[IL28B CC genotype: a protective factor and predictor of the response to interferon treatment in chronic hepatitis C virus infection]. / IL28B CC genotípus: védo tényezo és az interferonválasz prediktora krónikus hepatitis-C-vírus infekcióban.

INTRODUCTION: In chronic hepatitis C-virus infection the possible role of gene variants encoding cytokines has become the focus of interest. AIM: The aim of the study was to investigate the effect of IL28B polymorphisms on the outcome of chronic hepatitis C-virus genotype 1 infection in the Hungarian population. In addition, the association between IL28B genotypes and the Th1/Th2 cytokine production of activated peripheral blood monocytes and lymphocytes was evaluated. METHOD: Total of 748 chronic hepatitis C-virus genotype 1 positive patients (365 males and 383 females, aged between 18 and 82 years; mean age, 54±10 years) were enrolled, of which 420 patients were treated with pegylated interferon plus ribavirin for 24-72 weeks. Of the 420 patients, 195 patients (46.4%) achieved sustained virological response. The IL28B rs12979860 polymorphism was determined using Custom Taqman SNP Genotyping Assays (Applied Biosystems, Life Technologies, Foster, CA, USA). For cytokine studies, tumour necrosis factor-α, interleukin-2, interferon-γ, interleukin-2 and interleukin-4 production by LPS-stimulated monocytes and PMA-ionomycine activated lymphocytes were measured from the supernatant of the cells obtained from 40 hepatitis C-virus infected patients, using FACS-CBA Becton Dickinson test. The cytokine levels were compared in patients with different (CC, CT, TT) IL28B genotypes. RESULTS: The IL28B rs12979860 CC genotype occurred in lower frequency in hepatitis C-virus infected patients than in healthy controls (26.1% vs 51.4%, OR 0.333, p<0.001). Patients carried the T allele with higher frequency than controls (73.9%, vs 48.6%, OR 3.003, p<0.001). Pegylated interferon plus ribavirin treated patients with the IL28B CC genotype achieved higher sustained virological response rate than those with the CT genotype (58.6% vs 40.8%, OR 2.057, p = 0.002), and those who carried the T allele (41.8%, OR1.976, p = 0.002). LPS-induced TLR-4 activation of monocytes resulted in higher tumour necrosis factor-α production in patients with the IL28B CC genotype compared to non-CC individuals (p<0.01). Similarly, increased tumour necrosis factor-α, interleukin-2 and interferon-γ production by lymphocytes was found in the IL28B CC carriers (p<0.01) CONCLUSIONS: The IL28B CC genotype exerts protective effect against chronic hepatitis C-virus infection and may be a pretreatment predictor of sustained virological response during interferon-based antiviral therapy. The IL28B CC polymorphism is associated with increased Th1 cytokine production of activated peripheral blood monocytes and lymphocytes, which may play a role in interferon-induced rapid immune control and sustained virological response of pegylated interferon plus ribavirin treated patients.

Mitochondrial translocation of the glucocorticoid receptor in double-positive thymocytes correlates with their sensitivity to glucocorticoid-induced apoptosis.

Glucocorticoid receptor (GR) signaling plays an important role in the selection and apoptosis of thymocytes. Besides nuclear translocation, mitochondrial translocation of the ligand-bound GR in lymphoid cells was also shown, which might determine glucocorticoid (GC)-induced apoptosis sensitivity. In the present work, we followed the ligand-induced GR trafficking in CD4+CD8+ double-positive (DP) thymocytes. Using confocal microscopy, we found that upon short-term in vitro GC analog [dexamethasone (DX)] treatment, the GR translocates into the mitochondria but not into the nucleus in DP cells. We also analyzed the GR redistribution in cytosolic, nuclear and mitochondrial fractions of unseparated thymocytes by western blot and confirmed that in DX-treated cells a significant fraction of the GR translocates into the mitochondria. DX reduced the mitochondrial membrane potential of DP cells within 30 min, measured by flow cytometry, which refers to a direct modulatory activity of mitochondrial GR translocation. The abundant mitochondrial GR found in DP cells well correlates with their high GC-induced apoptosis sensitivity.

Developmental shift in TcR-mediated rescue of thymocytes from glucocorticoid-induced apoptosis.

