Sonja Gašić

Evaluation of the Immunomodulatory Activities of Royal Jelly Components In Vitro

In this work the effect of different components isolated from royal jelly (RJ) was studied using an in vitro rat T-cell proliferation assay. We found that lower concentrations of MEL 174 (final water extract of RJ) and MEL 147 (3-10-dihydroxydecanoic acid) stimulated T-cell proliferation, triggered by concanavalin A (Con-A) and the process was followed by an increase in the production of interleukin-2 (IL-2). Higher concentrations of MEL 174, MEL 247 (dry powder of RJ) and MEL 138 (trans-10-hydroxydec-2-enoic acid) inhibited T-cell proliferation. The inhibition of T-cell proliferation in the presence of MEL 174 was followed by a decrease in IL-2 production, which was partly abrogated by exogenous IL-2, a decrease in nitric oxide (NO) production and increased apoptosis. In conclusion, our results showed the complexity of biological activity of RJ and suggest that its water extract possesses the most potent immunomodulatory activity in vitro.

Interferon gamma alters the phenotype of rat thymic epithelial cells in culture and increases interleukin-6 production.

Rat thymic epithelial cells (TEC) in long-term culture were characterized by anticytokeratin monoclonal antibodies (mAbs) and electron microscopy. Phenotypic analysis performed by a large panel of mAbs showed that the highest percentage of these cells was of the subcapsular/medullary type. Recombinant rat interferon (IFN)-gamma up-regulated class-I and class-II MHC expression by TEC in culture as confirmed by immunohistochemistry and flow cytometry, but did not significantly alter other cell markers. TEC supernatants of IFN-gamma-treated cultures showed higher interleukin-6 (IL-6) activity, compared to the control, as determined by proliferation of the IL-6-sensitive B9-cell line. Increased IL-6 activity was probably not a consequence of increased TEC number in IFN-gamma-treated cultures because IFN did not significantly stimulate TEC proliferation in vitro. In contrast, IL-6 significantly stimulated TEC proliferation, indicating that this cytokine is not only a regulatory molecule for T-cell proliferation, but could also be an autocrine growth factor for thymic epithelium.

Interspecies differences in expression of cytokeratin polypeptides within thymic epithelium: A comparative immunohistochemical study

Cytokeratin (CK) polypeptide expression within the thymic epithelium of several mammalian species (mouse, rat, calf, pig, rabbit, and human) has been analyzed by the streptavidin-biotin immunoperoxidase method. Comparative analysis by a large panel of 17 monoclonal antibodies (mAbs) specific for individual CK polypeptides, pairs, or groups showed considerable heterogeneity of thymic epithelial cells (TEC) in each species. In addition, extreme interspecies difference in CK contents was observed. Four main phenotypic zones: the subcapsule/perivascular area, cortex, medulla, and Hassalls corpuscles (HC) were clearly identified, each characterized by different CK expression. Medullary TEC were more heterogenous and shared common CK polypeptides either with subcapsular/perivascular TEC, cortical TEC, or HC, in most species.

Different Roles of a Rat Cortical Thymic Epithelial Cell Line In Vitro on Thymocytes and Thymocyte Hybridoma Cells: Phagocytosis, Induction of Apoptosis, Nursing and Growth Promoting Activities

In this work, the interaction between a rat cortical thymic epithelial cell (TEC) line (R-TNC.1) with nursing activity and thymocytes as well as BWRT 8 thymocyte hybridoma (TH) cells has been studied. The R-TNC.1 cell line significantly bound thymocytes and TH. Binding was stronger during the first 30 min of cell incubation and was followed by a progressive deadhesion. Among adherent thymocytes the proportion of apoptotic cells increased with culture time which was a consequence of higher capacity of the line for binding of apoptotic than viable cells and induction of apoptosis in a subset of adherent thymocytes. Emperiopolesis activity of this thymic nurse cell (TNC) line was manifested by engulfment of thymocytes as well as TH cells. A subset of viable intra-TNC thymocytes has been triggered to die by apoptosis, whereas other internalized thymocytes have been stimulated to proliferate, as measured by an increase in the percentage of cells in mitosis and higher incorporation of bromodeoxyuridine (BrdU), in comparison to thymocytes cultivated alone. A significant stimulation of proliferation of engulfed TH cells was also observed. The R-TNC.1 cell line efficiently phagocytosed both apoptotic thymocytes and TH, and the process is followed by intra-TNC destruction of ingested cells. Cumulatively, these results suggest different role of the R-TNC.1 clone: phagocytosis of apoptotic cells; induction of apoptotic cell death in a subset of both bound and internalized thymocytes and stimulation of proliferation of a subset of intra-TNC thymocytes or TH cells.

