A kórokozók közvetlen kimutatásán alapuló mikrobiológiai diagnosztika idő- és munkaigényes folyamat. Nem csoda tehát, hogy megnőtt az igény a konvencionális módszereknél gyorsabb, nagy érzékenységű diagnosztikai eljárások kifejlesztése és bevezetése iránt, mint amilyenek a nukleinsav-kimutatáson alapuló módszerek. A molekuláris biológiai módszerek felgyorsítják a diagnosztikát, javítják a specificitást és lehetőséget nyújtanak a nehezen tenyészthető patogének azonosítására is. Az eredmények értékelésekor azonban figyelembe kell venni, hogy a kórokozó-specifikus nukleinsav kimutatása nem egyenértékű a szaporodni képes kórokozók jelenlétével! Az áramlási citometria legnagyobb előnye, a nukleinsav-detektáláson alapuló technikákkal szemben, a mikroorganizmusok egyedi, sejtszintű kimutatása. Gyorsasága révén kiemelt jelentőségre tehet szert a lassan növő mikrobák, a Mycobacterium törzsek és a gombák kimutatásában. Mivel a megfelelő minta-előkészítés megválasztásával alkalmas a patogének pontos azonosítására, így különösen jól hasznosítható a kevert populációk okozta fertőzések diagnosztikájában. Nem utolsósorban lehetővé teszi a fertőzött szervezet immunrendszerének monitorozását és az antimikrobiális kezelés nyomon követését is. Orv. Hetil., 2013, 154, 1207–1218.
The aim of the experiments was to study the regulation of triiodothyronine (T3) production in the unicellular Tetrahymena. Untreated and troph-hormone treated specimen were prepared and in different timepoints T3 content was measured and compared by immunocytochemical flow cytometry. 0.1 or 0.001 IU TSH in tryptone-yeast medium stimulated T3 synthesis at 10, 20, 30 min, but does not stimulate after 1 h. The overlapping gonadotropic hormone (GTH) also did it, however only at 10 min. In Losina salt solution (physiological for Tetrahymena) the effect was weaker, however outer amino acid source was not absolutely needed for the production of the hormone. The results show that the TSH regulation of thyroid hormone synthesis (storage, secretion) and troph-hormone overlap can be deduced to a unicellular level. This may allow the hypothesis that the endocrine mechanisms proved at a low level of phylogeny are preserved for the higher ranked organisms.
The immune cells of rat and man synthesize, store and secrete hormones, characteristic to the endocrine glands. In the present experiments female and male CD1 mice were treated with 10 IU/kg insulin sc. (the controls with normal saline) and after 30 min peritoneal fluid was gained. The cells of the peritoneal fluid (lymphocytes and the monocyte-granulocyte group) were studied by immunocytochemical flow-cytometry to adrenocorticotropic hormone (ACTH), triiodothyronine (T3), histamine and serotonin content. In the female mice each hormone level was significantly lower in the insulin-treated animals, except histamine in the monocyte-granulocyte group. In the insulin-treated male animals, the hormone levels were similar to the control. The results 1) support the previously hypothesized hormonal network in the immune system, 2) justify that the insulin effect is not species dependent and 3) call attention to the sex, species and organ differences in the response.
In earlier experiments single benzpyrene treatment of newborn rats caused strong alterations in the endorphin content of adult rats' immune cells. In the present experiments young (4-6 weeks old) male rats were studied for demonstrating the effect of the single neonatal or repeated (neonatally and at weanling) benzpyrene exposure on the serotonin content of immune cells (blood lymphocytes, monocytes, granulocytes; peritoneal fluid lymphocytes, mast cells, monocytes and granulocytes, thymic lymphocytes). Flow cytometric analysis showed that 50 µg benzpyrene treatment of five-week-old animals was ineffective after 5 days and this was the situation four weeks after single neonatal (20 µg) benzpyrene exposure. However, the repeated treatment of neonatally benzpyrene exposed 4 weeks old animals after 5 days resulted in elevated blood and thymic lymphocyte serotonin amount and in one index (peritoneal monocyte-granulocyte group) reduced serotonin content. This means that neonatal benzpyrene treatment does not influence directly the serotonin content (production or transport) of immune cells (unlike to the endorphin content) however, sensitizes them to a following benzpyrene exposure. The results widen the list of harmful effects (influencing steroid receptor binding, sexual behavior and immune cells' endorphin content) of perinatal benzpyrene exposure.
