The brain reacting to COVID-19: analysis of the cerebrospinal fluid proteome, RNA and inflammation.

Patients with COVID-19 can have a variety of neurological symptoms, but the active involvement of central nervous system (CNS) in COVID-19 remains unclear. While routine cerebrospinal fluid (CSF) analyses in patients with neurological manifestations of COVID-19 generally show no or only mild inflammation, more detailed data on inflammatory mediators in the CSF of patients with COVID-19 are scarce. We studied the inflammatory response in paired CSF and serum samples of patients with COVID-19 (n = 38). Patients with herpes simplex virus encephalitis (HSVE, n = 10) and patients with non-inflammatory, non-neurodegenerative neurological diseases (n = 28) served as controls. We used proteomics, enzyme-linked immunoassays, and semiquantitative cytokine arrays to characterize inflammatory proteins. Autoantibody screening was performed with cell-based assays and native tissue staining. RNA sequencing of long-non-coding RNA and circular RNA was done to study the transcriptome. Proteomics on single protein level and subsequent pathway analysis showed similar yet strongly attenuated inflammatory changes in the CSF of COVID-19 patients compared to HSVE patients with, e.g., downregulation of the apolipoproteins and extracellular matrix proteins. Protein upregulation of the complement system, the serpin proteins pathways, and other proteins including glycoproteins alpha-2 and alpha-1 acid. Importantly, calculation of interleukin-6, interleukin-16, and CXCL10 CSF/serum indices suggest that these inflammatory mediators reach the CSF from the systemic circulation, rather than being produced within the CNS. Antibody screening revealed no pathological levels of known neuronal autoantibodies. When stratifying COVID-19 patients into those with and without bacterial superinfection as indicated by elevated procalcitonin levels, inflammatory markers were significantly (p < 0.01) higher in those with bacterial superinfection. RNA sequencing in the CSF revealed 101 linear RNAs comprising messenger RNAs, and two circRNAs being significantly differentially expressed in COVID-19 than in non-neuroinflammatory controls and neurodegenerative patients. Our findings may explain the absence of signs of intrathecal inflammation upon routine CSF testing despite the presence of SARS-CoV2 infection-associated neurological symptoms. The relevance of blood-derived mediators of inflammation in the CSF for neurological COVID-19 and post-COVID-19 symptoms deserves further investigation.

Plasma concentrations of anti-inflammatory cytokine TGF-ß are associated with hippocampal structure related to explicit memory performance in older adults.

Human cognitive abilities, and particularly hippocampus-dependent memory performance typically decline with increasing age. Immunosenescence, the age-related disintegration of the immune system, is increasingly coming into the focus of research as a considerable factor contributing to cognitive decline. In the present study, we investigated potential associations between plasma levels of pro- and anti-inflammatory cytokines and learning and memory performance as well as hippocampal anatomy in young and older adults. Plasma concentrations of the inflammation marker CRP as well as the pro-inflammatory cytokines IL-6 and TNF-α and the anti-inflammatory cytokine TGF-ß1 were measured in 142 healthy adults (57 young, 24.47 ± 4.48 years; 85 older, 63.66 ± 7.32 years) who performed tests of explicit memory (Verbal Learning and Memory Test, VLMT; Wechsler Memory Scale, Logical Memory, WMS) with an additional delayed recall test after 24 h. Hippocampal volumetry and hippocampal subfield segmentation were performed using FreeSurfer, based on T1-weighted and high-resolution T2-weighted MR images. When investigating the relationship between memory performance, hippocampal structure, and plasma cytokine levels, we found that TGF-ß1 concentrations were positively correlated with the volumes of the hippocampal CA4-dentate gyrus region in older adults. These volumes were in turn positively associated with better performance in the WMS, particularly in the delayed memory test. Our results support the notion that endogenous anti-inflammatory mechanisms may act as protective factors in neurocognitive aging.

Serum and CSF cytokine levels mirror different neuroimmunological mechanisms in patients with LGI1 and Caspr2 encephalitis.

