Karina Guttek

Fully automated analysis of chemically induced γH2AX foci in human peripheral blood mononuclear cells by indirect immunofluorescence

Analysis of phosphorylated histone protein H2AX (γH2AX) foci is currently the most sensitive method to detect DNA double‐strand breaks (DSB). This protein modification has the potential to become an individual biomarker of cellular stress, especially in the diagnosis and monitoring of neoplastic diseases. To make γH2AX foci analysis available as a routine screening method, different software approaches for automated immunofluorescence pattern evaluation have recently been developed. In this study, we used novel pattern recognition algorithms on the AKLIDES® platform to automatically analyze immunofluorescence images of γH2AX foci and compared the results with visual assessments. Dose‐ and time‐dependent γH2AX foci formation was investigated in human peripheral blood mononuclear cells (PBMCs) treated with the chemotherapeutic drug etoposide (ETP). Moreover, the AKLIDES system was used to analyze the impact of different immunomodulatory reagents on γH2AX foci formation in PBMCs. Apart from γH2AX foci counting the use of novel pattern recognition algorithms allowed the measurement of their fluorescence intensity and size, as well as the analysis of overlapping γH2AX foci. The comparison of automated and manual foci quantification showed overall a good correlation. After ETP exposure, a clear dose‐dependent increase of γH2AX foci formation was evident using the AKLIDES as well as Western blot analysis. Kinetic experiments on PBMCs incubated with 5 μM ETP demonstrated a peak in γH2AX foci formation after 4 to 8 h, while a removal of ETP resulted in a strong reduction of γH2AX foci after 1 to 4 h. In summary, this study demonstrated that the AKLIDES system can be used as an efficient automatic screening tool for γH2AX foci analysis by providing new evaluation features and facilitating the identification of drugs which induce or modulate DNA damage.

Analysis of DNA Double-Strand Breaks and Cytotoxicity after 7 Tesla Magnetic Resonance Imaging of Isolated Human Lymphocytes

The global use of magnetic resonance imaging (MRI) is constantly growing and the field strengths increasing. Yet, only little data about harmful biological effects caused by MRI exposure are available and published research analyzing the impact of MRI on DNA integrity reported controversial results. This in vitro study aimed to investigate the genotoxic and cytotoxic potential of 7 T ultra-high-field MRI on isolated human peripheral blood mononuclear cells. Hence, unstimulated mononuclear blood cells were exposed to 7 T static magnetic field alone or in combination with maximum permissible imaging gradients and radiofrequency pulses as well as to ionizing radiation during computed tomography and γ-ray exposure. DNA double-strand breaks were quantified by flow cytometry and automated microscopy analysis of immunofluorescence stained γH2AX. Cytotoxicity was studied by CellTiter-Blue viability assay and [3H]-thymidine proliferation assay. Exposure of unstimulated mononuclear blood cells to 7 T static magnetic field alone or combined with varying gradient magnetic fields and pulsed radiofrequency fields did not induce DNA double-strand breaks, whereas irradiation with X- and γ-rays led to a dose-dependent induction of γH2AX foci. The viability assay revealed a time- and dose-dependent decrease in metabolic activity only among samples exposed to γ-radiation. Further, there was no evidence for altered proliferation response after cells were exposed to 7 T MRI or low doses of ionizing radiation (≤ 0.2 Gy). These findings confirm the acceptance of MRI as a safe non-invasive diagnostic imaging tool, but whether MRI can induce other types of DNA lesions or DNA double-strand breaks during altered conditions still needs to be investigated.

Stimulated human peripheral T cells produce high amounts of IL-35 protein in a proliferation-dependent manner.

