf dose-dependently caused ileal and colonic muscle contractions in vitro. A mechanistic study found that compound(s) other than caffeine contracted gut smooth muscle in a muscarinic receptor-dependent manner. In conclusion, coffee stimulates gut smooth muscle contractions via a muscarinic receptor-dependent mechanism and inhibits microbiota in a caffeine-independent manner.

Precision Lactobacillus reuteri therapy attenuates luminal distension-associated visceral hypersensitivity by inducing peripheral opioid receptors in the colon.

Luminal distension and abdominal pain are major clinical hallmarks of obstructive bowel disorders and functional bowel disorders linked to gut dysbiosis. Our recent studies found that chronic lumen distension increased visceral sensitivity and decreased abundance of gut commensal Lactobacillus reuteri in a rodent model of partial colon obstruction (OB). To establish causation, we performed precision microbial therapy to assess whether recolonization of L. reuteri prevents visceral hypersensitivity in lumen distension, and if so, to identify the gut-microbiota mechanism. Lumen distension was induced in Sprague-Dawley rats by implanting an OB band in the distal colon for up to 7 days. L. reuteri strains or vehicle were gavage ingested 1 × 10 colony-forming units/g daily starting 2 days before OB. L. reuteri rat strains that were able to recolonize obstructed colon significantly improved food intake and body weight in OB rats, and attenuated referred visceral hyperalgesia measured by the withdrawal response to von Frey filament applications to the abdomen. Mechanistically, L. reuteri treatment attenuated hyperexcitability of the dorsal root ganglia neurons projecting to the distended colon by promoting opioid receptor function in affected tissues. The expression of µ, δ, and κ opioid receptors was significantly downregulated in colonic muscularis externae and sensory neurons in OB rats. However, L. reuteri treatment prevented the loss of opioid receptors. Furthermore, administration of peripheral opioid receptor antagonist naloxone methiodide abolished the analgesic effect of L. reuteri in OB. In conclusion, precision L. reuteri therapy prevents lumen distension-associated visceral hypersensitivity by local bacterial induction of opioid receptors.

Novel Insights Into the Mechanisms of Abdominal Pain in Obstructive Bowel Disorders.

Obstructive bowel disorders (OBD) are characterized by lumen distention due to mechanical or functional obstruction in the gut. Abdominal pain is one of the main symptoms in OBD. In this article, we aim to critically review the potential mechanisms for acute and chronic pain in bowel obstruction (BO). While clustered contractions and associated increase of intraluminal pressure may account for colicky pain in simple obstruction, ischemia may be involved in acute pain in severe conditions such as closed loop obstruction. Recent preclinical studies discovered that visceral sensitivity is increased in BO, and visceral hypersensitivity may underlie the mechanisms of chronic abdominal pain in BO. Mounting evidence suggests that lumen distension, as a circumferential mechanical stretch, alters gene expression (mechano-transcription) in the distended bowel, and mechano-transcription of nociceptive and inflammatory mediators plays a critical role in the development of visceral hypersensitivity in BO. Mechano-transcription of nerve growth factor (NGF) in gut smooth muscle cells is found to increase voltage-gated Na+ channel (Nav) activity of the primary sensory neurons by up-regulating expression of TTX-resistant Nav1.8, whereas mechanical stretch-induced brain-derived neurotrophic factor (BDNF) reduces Kv currents especially A-type (IA) currents by down-regulating expression of specific IA subtypes such as Kv1.4. The NGF and BDNF mediated changes in gene expression and channel functions in the primary sensory neurons may constitute the main mechanisms of visceral hypersensitivity in OBD. In addition, mechanical stretch-induced COX-2 and other inflammatory mediators in the gut may also contribute to abdominal pain by activating and sensitizing nociceptors.

Microbiota dysbiosis and its pathophysiological significance in bowel obstruction.

Bowel obstruction (OB) causes local and systemic dysfunctions. Here we investigated whether obstruction leads to alterations in microbiota community composition and total abundance, and if so whether these changes contribute to dysfunctions in OB. Partial colon obstruction was maintained in rats for 7 days. The mid colon and its intraluminal feces - proximal to the obstruction - were studied. OB did not cause bacterial overgrowth or mucosa inflammation, but induced profound changes in fecal microbiota composition and diversity. At the phylum level, the 16S rRNA sequencing showed a significant decrease in the relative abundance of Firmicutes with corresponding increases in Proteobacteria and Bacteroidetes in OB compared with sham controls. Daily treatment using broad spectrum antibiotics dramatically reduced total bacterial abundance, but increased the relative presence of Proteobacteria. Antibiotics eliminated viable bacteria in the spleen and liver, but not in the mesentery lymph node in OB. Although antibiotic treatment decreased muscle contractility in sham rats, it had little effect on OB-associated suppression of muscle contractility or inflammatory changes in the muscle layer. In conclusion, obstruction leads to marked dysbiosis in the colon. Antibiotic eradication of microbiota had limited effects on obstruction-associated changes in inflammation, motility, or bacterial translocation.

