Possibility of mixed-species biofilms creation with Candida albicans and Porphyromonas gingivalis in periodontium diseases

StreszczenieWarunki panujące w jamie ustnej i okolicach przyzębia stanowią dogodne środowisko dla kolonizacji różnych gatunków patogenów. Infekcje przyzębia należą do najczęściej występujących chorób jamy ustnej związanych z formowaniem biofilmów. Biofilm jest złożoną strukturą zbudowaną z mikroorganizmów jednego lub wielu typów wraz z ich macierzą. Interakcje pomiędzy budującymi go organizmami wpływają na właściwości i stabilność biofilmu. Tworzenie tego typu układów zapewnia patogenom przetrwanie w niekorzystnych warunkach oraz zwiększa ich odporność na leczenie. Infekcje wywoływane przez bakterie i/lub grzyby przebiegające z udziałem biofilmu zalicza się do przewlekłych i trudnych w leczeniu.Celem niniejszej pracy było stworzenie modelu biofilmu mieszanego z wykorzystaniem drożdżaka C.albicans oraz beztlenowej bakterii gram ujemnej P. gingivalis zaangażowanych w patogenezę chorób przyzębia. Ponadto określenie wpływu czynników środowiska oraz czynników wirulencji bakterii na tworzenie i stabilność biofilmu.Przeprowadzono szereg eksperymentów służących standaryzacji metod prowadzenia biofilmów oraz metod barwienia. Stosując pomiar fluorescencji określano adhezję każdego z mikroorganizmów. Do oceny jakościowej wykorzystano mikroskopię fluorescencyjną. Wykazano, że dostępność tlenu, rodzaj stosowanej pożywki hodowlanej oraz kolejność zasiedlania powierzchni mają kluczowy wpływ na stabilność biofilmu. Potwierdzono udział poszczególnych czynników wirulencji P.gingivalis w formowanie biofilmu mieszanego zależny od wyżej wymienionych czynników środowiskowych. Lepsze poznanie mechanizmów zachodzących we wszystkich zaproponowanych w niniejszej pracy magisterskiej opcjach biofilmu może stanowić klucz do znalezienia skutecznych sposobów leczenia takich infekcji.AbstractThe ecological diversity of the oral cavity and periodontal environment constitute appropriate conditions for the colonization of diverse species of pathogens. Periodontal infections are among the most common oral diseases related with the biofilm formation. Biofilm is a complex structure composed of microorganisms of one or more types with their matrix. Interactions between the organisms that build it affect the properties and stability of the biofilm. Creation of such systems ensure that pathogens survive under unfavorable conditions and increase their resistance to treatment. Bacterial and / or fungal infections associated with biofilm are classified as being chronic and difficult to treat.The objective of this study was to develop a mixed biofilm model using C.albicans yeast and P. gingivalis anaerobic gram negative bacteria involved in pathogenesis of periodontal disease, as well as to determine the influence of environmental factors and bacterial virulence factors on the formation and biofilm stability. A number of experiments have been conducted to standardize biofilm culture methods and staining methods. The adhesion of each microorganism was evaluated by measuring fluorescence intensity. Fluorescence microscopy was used for qualitative assessment. It has been shown that the availability of oxygen, the type of media used and the order in which the surface is occupied have a key impact on the biofilm stability. It was also found that the above mentioned factors may influence the changes of involvement of individual viral factors P.gingivalis during the formation of mixed biofilm.This information may also be useful in proper selection of treatment. The impact of all factors in this study is tested on the types of infections with mixed biofilms. Better understanding of the mechanisms that occur in all of the biofilm reactions proposed in this paper may provide the key to find effective ways to treat such infections

Exogenous vitamins K exert anti-inflammatory effects dissociated from their role as substrates for synthesis of endogenous MK-4 in murine macrophages cell line