Changes in levels of cytokines or soluble receptors in biological fluids may provide information on immunological pathomechanisms underlying the respective diseases. Here, we studied cytokine patterns of patients with autoimmune encephalitis (AE) before and after immunosuppressive treatment in order to identify possible biomarker candidates and to look for putatively involved pathomechanisms. We performed measurements in Cerebrospinal fluid (CSF) and serum of 7 patients suffering from AE with antibodies (ab) against Leucine-rich glioma-inactivated-protein 1 (LGI1) and 9 AE patients with Contactin-associated protein-like 2 (Caspr2) ab recruited from two tertiary AE centers in Magdeburg and Berlin, Germany. In the Magdeburg samples before and after treatment were available for the measurements and in the Berlin cohort samples were collected after treatment was initiated. First, we used a human cytokine array comprising 36 cytokines or soluble receptors to screen for biomarkers in CSF samples of 8 AE (before and after treatment), 4 herpes-simplex virus meningoencephalitis patients and 4 controls without neuroinflammation. Next, CCL2, CXCl10, CXCl13, Il -6 and sICAM1 were chosen as candidates and measured in CSF and serum with specific ELISA systems in all 16 AE patients, 14 controls without neuroinflammation and 7 herpes-simplex virus meningitis patients. Clinical outcome was assessed via modified Rankin scale. LGI1 and Caspr2 abs from the Magdeburg cohort were purified by chromatography. IgG subclasses of these LGI1 or Caspr2 abs were identified by immunoblot analysis. The levels of most candidate parameters were higher in the CSF of Caspr2 than of LGI1 AE patients and controls, but there were no significant changes of cytokine concentrations before and after initiating treatment. Thus, these parameters seem unsuited as surrogate biomarkers of disease. Significantly higher levels were observed in the CSFs of Caspr2 AE patients (CXCL13 and sICAM1) as well as in the serum of Caspr2 (CXCL10) and LGI1 AE patients (CXCL13) in comparison to control samples. These results suggest that neuro-immunological pathomechanisms may differ between Caspr2 and LGI1 AE patients. Caspr2 AE seems to elicit a higher immune response than LGI1 AE, which has no correlation to the respective IgG subclass combination of AE specific abs involved in each type of disease.

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 1ß 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.

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..

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

TGFß1 (Transforming Growth Factor-beta1) is a versatile regulator of T cell immune responses. Depending on its context in the immunological environment, TGFß1 guides T cells toward specific activation programs including TH17 and regulatory T cell activities. Moreover, TGFß signals function in immune homeostasis by directly attenuating T cell effector activities. We uncovered a novel context under which TGFß1 stringently and reversibly silences activation responses of resting human T cells to TCR/CD28 stimulating surfaces:Using ligand-presenting beads, TGFß1 and TCR/CD28-activating signals were directed into defined plasma membrane domains of T cells. Selective targeting of TGFß1 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 TGFß1-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 TGFß1 and TCR-activating functions for the treatment of disease and pathological conditions which are caused by unwanted T cell activity.

Oral zinc aspartate treats experimental autoimmune encephalomyelitis.

The essential trace element zinc plays a critical role in the regulation of immune homeostasis. Zinc deficiency or excess can cause severe impairment of the immune response, which points to the importance of the physiological and dietary control of zinc levels for a functioning immune system. We previously reported that injection of zinc aspartate suppresses experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), as well as effector T cell functions in vitro. Among the preferred characteristics of novel therapeutics for the treatment of autoimmune diseases such as MS are oral availability and a tolerable effective dose to minimize side effects. In this study, we investigated whether oral administration of zinc aspartate, an approved drug to treat zinc deficiency in humans, is effective in controlling EAE at clinically approved doses. We show that oral administration of 6 µg/day [0.3 mg/kg body weight (BW)] or 12 µg/day [0.6 mg/kg BW] of zinc aspartate reduces clinical and histopathological signs during the relapsing remitting phase of the disease in SJL mice. The clinical effect in mice was accompanied by suppression of IFN-γ, TNF-α, GM-CSF and IL-5 production in stimulated human T cells and mouse splenocytes in a dose-dependent manner. Furthermore, a large array of proinflammatory cytokines was modulated by zinc aspartate exposure in vitro. These data suggest that administration of oral zinc aspartate may have beneficial effects on autoimmune diseases like MS.

