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

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

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

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

AbstractGenerally 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

<i>Mycoplasma agalactiae</i> Induces Cytopathic Effects in Infected Cells Cultured <i>In Vitro</i>

<div><p><i>Mycoplasma agalactiae</i> 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 <i>M</i>. <i>agalactiae</i> 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 <i>M</i>. <i>agalactiae</i> infection on the quality and viability of host cells <i>in vitro</i>. 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 <i>M</i>. <i>agalactiae</i>-infected cells. In compliance with these results, decreased viability and cell lysis of <i>M</i>. <i>agalactiae</i>-infected HeLa cells was also observed. Measurement of the amount of LDH released after <i>M</i>. <i>agalactiae</i> 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 <i>M</i>. <i>agalactiae</i> in inducing host cell lysis. This is the first study illustrating <i>M</i>. <i>agalactiae’s</i> induction of cytopathic effects in infected HeLa cells. Further detailed investigation of infected host tissue for apoptotic markers might demonstrate the association between <i>M</i>. <i>agalactiae</i>-induced host cell lysis and the tissue destruction observed during <i>M</i>. <i>agalactiae</i> natural infection.</p></div

DNA fluorescence staining showing chromatin condensation in <i>M</i>. <i>agalactiae</i>-infected HeLa cells 48 h p.i.

<p>Cells were permeabilized and stained with DAPI, and images were taken using a fluorescence microscope. As a positive control, HeLa cells were treated with 1 μM staurosporine for 3 h and stained with DAPI. Bars, 10 μm.</p

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

Percentage of cell lysis in <i>M</i>. <i>agalactiae</i>-infected HeLa cells.

<p>Both time- and dose- dependent increase (<i>p</i><0.05) was observed at 36 and 48 h p.i. with all tested MOIs compared to uninfected cells in absence of gentamicin. A significant decrease in cell lysis was observed in gentamicin-treated cells (+G) compared to gentamicin-untreated cells (-G) at 48 h p.i. (‘*’ indicates <i>p</i><0.05).</p