Glucocorticoid hormone (GC) production by thymic epithelial cells influences TcR signalling in DP thymocytes and modifies their survival. In the present work, we focused on exploring details of GC effects on DP thymocyte apoptosis with or without parallel TcR activation in AND transgenic mice, carrying TcR specific for pigeon cytochrome C, in vivo. Here we show that the glucocorticoid receptor (GR) protein level was the lowest in DP thymocytes, and it was slightly down-regulated by GC analogue, anti-CD3, PCC and combined treatments as well. Exogenous GC analogue treatment or TcR stimulation alone lead to marked DP cell depletion, coupled with a significant increase of early apoptotic cell ratio (AnnexinV staining), marked abrogation of the mitochondrial function in DP cells (CMXRos staining), and significant decrease in the Bcl-2(high) DP thymocyte numbers, respectively. On the other hand, the simultaneous exposure to these two proapototic signals effectively reversed all the above-described changes. The parallel analysis of CD4 SP cell numbers, AnnexinV, CMXRos, Bcl-2 and GR stainings revealed, that the GR and TcR signals were not antagonistic on the mature thymocytes. These data provide experimental evidence in TcR transgenic mice, in vivo, that when TcR activation and GR signals are present simultaneously, they rescue double positive thymocytes from programmed cell death. The two separate signalling pathways merge in DP thymocytes at such important apoptosis regulating points as the Bcl-2 and GR, showing that their balanced interplay is essential in DP cell survival.

Thienopyridine therapy influences late outcome after coronary stent implantation.

Clinical significance of resistance to aspirin and thienopyridine therapy is poorly defined. The authors aimed to evaluate whether more effective antiplatelet therapy is associated with better outcome in patients on dual-antiplatelet treatment. Using optical aggregometer, maximal platelet aggregation values were measured with induction of adenosine diphosphate, collagen, and adrenaline 30 +/- 5 days after coronary stent implantation in 134 patients. Markers of platelet activation were also analyzed with fluorescent immunoassay in 57 patients. After 10 months of follow-up, 33 patients reached the composite endpoint of cardiovascular death, myocardial infarction, and revascularisation. Adenosine diphosphate-induced maximal aggregation values were in significant relationship with the development of major adverse cardiac events (P < .01). Level of soluble P-selectin proved to be an independent risk factor of adverse outcome (P < .05). As efficacy of thienopyridine therapy showed significant relation with clinical outcome, the authors conclude that interindividual variability in response to adenosine diphosphate-receptor antagonists may be of substantial clinical importance.

A possible new bridge between innate and adaptive immunity: Are the anti-mitochondrial citrate synthase autoantibodies components of the natural antibody network?

Natural antibody (nAb) producing B-1 B cells are considered an intermediate stage of evolution between innate and adaptive immunity. nAbs are immunoglobulins that are produced without antigen priming. nAbs can recognize foreign targets and may serve in the first line of immune defense during an infection. Natural autoantibodies (nAAbs) present in the serum of both healthy humans and patients suffering from systemic autoimmune diseases recognize a set of evolutionarily conserved self-structures. Because of their endosymbiotic evolutionary origin, proteins compartmentalized into mitochondria represent an interesting transition from prokaryotic foreign (non-self) to essential (self) molecules. We investigated the possible overlap in recognized epitopes of innate and self-reactive nAbs and surveyed changes in physiological autoreactivity under pathological autoimmune conditions. Epitope mapping analysis of a mitochondrial inner membrane enzyme, citrate synthase (CS) (EC 2.3.3.1) by synthetic overlapping peptides and phage display libraries using sera from healthy individuals and from patients having systemic autoimmune disease revealed CS recognizing nAAbs with IgM isotype. We analyzed cross reactive epitopes on human CS, bacterial CS, and various standard autoantigens. The anti-CS nAAbs by participating in the nAb network, could function in innate defense mechanisms and at the same time recognize a target antigen (nucleosome) in a systemic autoimmune disease. Thus, at the level of recognized epitopes there is a possible new link between the innate like component and the adaptive-autoimmune arm of the humoral immune system.