A nucleoside analogue, 7-thia-8-oxoguanosine stimulates proliferation of thymocytes in vitro

7-thia-8-oxoguanosine (immunosine) is a nucleoside analogue with immunoenhancing activity. In this work, its effects on proliferation of thymocytes in vitro were studied. It was found that immunosine stimulated proliferation of thymocytes both of mice and rats. The stimulatory effect depended on antigen presenting cells (APC), since thymocytes depleted of accessory cells did not proliferate to immunosine. In addition, pretreatment of APC with immunosine for 24 h significantly increased proliferation of thymocytes. Immunosine stimulated interleukin 2 (IL-2) production and the expression of activation markers (CD25 and CD71). The upregulation of CD25 (alpha subunit of IL-2R) was detected both on thymocytes and thymic dendritic cells. Proliferation of thymocytes in the presence of immunosine was predominantly mediated by IL-2 since blocking IL-2Ralpha by specific monoclonal antibodies inhibited cell proliferation by 65-85%.

The effect of a new nitro-aspirin on apoptosis of neutrophil granulocytes.

Apoptosis of neutrophil granulocytes is a critical event in the resolution of inflammation. Neutrophils have a short lifespan which can be modulated by aspirin. In this work we studied the effect of a nitroaspirin (NCX4040) on apoptosis of inflammatory granulocytes. This nitro-aspirin has been synthesized in attempt to reduce the side effects of aspirin in the gastrointestinal tract. Inflammatory granulocytes have been isolated from polyvinyl sponges implanted under the skin of Albino Oxford (AO) rats. Inflammatory cells that were isolated 20 hours later were about 95% neutrophil granulocytes. The cells were cultivated 24h with different concentrations of NCX4040 ranging from 0.01 μM to 10μM. After that period, apoptosis of neutrophils was performed by using morphological criteria, as well as by flow cytometry (after staining the cells with propidium iodide). We found that NCX4040 at concentrations from 0.25 to 10 μM induced the apoptosis of rat inflammatory granulocytes in a dose-dependent manner. Also, in these concentrations NCX4040 decreased production of nitric oxide in the cells culture supernatants. In conclusion, our results suggest that antiinflammatory properties of NO-aspirins are additionally potentiated by their proapoptotic effect on granulocytes, which could be a novel mechanism of their action.

Comparison of signaling pathways involved in apoptosis of a thymocyte hybridoma triggered by a rat thymic medullary epithelial cell line, dexamethasone or T-cell receptor cross-linking

Using an in vitro co-culture assay we found that a rat medullary thymic epithelial cell (TEC) line (TE-R2.5) induces apoptosis of the BWRT8 thymocyte hybridoma (TH) (CD4(hi)CD8(low) alphabetaTCR(hi)). TH apoptosis induced by this TEC line was predominantly mediated by direct cell-cell contacts and was potentiated by cross-linking of the T cell receptor (TCR) by R73 monoclonal antibody (mAb). Dexamethasone (Dx) also triggered TH apoptosis but inhibited death of these cells induced by TE-R2.5 cells or immobilized R73 mAb. The TEC-induced apoptosis was independent of the LFA-1/ICAM-1 interaction but partly depended on a novel 29 kDa molecule expressed on TE-R2.5 cells. All three types of TH apoptosis were followed by the cleavage of poly-(ADP-ribose)-polymerase and were blocked by a caspase inhibitor Z-Val-Ala-Asp(OMe)-CH(2)F.PKC stimulation by phorbol myristate acetate interfered with the TH apoptosis induced by TE-R2.5 and Dx, but did not modulate the effect of R73 mAb. On the contrary, inhibition of calcineurin with cyclosporine A did not influence the apoptosis induced by TE-R2.5 and Dx, but completely prevented the R73-triggered TH cell death. The TE-R2.5-mediated BWRT8 apoptosis was suppressed by Na-orthovanadate, an inhibitor of protein tyrosine phosphatases (PTP) as well as by genistein, a protein tyrosine kinase (PTK) inhibitor, while both compounds potentiated the effect of Dx. Blocking PTP, but not PTK decreased the proapoptotic effect of R73 mAb. These results, including those using a BWRT8 subclone (BWRT8-MDP.2) which is resistant to TCR-triggered apoptosis, but sensitive to apoptosis stimulated by TE-R2.5 and Dx, indicate that TE-R2.5-induced TH apoptosis in our model is different from apoptosis in other TEC co-culture models, published so far.