The p35 subunit of IL-12 and the Epstein-Barr virus-induced gene 3 (EBI3) have been shown to form a heterodimeric cytokine, named interleukin-35 (IL-35). Recently, mRNA expression of both IL-12p35 and EBI3 was clearly shown in stimulated human T effector cells. Here, we investigated the production of IL-35 protein in human anti-CD3/CD28-stimulated pan T cells as well as T cell subpopulations using a specific human IL-35 ELISA system. We measured high concentrations of IL-35 (up to 3 ng/ml) in cell culture supernatants of stimulated pan T cells as well as CD4(+), CD8(+) and CD4(+)CD25(-) T cell subpopulations at 72 h after stimulation. Very low amounts of IL-35, in the range of 100pg/ml, were detectable in supernatants of resting T cells. These observations could be confirmed using a dot-blot assay for IL-12p35 and EBI3. High concentrations of IL-35 could be also measured in cell culture supernatants of both, resting and stimulated CD4(+)CD25(+) T cells. In order to learn more about the regulation of IL-35 production, we studied the effect of dexamethasone, cyclosporine A and rapamycin on IL-35 production of anti-CD3/CD28-stimulated human pan T cells as well as CD4(+) and CD8(+) T cell subpopulations. All three drugs significantly suppressed IL-35 production of these cells in a proliferation-dependent manner. In summary, we could show that stimulated human peripheral blood T cells of healthy donors produce high amounts of IL-35 protein. However, the biological function of this cytokine remains to be elucidated.

Assessment of modulated cytostatic drug resistance by automated γH2AX analysis

The efficacy of many chemotherapeutic agents relies on the preferential destruction of rapidly dividing cancer cells by inducing various kinds of DNA damage. The most deleterious type of DNA lesions are DNA double‐strand breaks (DSB), which can be detected by immunofluorescence staining of phosphorylated histone protein H2AX (γH2AX). Furthermore, γH2AX has been suggested as clinical pharmacodynamic biomarker in chemotherapeutic cancer treatment. A great challenge in treating neoplastic diseases is the varying response behavior among cancer patients. Thus, intrinsic or drug‐induced overexpression of efflux pumps often leads to multiple drug resistance (MDR) and treatment failure. In particular, inter‐individual differences in expression levels of efflux pumps, such as the permeability glycoprotein (P‐gp), were shown to correlate with cancer progression. Several efficient cytostatic drugs, including the DSB‐inducing agent etoposide (ETP) are known P‐gp substrates. In this respect, modulation of MDR by P‐gp inhibitors, like the immunosuppressives cyclosporine A (CsA) and rapamycin (Rapa) have been described. Here, we investigated the application of γH2AX focus assay to monitor the impact of CsA and Rapa on ETP‐induced cytotoxicity in human peripheral blood mononuclear cells. Evaluation of γH2AX foci was performed by the automated fluorescence microscopy and interpretation system AKLIDES. Compared to ETP treatment alone, our results revealed a significant rise in γH2AX focus number and percentage of DSB‐positive cells after cells have been treated with ETP in the presence of either CsA or Rapa. In contrast, DSB levels of cells incubated with CsA or Rapa alone were comparable to focus number of untreated cells. Our results successfully demonstrated how automated γH2AX analysis can be used as fast and reliable approach to monitor drug resistance and the impact of MDR modulators during treatment with DSB‐inducing cytostatics.

Insulin and Insulin-like growth factor-1 Can Activate the Phosphoinositide-3-kinase /Akt/FoxO1 Pathway in T Cells in vitro