Low glucocorticoid receptor (GR), high Dig2 and low Bcl-2 expression in double positive thymocytes of BALB/c mice indicates their endogenous glucocorticoid hormone exposure.

Several studies have shown that of the four major thymocyte subsets, the CD4/CD8 double positive (DP) thymocytes are the most sensitive to in vivo glucocorticoid hormone (GC)-induced apoptosis. Our aim was to analyse fine molecular differences among thymocyte subgroups that could underlie this phenomenon. Therefore, we characterised the glucocorticoid hormone receptor (GR) expression of thymocyte subgroups both at the mRNA and protein levels by real-time PCR and flow cytometry, and correlated these features to their apoptotic sensitivity. We also investigated the time-dependent effects of the GC agonist dexamethasone (DX) with or without GC antagonist (RU486) treatments on GR mRNA/protein expression. We also analysed the expression of two apoptosis-related gene products: dexamethasone-induced gene 2 (Dig2) mRNA and Bcl-2 protein. We found that DN thymocytes had the highest GR expression, followed by CD8 single positive (SP), CD4 SP and DP thymocytes in 4-week-old BALB/c mice, both at the mRNA and protein levels, respectively. In DP cells, the Dig2 expression was significantly higher, while the Bcl-2 expression was significantly lower than in DN, CD4 SP and CD8 SP thymocytes. Single high dose DX treatment caused time-dependent depletion of DP thymocytes due to their higher apoptosis rate, which could not be abolished with RU486 pretreatment. After a single high dose DX treatment, there was a transient, significant increase of the GR mRNA and protein level of unsorted thymocytes after 8 and 16 h, followed by a significant decrease at 24 h, respectively. The time-dependent GR expression changes after DX administration could not be inhibited by the GC antagonist RU486. Twenty-four hours after exposure to high dose DX the DN, CD4 SP and CD8 SP cells showed a significant decrease of GR mRNA and protein expression, whereas the DP thymocytes, showed no significant alteration of GR mRNA or protein expression. The kinetical analysis of GR expression and apoptotic marker changes upon single high dose GC analogue administration revealed a two-phase process in thymocytes: early events, within 4-8 h, include GR upregulation and early apoptosis induction, while the late events appear most prominently at 16-20 h, when the GR is already downregulated and apoptotic cell ratio reaches its peak, with marked DP cell depletion. The low GR, high Dig2 and low Bcl-2 expression, coupled with the absence of homologous downregulation of GR after exogenous GC analogue treatment, could contribute to the high GC sensitivity of DP thymocytes. The downregulated GR and Bcl-2 together with the upregulated Dig2 level in DP cells indicates the significance of intrathymic GC effects at this differentiation stage. Since GR expression changes and apoptotic events could not be completely inhibited by GC antagonist, we propose the involvement of non-genomic GR mechanisms in these processes.

Dexamethasone induces rapid tyrosine-phosphorylation of ZAP-70 in Jurkat cells.

Steroid hormones are known to mediate rapid non-genomic effects occurring within minutes, besides the classical genomic actions mediated by the nuclear translocation of the cytoplasmic glucocorticoid receptor (GR). The glucocorticoid hormone (GC) has significant role in the regulation of T-cell activation; however, the cross-talk between the GC and T-cell receptor (TcR) signal transducing pathways are still to be elucidated. We examined the rapid effects of GC exposure on in vitro cultured human T-cells. Our results showed that Dexamethasone (DX), a GC analogue, when applied at high dose (10 microM), induced rapid (within 5 min) tyrosine-phosphorylation events in Jurkat cells. Short DX pre-treatment strongly inhibited the tyrosine-phosphorylation stimulated by CD3 cross-linking. Furthermore, we also investigated the phosphorylation status of ZAP-70, an important member of tyrosine kinase mediated signalling pathway of TcR-elicited T-cell activation. Here, we demonstrate that high dose DX induced a rapid ZAP-70 tyrosine-phosphorylation in Jurkat T-cells. DX-induced ZAP-70 phosphorylation could be inhibited by RU486 (GR antagonist), suggesting that this process was GR mediated. DX-induced ZAP-70 phosphorylation did not occur in the absence of active p56-lck as examined in the p56-lck kinase-deficient Jurkat cell line JCaM1.6. Our results show that DX, at a high dose, can rapidly influence the initial tyrosine-phosphorylation events of the CD3 signalling pathway in Jurkat cells, thereby modifying TcR-derived signals. Lck and ZAP-70 represent an important molecular link between the TcR and GC signalling pathways.

