Mycoplasma pneumoniae infections, diagnosis and prevention

Bakalářská práce se zabývá patogenní bakterií Mycoplasma pneumoniae, vyvolávající časté infekce respiračního traktu u lidí. V práci jsou shrnuty dostupné informace o onemocněních, které uvedený mikroorganismus způsobuje. Dále jsou uvedeny postupy laboratorní diagnostiky, možnosti terapie a prevence. Pozornost je věnována rovněž i charakterizaci bakterie, kdy jsou zmíněny informace o její taxonomii, morfologii a patogenezi.This bachelor thesis deals with the pathogenic bacterium Mycoplasma pneumoniae, the causative agent of human respiratory tract infections. The work summarizes available information on diseases caused by this bacterium. Attention is paid also to bacterial characteristics which are summed up in the chapters Taxonomy, Morphology and Pathogenesis. Furthermore, the methods of identification, therapy and prevention are mentioned.Katedra biologických a biochemických věd1. Prezentace výsledků bakalářské práce. 2. Diskuze k posudku vedoucího bakalářské práce. 3. Studentka zodpověděla všechny dotazy a připomínky k BP

Biological Applications of Pd, Pt and Ru Complexes bearing Chiral Ligands

V rámci této diplomové práce bylo syntetizováno 7 originálních koordinačních sloučenin chelatovaných primárními a sekundárními aminy, které obsahují chirální benzthiazolový blok. Z dalších připravených sloučenin bylo upuštěno od použití sloučenin s Schiffovými bázemi s chirálním benzthiazolovým blokem z důvodu velmi nízkých výtěžků a nestálosti v dimethylsulfoxidu. Z výchozích palladnatých, platnatých a ruthenatých sloučenin za daných podmínek neprobíhaly pouze reakce s vybranými chirálními ligandy v případě ruthenatého komplexu, byly proto připraveny 4 palladnaté a 3 platnaté koordinační sloučeniny, a to reakcí v dichlormethanu s následnou krystalizací sloučenin ze směsi dichlormethan hexan. Takto připravené palladnaté a platnaté sloučeniny byly charakterizovány pomocí technik multinukleární NMR spektroskopie v roztocích. Následně byla u připravených sloučenin testována jejich antimikrobiální aktivita pomocí agarové difúzní metody za použití Mueller Hinton agaru. Testovanými kmeny byly Escherichia coli CCM 3954, zástupce gramnegativních (G-) bakterií, a Staphylococcus aureus CCM 2022, zastupující grampozitivní (G+) bakterie. Z testovaných sloučenin byla prokázána antimikrobiální aktivita platnatých sloučenin na kmen Staphylococcus aureus CCM 2022.Within the scope of this thesis, seven coordination metal complexes chelated by primary and secondary amines containing chiral benzothiazole block, have been synthesized. From used palladium(II), platinum(II) and ruthenium(II) starting complexes the appropriate reaction with chiral ligands does not perform in ruthenium(II) complex case only. Four Pd(II) and three Pt(II) coordination compounds have been prepared and characterized by the help of multinuclear NMR spectroscopy in solutions. Related Schiff base complexes bearing the same ligand skeleton were excluded from the further screening due to low chemical yields and instability in DMSO solution. Prepared compounds were tested for their antimicrobial activity by the agar diffusion method, using Mueller Hinton agar. The tested strains were Escherichia coli CCM 3954, a representative of gramnegative (G-) bacteria and Staphylococcus aureus CCM 2022, representing grampositive bacteria. Promising results were detected in the case of Staphylococcus aureus CCM 2022 strain inhibited by higher concentrations of platinum(II) complexes.Katedra biologických a biochemických věd1. Prezentace výsledků diplomové práce. 2. Diskuze k posudku vedoucího a oponenta bakalářské práce. 3. Studentka zodpověděla všechny dotazy a připomínky k DP

The use of anchored agonists of phagocytic receptors for cancer immunotherapy: B16-F10 murine melanoma model.

The application of the phagocytic receptor agonists in cancer immunotherapy was studied. Agonists (laminarin, molecules with terminal mannose, N-Formyl-methioninyl-leucyl-phenylalanine) were firmly anchored to the tumor cell surface. When particular agonists of phagocytic receptors were used together with LPS (Toll-like receptor agonist), high synergy causing tumour shrinkage and a temporary or permanent disappearance was observed. Methods of anchoring phagocytic receptor agonists (charge interactions, anchoring based on hydrophobic chains, covalent bonds) and various regimes of phagocytic agonist/LPS mixture applications were tested to achieve maximum therapeutic effect. Combinations of mannan/LPS and f-MLF/LPS (hydrophobic anchors) in appropriate (pulse) regimes resulted in an 80% and 60% recovery for mice, respectively. We propose that substantial synergy between agonists of phagocytic and Toll-like receptors (TLR) is based on two events. The TLR ligand induces early and massive inflammatory infiltration of tumors. The effect of this cell infiltrate is directed towards tumor cells, bearing agonists of phagocytic receptors on their surface. The result of these processes was effective killing of tumor cells. This novel approach represents exploitation of innate immunity mechanisms for treating cancer

Melanoma therapy based on the use of mannan covalently bound to tumor cell surface, synergy with LPS.