Immunosine (7-thia-8-oxoguanosine) acts as a cofactor for proliferation of T cells

Abstract— Immunosine (7‐thia‐8‐oxoguanosine) is a novel guanosine analogue showing immunostimulatory activity both in vivo and in vitro. This compound acts on different components of the immune system including B cells, natural killer (NK) cells and antigen‐presenting cells (APC). However, its influence on functions of T cells is poorly understood. In this work we studied the effect of immunosine on proliferation of total rat splenocytes and purified T cells triggered by different mitogens and the mechanisms involved. The results demonstrate that immunosine significantly stimulates proliferation of T cells. The effect was dose‐dependent and also depended on concentrations of specific stimulators. Maximal stimulation was seen using 250 μM immunosine. The stimulatory effect of immunosine on lymphocyte proliferation triggered by Concanavalin A (Con A) correlated with increased interleukin 2 (IL‐2) production and upregulation of the IL‐2 receptor α (IL‐2Rα) expression. The dependency of T‐cell proliferation on IL‐2/IL‐2R was confirmed using neutralizing anti‐IL‐2Rα monoclonal antibodies (mAbs). Higher concentrations of immunosine in the presence of optimal concentrations of Con A (5 μg/mL) inhibited proliferation of T cells. A similar stimulatory effect of immunosine on proliferation of purified T cells and IL‐2 production was observed using an anti‐T‐cell receptor (TCR) mAb and a combination of anti‐TCR mAb and IL‐2. However, the guanosine analogue did not significantly modulate proliferation of T cells triggered by IL‐2 alone. When the combination of phorbol myristate acetate (PMA) and ionomycin was used for T‐cell stimulation different results were obtained. Under lower cell stimulation immunosine significantly potentiated T‐cell proliferation, expression of IL‐2Rα and IL‐2 production. In the presence of suboptimal stimulation the compound stimulated T‐cell proliferation and IL‐2Rα expression, whereas under maximal stimulation an enhancing effect on IL‐2 production was seen. Since direct stimulatory effect of immunosine on T‐cell growth in culture was rather weak it can be postulated that the compound acts as a cofactor for T‐lymphocyte proliferation.

Timusne ćelije dadilje - specijalizovana mikrosredina timusa

Vise od dve decenije nakon prvog opisa, TNC su i dalje velika nepoznanica i potrebno je jos mnogo istraživanja pre nego sto budemo bili u mogucnosti da definisemo preciznu ulogu ovih celija u razvoju T-limfocita. Mnoga od dosadasnjih saznanja ukazuju da se timociti u kontaktu sa TNC nalaze na prekretnici u svom razvojnom putu: ili ce biti uklonjeni indukcijom apoptoze ili ce nastaviti svoj razvoj i dalje sazrevanje. Brojna pitanja su za sada bez odgovora, a među njima su dva posebno intrigantna. Koja je razlika između timocita koji se vezuju za TNC i onih koji to ne cine? Koja je razlika između populacije adherentnih timocita koji su selektivno internalizovani i onih koji su iskljuceni iz procesa internalizacije? Buduca istraživanja kretanja timocita ka, unutar i van TNC ce verujemo pružiti dragocene informacije o ovoj fazi u razvoju T-limfocita. Nezavisno od toga sta ce buducnost pokazati o pravoj ulozi TNC, jedinstven kompleks koji ove celije formiraju sa timocitima je vrlo neobican, uzbudljiv i zagonetan bioloski fenomen.