[Immunology of HCV infection: the causes of impaired cellular immune response and the effect of antiviral treatment]. / A hepatitis C-vírus-infekció immunológiája: az elégtelen celluláris immunválasz okai es az antivirális kezelés hatásai.

BACKGROUND: The outcome of HCV infection and the response to antiviral treatment depend on both viral and host factors. Host immune response contributes not only to viral control, clinical recovery and protective immunity, but also to chronic hepatitis and liver cirrhosis. Establishing immunological status and identifying pretreatment immunological factors associated with better response to therapy might be of importance in the understanding of the successful immune response and in the future of combination therapy to HCV infection. AIMS: The authors delivered a review on the immunology of HCV infection and characterized the cause of impaired cellular immune response in chronic HCV infection. Natural killer (NK) cell activity, perforin and the inhibitory CD81 HCV co-receptor expression, and Th1/Th2 cytokine production of the monocytes and lymphocytes have been investigated. PATIENTS AND METHODS: 42 patients with chronic hepatitis C, out of them 25 being on interferon (PEG-IFN) + ribavirin (RBV) therapy, 12 sustained virological responders, 26 HCV carriers with normal transaminase values and 22 healthy controls were studied. NK cell activity, perforin and CD81 expression, the IFNgamma, TNFalpha, IL-2 (Th1) and IL-4, IL-6, IL-10 (Th2) production of LPS stimulated monocytes and PMA + ionomycine stimulated lymphocytes were measured by flow-cytometry. RESULTS: In patients with chronic hepatitis C we demonstrated decreased NK cell activity associated with increased CD81 expression. The perforin expression of lymphocytes was also impaired in HCV patients. The pretreatment capacity of the macrophages to produce TNFalpha was predictive for sustained virological response. This increased TNFalpha production of the monocytes counteracted the observed impaired Th1 type cytokine production of the lymphocytes. IL-10 and IL-4 production showed positive correlation with HCV RNA levels, and negative correlation with histological activity index was noted. PEG-IFN + RBV treatment increased NK activity, perforin expression, Th1 type cytokine production of thr lymphocytes and downregulated CD81 expression inducing effective cellular immune response against HCV. The author's results provide further data to understand the causes of impaired cellular immune response in chronic HCV hepatitis and may be useful in the developement of immunotherapy as an adjunctive treatment to cure patients with chronic hepatitis C.

Sandwich type ELISA and a fluorescent cytometric microbead assay for quantitative determination of hepatitis B virus X antigen level in human sera.

The hepatitis B virus X protein (HBxAg) is responsible for severe complications of HBV infections including primary hepatocellular carcinoma. A sandwich type ELISA and a flow cytometric microbead assay for quantitative determination of serum levels of Hbx-Ag are introduced. We have previously developed monoclonal antibody families against well-conserved epitopes on HbxAg, characterized by different immunohistochemical and immunoserological techniques. Special selection of the antibody pairs provided highly sensitive and highly specific tools for quantitative immunoassay development. The resulting assays were tested on human sera (208 samples) collected from patients suffering from different clinical forms of HBV infection. The sensitivity range of the sandwich type ELISA was between 4 and 2000 ng/ml as measured on both the recombinant antigen and the sera of chronic hepatitis patients. A further flow cytometric microbead assay was established and tested in parallel with the ELISA. The quantitative results of these two immunoserological techniques were in strong correlation and they were found to be highly specific and sensitive on clinical samples. The HBxAg ELISA technique is applicable for routine clinical laboratory measurements, and our HBxAg microbead technique is recommended for complex multiparametric measurements combined with other markers.

Evidence for a novel protective role of the vanilloid TRPV1 receptor in a cutaneous contact allergic dermatitis model.