<p>Groups of 5 mice were treated starting the 12^th day after tumor transplantation.</p

Analysis of cell infiltrate in the tumor during therapy based on the use of f-MLFKK-BAM, LPS and their mixture. Granulocyte detection.

<p>Groups of 9 mice received a single dose of 0.5-MLFKK-BAM, LPS (0.5 mg/ml), mixture of 0.5 mM f-MLFKK-BAM and LPS (0.5 mg/ml), and PBS alone in 50 μl i.t. Preparation of cell suspension and granulocyte staining were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085222#pone-0085222-g003" target="_blank">Figure 3</a>.</p

Histology.

<p>Melanoma bearing mice were injected i.t. with 50 μl of BAM derivatives of agonists (0.2 mM laminarin-BAM, 0.2 mM mannan-BAM, 0.5 mM f-MLFKK-BAM), their mixtures with LPS (0.5 mg/ml), LPS and PBS alone. Two mice from each group were killed in 24 hours intervals (24, 48, 72 hrs). Excised tumors were fixed with 4% neutral solution of formaldehyde and paraffin blocks were prepared. Sections were stained with hematoxylin/eosin. A– PBS alone; B– effect of particular agonists of phagocytic receptors and LPS alone; C, D– synergistic effect of LPS combinations with particular agonists of phagocytic receptors. Aa – melanoblasts, Ab – necrotic focus with slight granulocyte infiltration, Ba – melanoblasts, Bb – necrotic focus with hemorrhage, Bc – granulation tissue, Bd – slacked edematous ligament, Ca – necrotic tissue with hemorrhage, Cb – negligible residue of tumor, Cc – edematous ligament with inflammatory infiltration, Da – slacked edematous ligament with inflammatory infiltration and hemorrhage foci, Db – bleeding necrosis.</p

Interaction of macrophages with melanoma cells labelled with phagocytic ligands. Formation of clusters.

<p>To the mixture of melanoma B16-F10 cells and macrophage cell line PMJ2R laminarin-BAM (A) or free laminarin (B) were added (0.05 mM final concentrations). Photos were taken after 1 hour incubation at 37°C. The experiment was repeated four times with similar results.</p

<i>In vitro</i> analysis of the effect of macrophages activated by LPS on melanoma cells bearing ligands of phagocytic receptors.

<p>Murine B16-F10 melanoma cells grown to confluency in 96 well tissue culture plate were incubated (30 min, 37°C) with solution of phagocytic receptor agonists (0.02 mM laminarin–BAM or 0.02 mM mannan-BAM or 0.05 mM f-MLFKK-BAM in culture medium) and subsequently washed. Cells of murine macrophage cell line PMJ2R were preincubated with LPS (1 μg/ml) for 2 hours at 37°C, washed, and added to B16-F10 in the ratio 5:1. This mixture was incubated for 4 hours at 37°C. After incubation, PMJ2R and dead cells were carefully washed off. Living B16-F10 melanoma cells were released by trypsinisation and calculated. (A) laminarin-BAM, (B) mannan-BAM, (C) mannan-BAM, cells cultured in medium with non-inactivated fetal calf serum, (D) f-MLFKK-BAM. *P≤0.05, **P ≤ 0.005 compared to B16-F10 <b>o</b>P≤0.05, <b>oo</b>P≤0.005, <b>ooo</b>P≤0.0005, <b>oooo</b>P≤0.00005 compared to B16-F10+ ligand. The experiment was repeated twice with similar results.</p

Analysis of cell infiltrate in the tumor during therapy based on the use of mannan-BAM, LPS and their mixture.

<p>Groups of 9 mice received a single dose of 0.2-BAM in PBS, LPS (0.5 mg/ml PBS), mixture of 0.2 mM mannan-BAM and LPS (0.5 mg/ml) in PBS, and PBS alone in 50 μl i.t. 3 mice from each group were killed in 12, 24 and 48 hours intervals, cells from excised tumors were prepared by enzymatic treatment (Liberase DL and DNase I) and analysed by flow cytometry. The following labelled antibodies were used: (A) anti-Mouse Ly-6G (Gr-1) Alexa Fluor 700 for granulocyte detection, (B) anti-Mouse CD19 APC for detection of B lymphocytes and (C) anti-Mouse NK1.1 PE for NK cells.</p

Synergy of laminarin-BAM with LPS, various regimes of application.

<p>Groups of 4–5 mice were treated starting the 12^th day after tumor transplantation.</p