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.

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.

Zinc aspartate induces proliferation of resting and antigen-stimulated human PBMC under high-density cell culture condition.

BACKGROUND: Zinc, one of the most important essential trace elements in the human body, regulates a wide range of cellular functions of immune cells, such as proliferation, differentiation and survival. Zinc deficiency affects both the innate and adaptive immune system. Zinc supplementation was discussed as possible therapy for infectious diseases and T cell-mediated autoimmune diseases. However, the influence of commercial zinc preparations on proliferation and cytokine production of resting and antigen-stimulated peripheral blood mononuclear cells (PBMC) has not yet been completely investigated. METHODS: Here, we examined whether zinc aspartate (Unizink®), an approved drug to treat zinc deficiency in patients, induces proliferation, cytokine production, and induction of apoptosis/caspase 3/7 activity of resting PBMC under high-density cell culture condition. In addition, we performed antigen-specific proliferation experiments, where PBMCs of healthy donors vaccinated against Influenza A (H1N1) and/or SARS-CoV-2 were stimulated with Influenza A (H1N1) peptides or SARS-CoV-2 peptides as well as the Mixed Lymphocyte Culture (MLC) in the presence of increasing concentrations of zinc aspartate. RESULTS: We observed a dose-dependent enhancement of proliferation and induction of cytokine production (IFN-γ, IL-5, GM-CSF and CXCL10) of resting PBMC in presence of zinc aspartate. The number of cells with active caspase 3/7 and, consecutively, the amount of cells undergoing apoptosis steadily decreased in presence of zinc aspartate. Moreover, zinc aspartate was capable of stimulating antigen-specific PBMC proliferation using MLC or influenza A (H1N1) and SARS-CoV-2 peptides in both a dose-dependent and a donor-specific manner. In the absence of zinc aspartate, we clearly could discriminate two groups of responders: low and high responders to antigenic stimulation. The addition of increasing concentration of zinc aspartate significantly stimulated the proliferation of PBMC from low responders, but not from high responders. CONCLUSION: Taken together, our results suggest that zinc aspartate induces the proliferation of resting and antigen-stimulated PBMCs under high-density cell culture conditions. Thus, zinc might represent a supportive treatment in patients suffering from infectious diseases.

Zinc aspartate suppresses T cell activation in vitro and relapsing experimental autoimmune encephalomyelitis in SJL/J mice.

Zinc is an essential trace element with a critical role in normal growth and development and in immune homeostasis. Zinc deficiency impairs both the innate and the adaptive immune system and can be normalized by zinc supplementation. On the other end of the spectrum, high dosages of zinc diminish immune cell functions similar to zinc deficiency. Here, we investigated the influence of zinc aspartate on proliferation and cytokine production of stimulated human T cells and mouse splenocytes in vitro. Furthermore, the effect of zinc aspartate was examined in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of Multiple Sclerosis (MS) with a Th1/Th17 T cell-mediated immunopathogenesis. Zinc aspartate suppressed proliferation as well as IL-2, IL-10 and IL-17 production in stimulated human T cells and mouse splenocytes. Importantly, administration of a medium range dose of 30 µg/day zinc aspartate [1.5 mg/kg body weight (BW)] in a therapeutic manner led to a significant reduction of the clinical severity of the EAE during the first relapse of the disease. A lower zinc aspartate dose (6 µg/day, 0.3 mg/kg BW) had no significant therapeutic effect on the severity of the EAE, while administration of higher zinc aspartate amounts (120 µg/day, 6 mg/kg BW) led to more severe disease. Taken together, our data suggest that zinc aspartate can modulate activation, proliferation and cytokine production of effector T cells in vitro and in vivo and that activated autoreactive T cells may be potential therapeutic targets of tightly controlled zinc supplementation in autoimmune diseases like MS.