ABSTRACT Hyper-glycemic food increases insulin-like growth factor 1 (IGF-1) and insulin signaling and regulates endocrine responses and thereby may modulate the course of acne. Inflammation and adaptive immune responses have a pivotal role in all stages of acne. Recent hypothesis suggests that hyperglycemic food reduces nuclear forkhead box-O1 (FoxO1) transcription factor and may eventually induces acne. The aim of our study was to investigate the role of IGF-1 and insulin on the phosphoinositide-3-kinase (PI3K)/Akt/FoxO1 pathway in human primary T cells and on the molecular functions of T cells in vitro. T cells were stimulated with 0.001 μM IGF-1 or 1 μM insulin +/− 20 μM PI3K inhibitor LY294002. T cells were also exposed to SZ95 sebocyte supernatants which were pre-stimulated with IGF-1 or insulin. We found that 0.001 µM IGF-1 and 1 µM insulin activate the PI3K pathway in T cells leading to up-regulation of p-Akt and p-FoxO1 at 15 and 30 minutes. Nuclear FoxO1 was decreased and FoxO transcriptional activity was reduced. 0.001 µM IGF-1 and 1 µM insulin increased T cell proliferation but have no significant effect on Toll-like receptor2/4 (TLR) expression. Interestingly, supernatants from IGF-1- or insulin-stimulated sebocytes activated the PI3K pathway in T cells but reduced T cell proliferation. Taken together, this study helps to support that high glycemic load diet may contribute to induce activation of the PI3K pathway and increase of proliferation in human primary T cells. Factors secreted by IGF-1- and insulin-stimulated sebocytes induce the PI3K pathway in T cells and reduce T cell proliferation, which probably can reflect a protective mechanism of the sebaceous gland basal cells.

Increased Cerebral Tff1 Expression in Two Murine Models of Neuroinflammation.

Background/Aims: The trefoil factor family (TFF) peptide TFF1 is a typical secretory product of the gastric mucosa and a very low level of expression occurs in nearly all regions of the murine brain. TFF1 possesses a lectin activity and binding to a plethora of transmembrane glycoproteins could explain the diverse biological effects of TFF1 (e.g., anti-apoptotic effect). It was the aim to test whether TFF expression is changed during neuroinflammation. Methods: Expression profiling was performed using semi-quantitative RT-PCR analyses in two murine models of neuroinflammation, i.e. Toxoplasma gondii-induced encephalitis and experimental autoimmune encephalomyelitis (EAE), the latter being the most common animal model of multiple sclerosis. Tff1 expression was also localized using RNA in situ hybridization histochemistry. Results: We report for the first time on a significant transcriptional induction in cerebral Tff1 expression in both T. gondii-induced encephalitis and EAE. In contrast, Tff2 and Tff3 expression were not altered. Tff1 transcripts were predominantly localized in the internal granular layer of the cerebellum indicating neuronal expression. Furthermore, also glial cells are expected to express Tff1. Characterization of both experimental models by expression profiling (e.g., inflammasome sensors, inflammatory cytokines, microglial marker Iba1, ependymin related protein 1) revealed differences concerning the expression of the inflammasome sensor Nlrp1 and interleukin 17a. Conclusion: The up-regulated expression of Tff1 is probably the result of a complex inflammatory process as its expression is induced by tumor necrosis factor α as well as interleukins 1β and 17. However on the transcript level, Tff1KO mice did not show any significant signs of an altered immune response after infection with T. gondii in comparison with the wild type animals.

Selective targeting of transforming growth factor-beta1 into TCR/CD28 signalling plasma membrane domains silences T cell activation

TGFβ1 (Transforming Growth Factor-beta1) is a versatile regulator of T cell immune responses. Depending on its context in the immunological environment, TGFβ1 guides T cells toward specific activation programs including TH17 and regulatory T cell activities. Moreover, TGFβ signals function in immune homeostasis by directly attenuating T cell effector activities. We uncovered a novel context under which TGFβ1 stringently and reversibly silences activation responses of resting human T cells to TCR/CD28 stimulating surfaces:Using ligand-presenting beads, TGFβ1 and TCR/CD28-activating signals were directed into defined plasma membrane domains of T cells. Selective targeting of TGFβ1 cytokine into TCR/CD28 signalling plasma membrane domains held back early response of TCR-proximal tyrosine phosphorylation and bead engulfment at activation sites. Consequently, downstream induction of proliferation and cytokine secretion were stringently attenuated. After extended incubation with TGFβ1-presenting beads, silenced T cells became receptive again to activation by renewed TCR/CD28-stimuli, indicating that the unresponsive state of T cells was reverted and did not reflect long-lasting anergy or decrease in T cell viability. These findings outline a new strategy of physically linking TGFβ1 and TCR-activating functions for the treatment of disease and pathological conditions which are caused by unwanted T cell activity.