The purpose of this study was to examine the involvement of the transient receptor potential vanilloid receptor 1 (TRPV1) in inflammatory processes observed in murine allergic contact dermatitis (ACD). Oxazolone-induced ACD evoked a significant ear swelling after 24-72 h. It was augmented in TRPV1 knockout mice at all time points and supported by histological analysis and measure of TNF-alpha. However, tissue swelling and cytokine generation was significantly reduced in both neurokinin 1 receptor and calcitonin gene-related peptide (CGRP) knockout mice. A protective involvement of the TRPV1 receptor was identified of contact dermatitis distinct from mechanisms involving the major pro-inflammatory neuropeptides.

Monoclonal antibodies identify four distinct annelid leukocyte markers.

This paper describes for the first time the production and characterization of a library of monoclonal antibodies (anti-EFCC clones) raised against coelomocyte (leukocyte) markers of Eisenia fetida earthworm. Leukocyte subgroups are components of earthworm innate immunity that require a more precise characterization using immunological markers. Flow cytometry, immunocytochemistry, and immunoprecipitation analyzed and confirmed the specificity of anti-EFCC clones. Anti-EFCC mAbs revealed different leukocyte subpopulations and various staining patterns on tissues. Two functional assays (e.g. phagocytosis and encapsulation) further characterized EFCC clusters revealing a common coelomocyte marker and three subpopulation-specific markers. No crossreactivity was found on human, mouse, rat or cells from Drosophila melanogaster but immunoreactivity was detected on snail (Planorbarius corneus) tissues. Immunohistochemical results suggest mesodermal origin of all coelomocyte subgroups that agree with classical morphological analyses.

Antigen and glucocorticoid hormone (GC) induce positive selection of DP thymocytes in a TcR transgenic mouse model.

Thymocyte maturation in the thymus is controlled by stromal and humoral components. Among the humoral regulators locally produced glucocorticoids (GCs) seem to have a key role in the positive selection of thymocytes. Our previous studies have shown that the administration of GCs or the stimulation through the CD3 complex can induce apoptosis of double positive (DP) cells, but the combined presence of these stimuli induces positive selection. In this work our aim was to investigate the effects of antigen exposure and synthetic GC hormone (dexamethasone, DX) administration on the selection processes of DP cells in TcR transgenic mice. In our model, AND-pigeon cytochrome c (PCC)-specific I-E(k) (MHC-II) restricted Vbeta3, Valpha11 TcR expressing transgenic mice were treated with PCC, with high or low dose DX, or with PCC and DX together, followed by the analysis of total thymocyte numbers, thymocyte composition, with regard to their CD69, Vbeta3 and Annexin V expression. The administration of PCC and/or DX for 2 days resulted in a decreased DP cell number and a significantly increased CD4 SP cell ratio. However, in both cases the total thymocyte numbers decreased. CD69 expression increased on both DP and CD4 SP cells after PCC and/or DX treatments. We found that after DX or combined treatment, the percentage of Annexin V positive cells increased. The ratio of Vbeta3 TcR bearing DP thymocytes showed no change after DX or PCC administrations alone, but it decreased significantly after combined treatment. MHC-II bound PCC peptides in the presence of GCs enhanced the maturation of Vbeta3+ DP cells into CD4 SP stage, therefore, the Vbeta3- cells remained mostly in the DP immature stage. These data indicate that both antigen and low dose GC alone are capable of inducing positive selection of DP cells, but together they gave a stronger effect in promoting positive selection. From these we conclude that GCs influence the maturation and selection processes of thymocytes.

Increased baseline proinflammatory cytokine production in chronic hepatitis C patients with rapid virological response to peginterferon plus ribavirin.