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.

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.

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.

Immune Cell-Type Specific Ablation of Adapter Protein ADAP Differentially Modulates EAE.

The cytosolic adhesion and degranulation-promoting adapter protein ADAP is expressed in various hematopoietic cells including T cells, NK cells, myeloid cells, and platelets but absent in mature B cells. The role of ADAP in T cell activation, proliferation and integrin activation is well-accepted. We previously demonstrated that conventional ADAP knockout mice show a significantly attenuated course of experimental autoimmune encephalomyelitis (EAE). To dissect the impact of different ADAP expressing cell populations on the reduced EAE severity, here, we generated lineage-specific conditional knockout mice. ADAP was deleted in T cells, myeloid cells, NK cells and platelets, respectively. Specific loss of ADAP was confirmed on the protein level. Detailed immunophenotyping was performed to assess the consequence of deletion of ADAP with regard to the maturation and distribution of immune cells in primary and secondary lymphoid organs. The analysis showed equivalent results as for conventional ADAP knockout mice: impaired thymocyte development in ADAPfl/fl Lck-Cre mice, normal NK cell and myeloid cell distribution in ADAPfl/fl NKp46-Cre mice and ADAPfl/fl LysM-Cre mice, respectively as well as thrombocytopenia in ADAPfl/fl PF4-Cre mice. Active EAE was induced in these animals by immunization with the myelin oligodendrocyte glycoprotein35-55 peptide. The clinical course of EAE was significantly milder in mice with loss of ADAP in T cells, myeloid cells and NK cells compared to ADAP-sufficient control littermates. Surprisingly, specific deletion of ADAP in platelets resulted in a more exacerbated disease. These data show that T cell-independent as well as T cell-dependent mechanisms are responsible for the complex phenotype observed in conventional ADAP knockout mice.

Characterization of Mice with a Platelet-Specific Deletion of the Adapter Molecule ADAP.

The adhesion and degranulation-promoting adapter protein (ADAP) is expressed in T cells, NK cells, myeloid cells, and platelets. The involvement of ADAP in the regulation of receptor-mediated inside-out signaling leading to integrin activation is well characterized, especially in T cells and in platelets. Due to the fact that animal studies using conventional knockout mice are limited by the overlapping effects of the different ADAP-expressing cells, we generated conditional ADAP knockout mice (ADAPfl/fl PF4-Cretg) (PF4, platelet factor 4). We observed that loss of ADAP restricted to the megakaryocytic lineage has no impact on other hematopoietic cells even under stimulation conditions. ADAPfl/fl PF4-Cretg mice showed thrombocytopenia in combination with reduced plasma levels of PF4 and transforming growth factor ß1 (TGF-ß1). In vitro, platelets from these mice revealed reduced P-selectin expression, lower levels of TGF-ß1 release, diminished integrin αIIbß3 activation, and decreased fibrinogen binding after stimulation with podoplanin, the ligand of C-type lectin-like receptor 2 (CLEC-2). Furthermore, loss of ADAP was associated with impaired CLEC-2-mediated activation of phospholipase Cγ2 (PLCγ2) and extracellular signal-regulated kinase 1/2 (ERK1/2). Induction of experimental autoimmune encephalomyelitis (EAE) in mice lacking ADAP expression in platelets caused a more severe disease. In vivo administration of TGF-ß1 early after T cell transfer reduced EAE severity in mice with loss of ADAP restricted to platelets. Our results reveal a regulatory function of ADAP in platelets in vitro and during autoimmune disease EAE in vivo.

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.

Insulin and Insulin-like growth factor-1 can activate the phosphoinositide-3-kinase /Akt/FoxO1 pathway in T cells in vitro.

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.

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.