Pitavastatin Is a Highly Potent Inhibitor of T-Cell Proliferation.

Repositioning of approved drugs is an alternative time- and cost-saving strategy to classical drug development. Statins are 3-hydroxy-3-methylglutaryl-CoA (HMG CoA) reductase inhibitors that are usually used as cholesterol-lowering medication, and they also exhibit anti-inflammatory effects. In the present study, we observed that the addition of Pitavastatin at nanomolar concentrations inhibits the proliferation of CD3/CD28 antibody-stimulated human T cells of healthy donors in a dose-dependent fashion. The 50% inhibition of proliferation (IC50) were 3.6 and 48.5 nM for freshly stimulated and pre-activated T cells, respectively. In addition, Pitavastatin suppressed the IL-10 and IL-17 production of stimulated T cells. Mechanistically, we found that treatment of T cells with doses <1 µM of Pitavastatin induced hyperphosphorylation of ERK1/2, and activation of caspase-9, -3 and -7, thus leading to apoptosis. Mevalonic acid, cholesterol and the MEK1/2 inhibitor U0126 reversed this Pitavastatin-mediated ERK1/2 activation and apoptosis of T cells. In summary, our results suggest that Pitavastatin is a highly potent inhibitor of T-cell proliferation, which induces apoptosis via pro-apoptotic ERK1/2 activation, thus representing a potential repositioning candidate for the treatment of T-cell-mediated autoimmune diseases.

Zinc Aspartate Induces IL-16 Secretion and Apoptosis in Human T Cells.

T cell activation mediates immunity to pathogens. On the flipside, T cells are also involved in pathological immune responses during chronic autoimmune diseases. We recently reported that zinc aspartate, a registered drug with high bioavailability, dose-dependently inhibits T cell activation and Th1/Th2/Th17 cytokine production of stimulated human and mouse T cells. To understand the suppressive effect of zinc on T cell function, we here investigated the influence of zinc aspartate on human T cells focusing on the secretion of immunosuppressive cytokines, induction of apoptosis, and caspase 3/7 activity. To this end, we monitored either freshly stimulated or pre-activated human T cells in the presence of zinc aspartate from 40-140 µM over a period of 72 h. Under both experimental conditions, we observed a dose-dependent suppression of human T cell proliferation. While IL-1ra, latent TGF-ß1, and IL-10 were dose-dependently reduced, we, unexpectedly, detected elevated levels of IL-16 upon zinc supplementation. In addition, the number of cells with active caspase 3/7 and, consecutively, the amount of cells undergoing apoptosis, steadily increased at zinc aspartate concentrations exceeding 100 µM. Taken together, our findings suggest that zinc aspartate impairs T cell fitness and might be beneficial for the treatment of T cell-mediated autoimmune diseases.

Impact of Different JAK Inhibitors and Methotrexate on Lymphocyte Proliferation and DNA Damage.

Janus kinase inhibitors (JAKis) represent a new strategy in rheumatoid arthritis (RA) therapy. Still, data directly comparing different JAKis are rare. In the present in vitro study, we investigated the immunomodulatory potential of four JAKis (tofacitinib, baricitinib, upadacitinib, and filgotinib) currently approved for RA treatment by the European Medicines Agency. Increasing concentrations of JAKi or methotrexate, conventionally used in RA therapy, were either added to freshly mitogen-stimulated or preactivated peripheral blood mononuclear cells (PBMC), isolated from healthy volunteers. A comparable, dose-dependent inhibition of lymphocyte proliferation was observed in samples treated with tofacitinib, baricitinib, and upadacitinib, while dosage of filgotinib had to be two orders of magnitude higher. In contrast, antiproliferative effects were strongly attenuated when JAKi were added to preactivated PBMCs. High dosage of upadacitinib and filgotinib also affected cell viability. Further, analyses of DNA double-strand break markers γH2AX and 53BP1 indicated an enhanced level of DNA damage in cells incubated with high concentrations of filgotinib and a dose-dependent reduction in clearance of radiation-induced γH2AX foci in the presence of tofacitinib or baricitinib. Thereby, our study demonstrated a broad comparability of immunomodulatory effects induced by different JAKi and provided first indications, that (pan)JAKi may impair DNA damage repair in irradiated PBMCs.

Screening of FDA-Approved Drug Library Identifies Adefovir Dipivoxil as Highly Potent Inhibitor of T Cell Proliferation.

Repositioning of approved drugs for identifying new therapeutic purposes is an alternative, time and cost saving strategy to classical drug development. Here, we screened a library of 786 FDA-approved drugs to find compounds, which can potentially be repurposed for treatment of T cell-mediated autoimmune diseases. Investigating the effect of these diverse substances on mitogen-stimulated proliferation of both, freshly stimulated and pre-activated (48 h) peripheral blood mononuclear cells (PBMCs), we discovered Adefovir Dipivoxil (ADV) as very potent compound, which inhibits T cell proliferation in a nanomolar range. We further analyzed the influence of ADV on proliferation, activation, cytokine production, viability and apoptosis of freshly stimulated as well as pre-activated human T cells stimulated with anti-CD3/CD28 antibodies. We observed that ADV was capable of suppressing the proliferation in both T cell stimulation systems in a dose-dependent manner (50% inhibition [IC50]: 63.12 and 364.8 nM for freshly stimulated T cells and pre-activated T cells, respectively). Moreover, the drug impaired T cell activation and inhibited Th1 (IFN-γ), Th2 (IL-5), and Th17 (IL-17) cytokine production dose-dependently. Furthermore, ADV treatment induced DNA double-strand breaks (γH2AX foci expression), which led to an increase of p53-phospho-Ser15 expression. In response to DNA damage p21 and PUMA are transactivated by p53. Subsequently, this caused cell cycle arrest at G0/G1 phase and activation of the intrinsic apoptosis pathway. Our results indicate that ADV could be a new potential candidate for treatment of T cell-mediated autoimmune diseases. Prospective studies should be performed to verify this possible therapeutic application of ADV for such disorders.