Stress caused by 48 h food and water deprivation provoked significant changes in T3 and serotonin content of lymphocytes. The concentration of these hormones decreased in the last hour of stress. However, 48 h later there was no difference between the hormone content of immune cells of stressed and control animals. Since in earlier experiments three weeks after exposed to stress a significant difference between the control and stressed animals was found, this means that an imprinting-like phenomenon happened with consequences manifested later. The most sensitive cells to acute stress are lymphocytes, however the imprinting influences all types of of the immune cells.
Histone deacetylases can also influence acetylation of tubulin. In the present experiments, after 60 min of 10 μM trichostatin (TSA) treatment the structure and amount of tubulin and acetylated-tubulin were studied immunocytochemically, by using confocal microscopy and flow cytometry. In TSA-treated
cells deep fibres were never labeled with antibody to acetylated tubulin. Flow cytometry with anti acetylated-tubulin antibody demonstrated that in the control cell populations there were weaker and stronger labelled parts. After TSA treatment in the weaker labeled part the cell number decreased, and in the stronger labeled part increased significantly: this means that after the histone deacetylase inhibitor TSA treatment the amount of acetylated-tubulin in numerous
cells is significantly elevated. Labeling with anti-tubulin antibody was not changed significantly. On the basis of these results we postulate that histone deacetylase also in
influences the acetylation of tubulin, and this enzyme is sensitive to TSA treatments.
White blood cells of rats (lymphocytes, monocytes, macrophages, granulocytes and mast cells) contain b-endorphin. Two months after a single neonatal benzpyrene treatment (imprinting) there is an elevated level of immunoreactive endorphin in the blood and peritoneal cells of female animals and blood cells of males. The endorphin content decreased in the peritoneal cells of males. In the blood, the granulocytes of female, and the lymphocytes of male rats contained the highest amount of endorphin. In the peritoneal fluid also the granulocytes of females contained the highest amount of endorphin, in contrast to males, where the endorphin content of cells decreased and the lowest level of it was present in the lymphocytes. The experiments justify that benzpyrene treatment can durably influence endorphin levels of white blood cells and gives new data to the already known lifelong health destroying effects of perinatal benzpyrene exposition (alterations of hormone receptor binding capacity and sexual behavior).
In order to approach their natural conditions, populations of Tetrahymena were kept in Losina-Losinky’s salt solution for 1 h, than in the tryptone+yeast medium. During this time they were treated with histamine, serotonin or insulin, or with the combinations of these hormones. Effect of the combined treatments on the production of serotonin (5HT), or adrenocorticotropic hormone (ACTH) or triiodothyronine (T3) by the cells was compared to the effect of single-hormone treatments. Significant differences were seen between the results obtained following the single or combined treatments. There was no summation of the effects, however an elevation or diminution of the hormone production was observed after the combined treatment, as compared with the untreated controls or with the use of one of the hormones in the samples. The experiments demonstrate that there is a hormonal regulation between the Tetrahymena cells and the hormones influence each other’s effect.
In a previous experiment thyrotropin (TSH) increased the triiodothyronine (T3) production of Tetrahymena and chorionic gonadotropin (HCG) moderately overlapped the effect. At present the production of three amino acid type (histamine, serotonin, epinephrine) and one peptide (endorphin) hormones were studied under the effect of TSH or HCG, in tryptone-yeast (TY) or salt (Losina-Losinsky) medium. The duration of the effect was 10 min. TSH significantly (with almost 20%) decreased epinephrine production in TY medium and HCG similarly decreased epinephrine and increased histamine level. In salt solution TSH as well as HCG decreased the level of serotonin. The results show that at this low level of phylogeny TSH effect is not completely thyroxine-specific, however it is not general. HCG overlaps TSH effect on epinephrine and serotonin production, however its effect is broader. The experiments also demonstrate that the effect of pituitary trop-hormones can be bidirectional in Tetrahymena, as histamine level was increased and epinephrine level was decreased by HCG, in the same cells.
Authors:Eszter Lajkó, Éva Pállinger and György Csaba
The unicellular Tetrahymena pyriformis was stressed by 37°C heat for 1 h and its hormone (serotonin, histamine, triiodothyronine) content was measured by immunocytochemical flow cytometry in different time points (immediately after treatment and after 1, 2, 8, 16 weeks). The treatment increased each hormone level for two weeks, however, after 8 weeks the hormone concentration inside the cells decreased and in case of serotonin this was similar in the 16th week, while the other two hormones’ level was similar to the control. Insulin further increased the hormone production during treatment, but this effect was not durable. After one week the cells behave similar to those, subjected to heath shock only. The results show that a single stress causes deep and durable changes in the hormone household of Tetrahymena which is influenced by exogenously given insulin only in the acute phase.