Microsomal Prostaglandin E Synthase-1 Plays a Critical Role in Long-term Motility Dysfunction after Bowel Obstruction.

Motility dysfunction is present not only during bowel obstruction (BO), but after obstruction is resolved. Previous studies found that lumen distension associated mechano-transcription of COX-2 and production of PGE2 in gut smooth muscle cells (SMC) account for motility dysfunction during obstruction. We hypothesized that PGE2 may exert autocrine effect in SMC to induce microsomal prostaglandin E synthase-1 (mPGES-1), which contributes to motility dysfunction after obstruction is resolved. Partial colon obstruction was induced in rats with an obstruction band, which was released 7 days later. Rats were further studied in the post-BO state. Circular muscle contractility of the mid colon (previously distended during obstruction) remained suppressed, and colon transit was impaired in the post-BO state. The COX-2, mPGES-1, and PGE2 levels were all increased in the distended bowel during obstruction. However, after obstruction was resolved, COX-2 expression returned to normal, whereas mPGES-1 and PGE2 levels remained increased. Expression of mPGES-1 in colon SMC was inducible by stretch or PGE2. Administration of mPGES-1 inhibitor Cay 10526 either before or after the release of obstruction normalized PGE2 levels and improved motility in the post-BO rats. In conclusion, mPGES-1 plays a critical role in the continuous suppression of motor function in the post-BO state.

Mycoplasma agalactiae Induces Cytopathic Effects in Infected Cells Cultured In Vitro.

Mycoplasma agalactiae is the etiological agent of the contagious agalactia syndrome in sheep and goats and causes significant economic losses worldwide. Yet the mechanism of pathogenesis is largely unknown. Even whole-genome sequence analysis of its pathogenic type strain did not lead to any conclusions regarding its virulence or pathogenicity factors. Although inflammation and tissue destruction at the local site of M. agalactiae infection are largely considered as effects of the host immune response, the direct effect of the agent on host cells is not completely understood. The aim of this study was to investigate the effect of M. agalactiae infection on the quality and viability of host cells in vitro. Changes in cell morphology including cell elongation, cytoplasm shrinkage and membrane blebbing were observed in infected HeLa cells. Chromatin condensation and increased caspase-3 cleavage in infected HeLa cells 48 h after infection suggests an apoptosis-like phenomenon in M. agalactiae-infected cells. In compliance with these results, decreased viability and cell lysis of M. agalactiae-infected HeLa cells was also observed. Measurement of the amount of LDH released after M. agalactiae infection revealed a time- and dose-dependent increase in HeLa cell lysis. A significant decrease in LDH released after gentamicin treatment of infected cells confirmed the major role of cytadherent M. agalactiae in inducing host cell lysis. This is the first study illustrating M. agalactiae's induction of cytopathic effects in infected HeLa cells. Further detailed investigation of infected host tissue for apoptotic markers might demonstrate the association between M. agalactiae-induced host cell lysis and the tissue destruction observed during M. agalactiae natural infection.

Sheep primary cells as in vitro models to investigate Mycoplasma agalactiae host cell interactions.

Appropriate infection models are imperative for the understanding of pathogens like mycoplasmas that are known for their strict host and tissue specificity, and lack of suitable cell and small animal models has hindered pathogenicity studies. This is particularly true for the economically important group of ruminant mycoplasmas whose virulence factors need to be elucidated for designing effective intervention strategies. Mycoplasma agalactiae serves as a useful role model especially because it is phylogenetically very close to M. bovis and causes similar symptoms by as yet unknown mechanisms. Here, we successfully prepared and characterized four different primary sheep cell lines, namely the epithelial and stromal cells from the mammary gland and uterus, respectively. Using immunohistochemistry, we identified vimentin and cytokeratin as specific markers to confirm the typical cell phenotypes of these primary cells. Furthermore, M. agalactiae's consistent adhesion and invasion into these primary cells proves the reliability of these cell models. Mimicking natural infections, mammary epithelial and stromal cells showed higher invasion and adhesion rates compared to the uterine cells as also seen via double immunofluorescence staining. Altogether, we have generated promising in vitro cell models to study host-pathogen interactions of M. agalactiae and related ruminant pathogens in a more authentic manner.