Vitamins K exert a range of activities that extend far beyond coagulation and include anti-inflammatory effects, but the mechanisms involved in anti-inflammatory action remain unclear. In the present study, we showed that various forms of exogenous vitamins—K(1), K(3), K(2) (MK-4, MK-5, MK-6 and MK-7)—regulated a wide scope of inflammatory pathways in murine macrophages in vitro, including NOS-2, COX-2, cytokines and MMPs. Moreover, we demonstrated for the first time that macrophages are able to synthesise endogenous MK-4 on their own. Vitamins with shorter isoprenoid chains—K(1), K(3) and MK-5—exhibited stronger anti-inflammatory potential than vitamins with longer isoprenoid chains (MK-6 and MK-7) and simultaneously were preferably used as a substrate for MK-4 endogenous production. Most interesting, atorvastatin pretreatment inhibited endogenous MK-4 production but had no impact on the anti-inflammatory activity of vitamins K. In summary, our results demonstrate that macrophages are able to synthesise endogenous MK-4 using exogenous vitamins K, and statin inhibits this process. However, the anti-inflammatory effect of exogenous vitamins K was independent of endogenous MK-4 synthesis

Systemic administration of insulin receptor antagonist results in endothelial and perivascular adipose tissue dysfunction in mice

Hyperglycemia linked to diabetes results in endothelial dysfunction. In the present work, we comprehensively characterized effects of short-term hyperglycemia induced by administration of an insulin receptor antagonist, the S961 peptide, on endothelium and perivascular adipose tissue (PVAT) in mice. Endothelial function of the thoracic and abdominal aorta in 12-week-old male C57Bl/6Jrj mice treated for two weeks with S961 infusion via osmotic pumps was assessed in vivo using magnetic resonance imaging and ex vivo by detection of nitric oxide (NO) production using electron paramagnetic resonance spectroscopy. Additional methods were used to analyze PVAT, aortic segments and endothelial-specific plasma biomarkers. Systemic disruption of insulin signaling resulted in severe impairment of NO-dependent endothelial function and a loss of vasoprotective function of PVAT affecting the thoracic as well as abdominal parts of the aorta, however a fall in adiponectin expression and decreased uncoupling protein 1-positive area were more pronounced in the thoracic aorta. Results suggest that dysfunctional PVAT contributes to vascular pathology induced by altered insulin signaling in diabetes, in the absence of fat overload and obesity

Thrombin inhibition prevents endothelial dysfunction and reverses 20-HETE overproduction without affecting blood pressure in angiotensin II-induced hypertension in mice

Angiotensin II (Ang II) induces hypertension and endothelial dysfunction, but the involvement of thrombin in these responses is not clear. Here, we assessed the effects of the inhibition of thrombin activity by dabigatran on Ang II-induced hypertension and endothelial dysfunction in mice with a particular focus on NO- and 20-HETE-dependent pathways. As expected, dabigatran administration significantly delayed thrombin generation (CAT assay) in Ang II-treated hypertensive mice, and interestingly, it prevented endothelial dysfunction development, but it did not affect elevated blood pressure nor excessive aortic wall thickening. Dabigatran’s effects on endothelial function in Ang II-treated mice were evidenced by improved NO-dependent relaxation in the aorta in response to acetylcholine in vivo (MRI measurements) and increased systemic NO bioavailability (NO2− quantification) with a concomitant increased ex vivo production of endothelium-derived NO (EPR analysis). Dabigatran treatment also contributed to the reduction in the endothelial expression of pro-inflammatory vWF and ICAM-1. Interestingly, the fall in systemic NO bioavailability in Ang II-treated mice was associated with increased 20-HETE concentration in plasma (UPLC-MS/MS analysis), which was normalised by dabigatran treatment. Taking together, the inhibition of thrombin activity in Ang II-induced hypertension in mice improves the NO-dependent function of vascular endothelium and normalises the 20-HETE-depedent pathway without affecting the blood pressure and vascular remodelling