Browsing by Subject "zebrafish"

Mycobacterium tuberculosiksen aiheuttama tuberkuloosi on yksi yleisimmistä kuolemaan johtavista sairauksista kehitysmaissa. M. tuberculosis-bakteerin tiedetään kykenevän väistämään potilaan immuunipuolustusta ja hyödyntämään makrofagien sekä muiden puolustussolujen ominaisuuksia. M. tuberculosis kykenee lisääntymään makrofagien sisällä. Eksosomit ovat solunulkoisia rakkuloita, joita kaikki solut erittävät. Eksosomeilla tiedetään olevan myös immunomodulatorisia vaikutuksia. Tutkimuksen tarkoituksena oli kehittää in vivo-malli eksosomien immunomodulatoristen ominaisuuksien mallintamiseen seeprakaloissa, hyödyntämällä seeprakalojen omaa Mycobacterium marinum-patogeeniä. Tutkimuksessa käytettiin ihmisen monosyyttilinjan soluja (THP-1), lämpötapettuja M. marinum-bakteereja ja seeprakalojen poikasia. THP-1 -soluja käsiteltiin lämpötapetuilla M. marinum bakteereilla viidellä eri bakteerikonsentraatiolla. Lisäksi tutkimuksessa oli yksi bakteereilla käsittelemätön THP-1 -soluviljelmä. Kaikista THP-1 -soluviljelmistä eristettiin ultrasentrifugaatiolla kyseisten solujen tuottamia eksosomeja. Eristettyä eksosomeja mikroinjektoitiin kuorittujen seeprakalan poikasten ruskuaispussin blood circulation valley -suoneen, jotta nähtäisiin aiheuttavatko ne muutoksia kalojen tulehduksenvälittäjäaineiden geenien ilmentymisessä eli geeniekspressiossa. Noin puolen vuorokauden jälkeen kalaryhmästä poistettiin kuolleet yksilöt ja elossa olevat kerättiin talteen RNA-eristystä varten. Eristetty RNA käännettiin DNA:ksi ja reaaliaikaisella polymeraasiketjureaktiolla, qPCR:llä, mitattiin kohdegeenien ekspressioita. Tutkimuksessa havaittiin valittujen tulehdusvälittäjäaineiden geenien ilmentymisen lisääntymistä seeprakaloissa. Geenin ilmentymstaso oli sitä suurempi, mitä suuremmalle bakteerimäärälle THP-1 -solut oli altistettu ennen eksosomien eristystä. Kaikkein korkeimmat tulehdusvälittäjäaineiden geeniekspressiotasot nähtiin altistamattomien solujen eksosomeilla injektoitujen kalojen ryhmissä. Kontrolliksi valitussa geenissä ei havaittu vastaavaa geeniekspression kasvua. Näiden tutkimustulosten perusteella lämpötapetulla M. marinumilla käsiteltyjen THP-1 -solujen eksosomeilla näyttäisi olevan kyky aiheuttaa samankaltaisia muutoksia tulehdusvälittäjäaineiden geeniekspressiossa kuin seeprakalojen suoralla M. marinum infektiolla. Tulosten vahvistamiseen tarvitaan vielä lisätutkimuksia.

Catastrophic childhood epilepsies are characterized by persistent seizures and are frequently associated with cognitive and developmental impairments. Many, approximately 30%, of these epilepsies are rare genetic disorders that do not have effective therapeutic options. The bench to drug process is lengthy and expensive, and thus it is critical to find more affordable drug screening options. Zebrafish are an ideal model organism for screening studies as they share considerable (70%) genetic similarities with humans and are cheap to maintain with efficient breeding capabilities. In the present study, 37 zebrafish lines were screened for epileptic brain activity to identify high priority genes for future pharmacology studies. Each zebrafish line, generated by CRISPR-Cas9 represents a single gene loss of function mutation associated with 3 epilepsy based on genome wide association studies. Larval zebrafish were screened for spontaneous seizure activity using electrophysiological assays. The following 8 genes were significantly associated with spontaneous seizure activity in zebrafish: EEF1A, ARX, GRIN1, GABRB3, PNPO, STRADA, SCN1A, and STXBP1. There is now an open-source database for all 37 zebrafish lines, which contains sequencing information, survival curves, behavioral profiles, and electrophysiological data. The findings reveal novel target genes for future drug development and discovery. Future work is needed to explore whether zebrafish also model co-morbidities commonly seen in human patients with epilepsy.

Tuberculosis (TB) still ranks as one of the most dangerous infectious diseases around the world and it is accountable for over 1.5 million deaths every year. World Health Organization has estimated that one fourth of the world’s population is infected with Mycobacterium tuberculosis. The current treatment against TB has drawbacks and the only available vaccine against TB does not provide sufficient protection against the disease and therefore new treatments are much needed. There has also been a lack of good animal models, but the zebrafish (Danio rerio) have been recently found to be a good model to study especially granuloma formation, latency, and reactivation of TB. Their natural pathogen, Mycobacterium marinum causes similar infection in the fish than M. tuberculosis in humans. One characteristic of TB is the formation of granulomas, which are aggregates of immune cells that contain the bacteria. However, M. tuberculosis can escape the granuloma and in such a way spread in the host. The inflammasome is an innate immune system mechanism that activates the immunological response in an infection and has a role in the formation of granulomas. PYCARD is an adaptor protein that has a role in inflammasome activation, which makes it an interesting target when studying the immunological response against M. tuberculosis infection. In this study, granuloma formation in pycard-/- and pycard+/+ zebrafish were compared. The granulomas were studied for their size, location, structure and hypoxicity, and the number of granulomas in each fish was counted. Also, the number of free bacteria was assessed. No significant differences were found in any of these aspects between pycard-/- and pycard+/+ fish. Variation between individual fish was great in both groups.

Histamine and hypocretin/orexin are neuromodulators important for regulation of alertness and wakefulness. These systems project to major areas of the brain, are highly conserved among vertebrates and they significantly innervate each other. Different studies have indicated an interaction between the histaminergic and orexin systems, however the role of histamine in this interaction is still not well-established. The goal of this study was to examine possible changes in orexin neurons development and larvae behaviour, after genetic loss of histamine decarboxylase (hdc), the histamine-synthesizing enzyme. Using whole-mount in-situ hybridization and immunofluorescence staining we observed a significant reduction in the expression of the hcrt mRNA and the orexin A peptide in 6 dpf hdcKO zebrafish larvae. However, KO of hdc had no effect on startle response, dark flash response and sleeping behaviour of 6 dpf larvae. To further investigate the regulatory role of the histaminergic system, we employed treatment of hdcWT and KO larvae with ciproxifan, a histamine H3 receptor inverse agonist. Ciproxifan treatment increased darkness habituation in 7 dpf hdcWT and KO larvae but reduced the intensity of the dark flash response only on hdcWT larvae. Furthermore, ciproxifan treatment differentially affected the expression of the orexin A peptide in 7 dpf hdcWT and KO larvae but had no effect on the expression levels of the hcrt mRNA. Collectively, these findings suggest the significance of histaminergic signaling for normal development of orexin neurons and the implication of histamine in the execution of the dark flash response. Lastly, this study indicates the complex role of the histamine H3 receptor and the requirement of further studies for better characterization of its function.

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.