BACKGROUND: Chronic hepatitis C (CHC) patients achieving rapid virological response (RVR) on PEG-IFN/ribavirin (P/R) therapy have high chance of sustained virological response (SVR). To analyze host immunological factors associated with RVR, viral kinetics, phenotype distribution and Th1/Th2 cytokine production by peripheral blood mononuclear cells (PBMC) were studied prior to and during P/R therapy. METHODS: TNF-α, IFN-γ, IL-2, IL-6, IL-4 and IL-10 production by PBMC were measured after Toll-like receptor 4 (TLR-4) or phorbol myristate acetate/Ionomycin stimulation in 20 healthy controls and in 50 CHC patients before receiving and during P/R therapy. RVR was achieved by 14, complete early virological response (cEVR) by 19 patients and 17 patients were null-responders (NR). RESULTS: Patients with RVR showed an increased baseline TNF-α and IL-6 production by TLR-4 activated monocytes and increased IFN-γ, decreased IL-4 and IL-10 production by lymphocytes compared to non-RVR patients. SVR was also associated with increased baseline TNF-α production and decreased IL-10 levels compared to patients who did not achieve SVR. Baseline IL-2 production was higher in cEVR compared to NR patients. Antiviral treatment increased TNF-α, IL-6 production by monocytes and IFN-γ secretion by lymphocytes and decreased IL-4 and IL-10 production by lymphocytes in cEVR compared to NR patients. CONCLUSION: RVR was associated with increased baseline proinflammatory cytokine production by TLR-4 stimulated monocytes and by activated lymphocytes. In null-responders and in patients who did not achieve SVR both TLR-4 sensing function and proinflammatory cytokine production were impaired, suggesting that modulation of TLR activity and controlled induction of inflammatory cytokine production may provide further therapeutic strategy for CHC patients non-responding to P/R treatment.

Emerging pathways of non-genomic glucocorticoid (GC) signalling in T cells.

In the last decade new glucocorticoid (GC)-signalling mechanisms have emerged. The evolving field of non-genomic GC actions was precipitated from two major directions: (i) some rapid/acute clinical GC applications could not be explained based on the relatively slowly appearing genomic GC action and (ii) accumulating evidence came to light about the discrepancy in the apoptosis sensitivity and GR expression of thymocytes and other lymphoid cell types. Herein, we attempt to sample the latest information in the field of non-genomic GC signalling in T cells, and correlate it with results from our laboratory. We discuss some aspects of the regulation of thymocyte apoptosis by GCs, paying special interest to the potential role(s) of mitochondrial GR signalling. The interplay between the T cell receptor (TcR) and glucocorticoid receptor (GR) signalling pathways is described in more detail, focusing on ZAP-70, which is a novel target of rapid GC action.

Intermolecular relations between the glucocorticoid receptor, ZAP-70 kinase, and Hsp-90.

The glucocorticoid receptor (GR) participates in both genomic and non-genomic glucocorticoid hormone (GC) actions by interacting with other cytoplasmic signalling proteins. Previously, we have shown that high dose Dexamethasone (DX) treatment of Jurkat cells causes tyrosine phosphorylation of ZAP-70 within 5 min in a GR-dependent manner. By using co-immunoprecipitation and confocal microscopy, here we demonstrate that the liganded GR physically associates with ZAP-70, in addition to its phosphorylation changes. The association of the ligand-bound GR and ZAP-70 was also observed in HeLa cells transfected with ZAP-70, suggesting that this co-clustering is independent of lymphocyte specific factors. Furthermore, the ZAP-70 was found to also co-precipitate with Hsp-90 chaperone both in Jurkat and transgenic HeLa cells, independent of the presence of DX. These findings raise the possibility that ZAP-70 may serve as an important link between GC and TcR-induced signaling, thereby transmitting non-genomic GC action in T-cells.

Effects of mistletoe extract on murine thymocytes in vivo and on glucocorticoid-induced cell count reduction.

BACKGROUND: Mistletoe extracts are widely used in cancer patients due to their cytostatic and immunomodulatory effects. Essential components include mistletoe lectins which act as biomodulators with proinflammatory and apoptosisinducing effects. This study investigates the acute and longterm effects of standardized mistletoe extract (Iscador(R) M spec 5 mg) on thymocyte subpopulations and peripheral T-cells using a murine (Balb/c) model. MATERIALS AND METHODS: Using cell surface CD4/CD8 staining and flow cytometry, we followed the changes in CD4-CD8- double-negative (DN), CD4(+)CD8(+) double-positive (DP) and CD4(+) or CD8(+) single-positive (SP) T-cells 24 h after single or repeated injections of 3 different dilutions (1:12, 1:60, 1:300) corresponding to 2.1, 0.42 and 0.08 mg/kg of Iscador. Thymocyte apoptosis was detected by flow cytometry using Annexin V and propidium iodide. RESULTS: 24 h after a single injection of the 2 lower doses, the number of DN thymocytes increased significantly with an enhanced ratio of apoptotic cells. Following administration of the lowest dose, in peripheral blood the CD4(+)/CD8(+) ratio was elevated. In the long-term trial, Balb/c mice were treated twice a week with 3 different doses of Iscador +/- 20 mg/kg of dexamethasone (DX), resulting in significantly enhanced DN thymocytes and elevated levels of apoptotic cells after treatment with the 2 lower doses. Iscador also inhibited the DX-induced reduction in the thymic DN cell count, as well as the DX-induced decrease in the CD4(+)/CD8(+) ratio and CD4(+) in the peripheral blood. CONCLUSION: Our results suggest that standardized mistletoe extract modulates proliferation and apoptosis of thymocytes in a dose-dependent manner and may act lymphoprotective during DX treatment.

