Browsing by Subject "lipopolysaccharide"

Yersinia enterocolitica is an important model organism for gram-negative virulent bacteria. It is also a causative agent of yersiniosis, a common infectious disease related mostly to gastroenteritis, with the serotype O:3 (Ye O:3) being diagnosed in most cases. The lipopolysaccharide (LPS) of Ye O:3 comes with many peculiarities, and with the temperature-regulated formation of its components (e.g O-antigen) being the most remarkable. This interplay between the bacteria and their surrounding environment serves as a good platform to investigate the molecular mechanisms involved in both sensing and responding to the changes. In our study, we looked for the best gene candidates taking part in the O-antigen temperature-regulation based on the RNAseq data, and consequently we tried to reveal the metabolic pathways possibly shaping up the O-antigen structure. Our initial findings helped us formulate a hypothesis about internal competition between the LPS constituents for the common intermediates. Different availability of some of those precursors, namely UPD-N-acetyl-D-glucosamine and its lipid carrier – undecaprenyl phosphate, at different temperatures, would trigger the observed alternations in the O-antigen abundancy. Indeed, our functional assay using the overexpression strain grown in defined media, confirmed a reversible shortage of at least one of those two precursor molecules required for the O-antigen assembly in the bacteria grown at 37 degrees Celsius.

Background: Alcohol dependence is a chronic severe substance use disorder that has devastating personal and public health consequences. The efficacy of the current pharmacotherapy options for the treatment of alcohol dependence are modest at best, therefore better alternatives are greatly needed. Lysergic acid diethylamide (LSD) has shown promise in treatment of alcohol dependence in several clinical trials. A sigle high dose of LSD has been suggested to have a treatment effect that last for at least six months, indicating a remarkably better efficacy than the currently available methods. LSD itself has been reported to have a low addiction potential. In mouse models, acute LSD has been demonstrated to reduce ethanol consumption. Yet, the mechanism of action behind these effects has remained largely unknown. LSD is an agonist of serotonin’s 5-HT2A and 5-HT2C receptors which have been shown to modulate the dopaminergic activity of the reward circuitry, a crucial brain area in the initiation of addiction. Intracranial self-stimulation (ICSS) is a procedure for a quantitative assessment of reward behavior in animal models. In ICSS, laboratory rodents self-administer electric stimulation to the dopaminergic pathways of the reward circuitry inducing a reinforcing effect similar to drug reward. Aim: The aim of the current body of work was to use ICSS to assess the acute effects of LSD on reward behavior in C57BL/6JRj mice. This was done to improve the understanding of the mechanism of action of LSD and to evaluate whether the ethanol-consumption-reducing effect of LSD in mice is mediated through the reward mechanism. Methods: Bipolar electrodes targeting the medial forebrain bundle were implanted in the brains of C57BL/6JRj mice in a stereotaxic surgery. The animals were trained to acquire the self-stimulation in the discrete-trial current-intensity procedure. First, the possible dose-dependent acute effects were tested with three different doses of LSD. Next, the acute effect of LSD on amphetamine-induced changes in ISCC were tested. Lastly, a small preliminary test on the effects of LSD on lipopolysaccharide (LPS) -induced changes on ICSS were conducted. Results and conclusions: Acute LSD did not affect reward behavior in ICSS on any of the tested doses. Accordingly, LSD did not affect the facilitation of ICSS induced by acute amphetamine. The results of the LPS experiment were likely to be skewed by the development of tolerance to LPS, therefore the evaluation of the possible effect of LSD was not possible. These findings suggest that the previously reported LSD-induced reduction in ethanol consumption in mice, is not mediated through alteration of the reward mechanism. At the same time, these findings provide further evidence supporting the suggestion that LSD itself does not induce facilitation of the reward circuitry needed for the development of addiction.

Gut inflammation and permeability is speculated to play a major role in the pathophysiology of several human diseases. Signs of a low-grade gut inflammation in patients with type 1 diabetes (T1D) have been found. Focus of this study was to understand the role of gut inflammation and increased gut permeability in the development of diabetic complications, especially nephropathy. Approximately, one-third of Finnish patients with T1D develop kidney disease during their lifetime. Inflammatory mechanisms may have an essential role in the pathophysiology of the disease. Lipopolysaccharide, LPS, is found in the outer membrane of gram-negative bacteria. LPS activates innate immune system and triggers the activation of inflammatory cytokines, neutrophils and macrophages as well as many pathophysiological processes in vivo, for instance fever and endotoxic shock. Aim of this study was to establish a zebrafish gut inflammation model using fluorophore conjugated endotoxin, LPS. We hypothesized that delivery of LPS in addition to EDTA in the gut of zebrafish triggers inflammation and increased gut permeability which may lead to leakage of LPS to blood stream and potentially kidney injury. This novel zebrafish inflammation model could possibly be used for studying the pathophysiological mechanisms of gut inflammation and possible kidney injury as well as for screening new anti-inflammatory drugs. In addition, this animal model can be used for studying intestinal alkaline phosphatase (IAP) in reducing gut permeability and LPS-mediated kidney damage. IAP is an enzyme produced in small-intestinal epithelium. IAP can detoxify several bacterial endotoxins including LPS and thus protect against the induction of intestinal inflammation. LPS and EDTA were delivered in the gut of 6 days old zebrafish larvae using microgavage injection. Fluorescence microscopy imaging of live zebrafish enabled following the same individual at different timepoints after injections. Paraffin sectioning of the small larvae was promising for investigating the morphology and permeability of the gut as well as possible immunostaining for detection of IAP. L-phenylalanine was used for inhibition of IAP enzyme. Using the novel method of microinjection to gut on zebrafish larvae the timing and amount of delivered materials to gut can be controlled well. The anatomy and function of the gut in zebrafish is very similar to small intestine of mammals and the highly developed vertebrate immune system makes zebrafish an interesting model organism for studying gut inflammation and permeability. In addition, inflammatory processes can be visualized in live, intact transparent zebrafish larvae. However, the technique has a lot of challenges including small size of the fish and possible tissue damage of the fish while performing injection. More experiments need to be carried out to establish the model for drug screening. Also, along with microscopy images, a more precise way for quantification the gut permeability is needed. Based on the images it’s not yet possible to conclude whether LPS increased gut permeability or if IAP inhibition with L-phenylalanine worked in zebrafish larvae. Using adult zebrafish in the future will give more information about the chronic gut inflammation and development of possible kidney injury.