Zinc aspartate suppresses proliferation and Th1/Th2/Th17 cytokine production of pre-activated human T cells in vitro

The essential trace element zinc, necessary for many biological processes of living organisms, plays a regulatory role in the maintenance of immune functions. Zinc deficiency affects components of both the innate and the adaptive immune system. On the other side, zinc is capable of suppressing activation and proliferation of human T cells. In the present study, we investigated the effect of zinc aspartate (Unizink®), an approved drug to treat zinc deficiency, on pre-activated human T cells (T cell blasts) in vitro. T cells of healthy donors were stimulated for 48 h with anti-CD3/CD28 antibodies. After this time period, zinc aspartate or the immunosuppressive drugs cyclosporin A, dexamethasone, and rapamycin were added for additional 24 h to these cell cultures. Subsequently, T cell proliferation and cytokine production was measured. In contrast to cyclosporine A and dexamethasone, only zinc aspartate and rapamycin were capable of suppressing the proliferation and Th1 (IFN-γ), Th2 (IL-5), and Th17 (IL-17) cytokine production of pre-activated T cells. This data suggest that zinc aspartate has the capacity to suppress proliferation and cytokine production of pre-activated human T cells in vitro. Thus, administration of zinc aspartate may have beneficial effects on T cell-mediated autoimmune diseases.

Der Einfluss des Spurenelements Zink auf das Immunsystem

Zusammenfassung Klinische und experimentelle Untersuchungen haben gezeigt, dass enge Wechselwirkungen zwischen dem essentiellen Spurenelement Zink und dem Immunsystem existieren. Zink beeinflusst die zellulären und humoralen Komponenten sowohl des angeborenen als auch des erworbenen Immunsystems. Zinkmangelzustände beim Menschen sind häufig mit einer gestörten Immunfunktion, also einem sekundären Immundefekt vergesellschaftet. Je nach zugrundeliegender Ursache des Zinkmangels führt eine kontrollierte Zinksubstitution zu einer Normalisierung des Serumzinks, der Zinkhomöostase des Körpers und zu einer Verbesserung der Immunfunktion. In vitro wirken geringe Zinkkonzentrationen stimulierend auf funktionelle Parameter von Immunzellen, höhere Zinkkonzentrationen sind supprimierend oder zytotoxisch für diese Zellen. Arbeiten der letzten Jahre belegen die immunsuppressive Wirkung von Zink in T-Zell-vermittelten autoimmunen Tiermodellen, wie der Experimentellen Autoimmunen Enzephalomyelitis (EAE). Zudem wurde bei einer Reihe von Autoimmunerkrankungen eine Abnahme der Serum-/Plasma-Zinkkonzentrationen nachgewiesen. In zukünftigen klinischen Studien sollte bei diesen Erkrankungen der mögliche Einsatz einer kontrollierten immunsuppressiven Zinktherapie geprüft werden.

The influence of the trace element zinc on the immune system

Abstract Clinical and experimental examinations showed a close relationship between zinc as an essential trace element and the immune system. Thus, cellular and humoral components from both the innate and the adaptive immune system are affected by zinc. Human zinc deficiencies are frequently connected with disturbed immune functions. Controlled zinc substitution results in a normalization of zinc serum levels, zinc homeostasis, and the immunological parameters. As shown in in vitro experiments, low zinc concentrations stimulate functional parameters of immune cells, but high zinc concentrations are suppressive or cytotoxic for these cells. Recently, the immunosuppressive effect of zinc was demonstrated in animal models of T-cell-dependent autoimmune diseases, like experimental autoimmune encephalomyelitis. Moreover, decreased serum/plasma zinc concentrations have been detected in patients with different autoimmune diseases. Prospective studies should verify the possibility of controlled immunosuppressive zinc therapies for these diseases.