Investigation of the effect of mistletoe (Viscum album L.) extract Iscador on the proliferation and apoptosis of murine thymocytes.

Mistletoe (Viscum album L.) extracts (ME) have been shown to exhibit a bell-shaped curve of immunological efficacy and mistletoe lectins (MLs) were found to play an important role in this phenomenon. The aim of present in vivo study was to investigate the acute- and long-term effect of a standardized ME (Iscador M special) on thymocyte subpopulations and peripheral T cells using a murine (Balb/c) model. In thymus CD4-CD8- double negative (DN), CD4+CD8+ double positive (DP), CD4+ or CD8+ single positive T cells were determined 24 h after a single injection or following a long-term treatment (twice a week for 4 weeks) with three different dilutions of ME which are corresponding to 4.5 ng/ kg, 22.5 ng/kg and 112.5 ng/kg doses of MLs. The apoptosis of the thymocytes was also tested by flow cytometry using Annexin V and propidium iodide. 24 h after a single injection of ME only the lowest dose caused in the blood samples an elevated CD4+/CD8+ ratio and in thymus an enhanced proliferation of DN thymocytes indicating a similar bell-shaped curve of immunological efficacy. After a treatment for four weeks these responses were less intensive indicating that none of the three doses are immunologically optimal. Surprisingly, both in the acute and in the long-term trial only the lower doses induced significant enhancements in the ratio of apoptotic thymocytes. In addition, ME inhibited the dexamethasone (DX)-induced reduction of DN cell count in thymus, as well as the DX-induced decrease of CD4+/CD8+ ratio and CD4+ cell level in peripheral blood. These in vivo results suggest that investigation of thymocytes in vivo can be helpful in the immunological dose-finding since standardized ME is able to modulate the proliferation and apoptosis of thymocytes with a bell-shaped curve of efficacy. In addition, ME may act lymphoprotectively during DX treatment.

Glucocorticoid (GC) sensitivity and GC receptor expression differ in thymocyte subpopulations.

Positive and negative selection steps in the thymus prevent non-functional or harmful T cells from reaching the periphery. To examine the role of glucocorticoid (GC) hormone and its intracellular receptor (GCR) in thymocyte development we measured the GCR expression in different thymocyte subpopulations of BALB/c mice with or without previous dexamethasone (DX), anti-CD3 mAb, RU-486 and RU-43044 treatment. Four-color labeling of thymocytes allowed detection of surface CD4/CD8/CD69 expression in parallel with intracellular GCR molecules by flow cytometry. Double-positive (DP) CD4+CD8+ thymocytes showed the lowest GCR expression compared to double-negative (DN) CD4-CD8- thymocytes and mature single-positive (SP) cells. DX treatment caused a concentration-dependent depletion of the DP cell population and increased appearance of mature SP cells with reduced GCR levels. GCR antagonists (RU-486 or RU-43044) did not influence the effect of DX on thymocyte composition; however, RU-43044 inhibited the high-dose GC-induced GCR down-regulation in SP and DN cells. GCR antagonists alone did not influence the maturation of thymocytes and receptor numbers. Combined low-dose anti-CD3 mAb and DX treatment caused an enhanced maturation (positive selection) of thymocytes followed by the elevation of CD69+ DP cells. The sensitivity of DP thymocytes with a GCRlow phenotype to GC action and the ineffectiveness of the GCR antagonist treatment may reflect a non-genomic GC action in the thymic selection steps.