The potential toxic impact of different gadolinium-based contrast agents combined with 7-T MRI on isolated human lymphocytes.

BACKGROUND: To investigate a potentially amplifying genotoxic or cytotoxic effect of different gadolinium-based contrast agents (GBCAs) in combination with ultra-high-field 7-T magnetic resonance imaging (MRI) exposure in separated human peripheral blood lymphocytes. METHODS: This in vitro study was approved by the local ethics committee and written informed consent was obtained from all participants. Isolated lymphocytes from twelve healthy donors were incubated with gadobutrol, gadoterate meglumine, gadodiamide, gadopentetate dimeglumine, or gadoxetate either alone or combined with 7-T MRI (1 h). Deoxyribonucleic acid (DNA) double-strand breaks were assessed 15 min after MRI exposure by automated γH2AX foci quantification. Cytotoxicity was determined at later endpoints by Annexin V/propidium iodide apoptosis assay (24 h) and [3H]-thymidine proliferation test (72 h). As a reference, lymphocytes from four different donors were exposed analogously to iodinated contrast agents (iomeprol, iopromide) in combination with computed tomography. RESULTS: Baseline γH2AX levels (0.08 ± 0.02 foci/cell) were not significantly (p between 0.135 and 1.000) enhanced after administration of GBCAs regardless of MRI exposure. In contrast to the two investigated macrocyclic GBCAs, lymphocytes exposed to the three linear GBCAs showed a dose-dependent increase in apoptosis (maximum 186% of unexposed control, p < 0.001) and reduced proliferation rate (minimum 0.7% of unexposed control, p < 0.001). However, additional 7-T MRI co-exposure did not alter GBCA-induced cytotoxicity. CONCLUSIONS: Exposure of lymphocytes to different GBCAs did not reveal significant induction of γH2AX foci, and enhanced cytotoxicity was only observed in lymphocytes treated with the linear GBCAs used in this study, independent of additional 7-T MRI co-exposure.

Impact of in Vivo High-Field-Strength and Ultra-High-Field-Strength MR Imaging on DNA Double-Strand-Break Formation in Human Lymphocytes.

Purpose To determine the impact of different magnetic field strengths (1, 1.5, 3, and 7 T) and the effect of contrast agent on DNA double-strand-break (DSB) formation in patients undergoing magnetic resonance (MR) imaging. Materials and Methods This in vivo study was approved by the local ethics committee, and written informed consent was obtained from each patient. To analyze the level of DNA DSBs, peripheral blood mononuclear cells were isolated from blood samples drawn directly before, as well as 5 minutes and 30 minutes after MR imaging examination. After performing γH2AX immunofluorescence staining, DSBs were quantified with automated digital microscopy. MR group consisted of 43 patients (22 women, 21 men; mean age, 46.1 years; range, 20-77 years) and was further subdivided according to the applied field strength and administration of contrast agent. Additionally, 10 patients undergoing either unenhanced or contrast material-enhanced computed tomography (CT) served as positive control subjects. Statistical analysis was performed with Friedman test. Results Whereas DSBs in lymphocytes increased after CT exposure (before MR imaging: 0.14 foci per cell ± 0.05; 5 minutes after: 0.26 foci per cell ± 0.07; 30 minutes after: 0.24 foci per cell ± 0.07; P ≤ .05), no alterations were observed in patients examined with MR imaging (before MR imaging: 0.13 foci per cell ± 0.02; 5 minutes after: 0.12 foci per cell ± 0.02; 30 minutes after: 0.11 foci per cell ± 0.02; P > .05). Differentiated analysis of MR imaging subgroups again revealed no significant changes in γH2AX level. Conclusion Analysis of γH2AX foci showed no evidence of DSB induction after MR examination, independent of the applied field strength and administration of gadolinium-based contrast agent.

DNA double-strand breaks and micronuclei in human blood lymphocytes after repeated whole body exposures to 7T Magnetic Resonance Imaging.

PURPOSE: To examine the extent of genetic damage, assessed from deoxyribonucleic acid (DNA) double-strand breaks (DSBs) and micronuclei (MN) in peripheral blood mononuclear cells obtained from individuals repeatedly exposed to 7T Magnetic Resonance Imaging (MRI). MATERIALS AND METHODS: The study protocol was approved by the local ethics committee. Informed consent was obtained from 22 healthy, non-smoking, non-alcoholic male individuals, who had never undergone radio-/chemo-therapy, scintigraphy, and had not undergone X-ray examination one year prior blood withdrawal. Eleven participants were repeatedly exposed to 7T and 3T MRI while working with/around scanners or frequently participating as 7T and lower field MRI research subjects (mean age 34±7years). The other half was never exposed to 7T or lower field MRI and served as controls (mean age 33±9years). The damage in lymphocytes was assessed using anti-γH2AX immunofluorescence staining of DNA DSBs and by quantification of MN. Isolated cells were further exposed in vitro to 7T MRI either alone or in the presence of the DNA damaging drug etoposide, to determine if there is any additional combined effect. The kinetics of DNA damage repair were examined. RESULTS: The mean base-level of γH2AX foci/cell and incidence of MN between repeatedly exposed and control group were not significantly different (P=0.618 and P=0.535, respectively). The additional in vitro exposure of cells to 7T MRI had no significant impact on MN frequencies and γH2AX foci at 1, 20 and 72h after exposure. CONCLUSION: Frequently repeated 7T MRI exposure did not result in a detectable increase in genotoxicity indices and alterations of DNA repair kinetics.

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.

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..