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Here we show that an approach of in-vitro transcribed mRNA nucleofection expands the range of transfection of primary human T cells. It represents a reproducible and time-efficient technology, and is thus an ideal tool in basic research involving highly controlled in-vitro experiments with a gene of interest aiming at identifying its biological human T cell function., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The hair follicle (HF) is a continuously remodeled mini organ that cycles between growth (anagen), regression (catagen), and relative quiescence (telogen). As the anagen-to-catagen transformation of microdissected human scalp HFs can be observed in organ culture, it permits the study of the unknown controls of autonomous, rhythmic tissue remodeling of the HF, which intersects developmental, chronobiological, and growth-regulatory mechanisms. The hypothesis that the peripheral clock system is involved in hair cycle control, i.e., the anagen-to-catagen transformation, was tested. Here we show that in the absence of central clock influences, isolated, organ-cultured human HFs show circadian changes in the gene and protein expression of core clock genes (CLOCK, BMAL1, and Period1) and clock-controlled genes (c-Myc, NR1D1, and CDKN1A), with Period1 expression being hair cycle dependent. Knockdown of either BMAL1 or Period1 in human anagen HFs significantly prolonged anagen. This provides evidence that peripheral core clock genes modulate human HF cycling and are an integral component of the human hair cycle clock. Specifically, our study identifies BMAL1 and Period1 as potential therapeutic targets for modulating human hair growth.

The enzyme-triggered release of the antimicrobial agent octenidine out of poly(l-lactide)-based nanoparticles (PLLA-NPs) and their in vitro antibacterial activities in the presence of gram-positive and gram-negative bacteria are presented. The formation of the nanoparticles was achieved using a combination of the solvent evaporation and the miniemulsion approach. For the stabilization of the polymeric nanoparticles, non-ionic polymers (polyvinylalcohol [PVA], hydroxyethyl starch [HES], human serum albumin [HSA]) were successfully used for enzymatic degradation; ionic surfactants such as sodium dodecyl sulfate and cetyltrimethylammonium chloride inhibited the enzymatic degradation. The change in pH, size, size distribution and morphology during the degradation process of PLLA-NPs and the release of the antimicrobial agent was studied. The influence of the different amounts of octenidine and of the different stabilizers on the NPs' stability, size, size distribution, morphology, zeta potential and on the surface group's density is discussed. Fluorescently labeled HES-stabilized PLLA-NPs are immobilized by colloidal electrospinning. The observed data from HPLC measurements show that octenidine is released out of PLLA-NPs which are stabilized with PVA, HES or HSA. In bacteria tests the PLLA nanoparticles showed a greater ability to inhibit the growth of Staphylococcus aureus compared to Escherichia coli., From the Clinical Editor: This article discusses the enzyme-triggered release and antibacterial effects of octenidine from poly(l-lactide)-based nanoparticles demonstrating the viability of this approach for potential future antibacterial therapy., (© 2013.)

Dexamethasone (DXM) is a synthetic glucocorticoid with anti-inflammatory properties. Targeted delivery of dexamethasone to inflammatory cells, e.g. macrophages and Kupffer cells represents a promising approach to minimize side effects. The aim of the present study was to induce a targeted transport of novel DXM-based biodegradable nanocapsules to phagocytic cells. Nanocapsules (NCs) consisting of a hydroxyethylated glucose polymer (hydroxyethyl starch, HES) shell with encapsulated DXM and NCs synthesized exclusively in inverse miniemulsion out of DXM were investigated. Non-parenchymal murine liver cells served as target cells. HES-DXM NCs were predominantly incorporated by Kupffer cells (KCs). In contrast, DXM NCs were phagocytized by KCs and endothelial cells. The release of the NC-content was confirmed by incorporation of CellTracker™ into the NCs. Uptake of DXM NCs by Kupffer cells reduced significantly the release of inflammatory cytokines in response to LPS stimulation. Importantly, the DXM NCs consisting exclusively out of a dexamethasone shell offer the potential to serve as carriers for additional therapeutics., From the Clinical Editor: In this paper, nanocapsule-based targeted delivery of dexamethasone to inflammatory cells is presented as a promising approach to minimize side effects and increase efficacy of this anti-inflammatory clinically used corticosteroid., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Drug-eluting-stents (DES) reduce clinical restenosis, but have mostly failed to demonstrate a reduction in death or myocardial infarction. The aim of this study was to evaluate the prognostic impact of the introduction of DES in patients undergoing multivessel percutaneous coronary intervention (MV-PCI)., Methods: Survival was assessed in 679 consecutive patients, who underwent PCI in at least two main vessels. Follow-up was available in 667 patients (98%) with a mean follow-up of 4.8 ± 2.5 years. We compared several scenarios: firstly, patients receiving at least one DES (≥ 1 DES group) vs. bare metal stent (BMS)-only patients (BMS only); secondly, the population was divided into a pre-DES-era (2000-2003; N=257) and a DES-era (2004-2006; N=422)., Results: 316 patients (47%) were treated for acute myocardial infarction (MI; N=176 ST-elevation MI; N=140 non-ST-elevation MI). On average, 2.2 ± 0.4 vessels were treated and 212 patients received at least one DES. The DES group showed a higher number of diseased (2.5 ± 0.6 vs. 2.4 ± 0.5; p=0.02) and treated vessels (2.2 ± 0.5 vs. 2.1 ± 0.3; p<0.01) and received more stents (3.3 ± 1.4 vs. 3.0 ± 1.1; p<0.01). The BMS group presented more frequently with acute MI (55% vs. 29%; p<0.01). The DES group showed more complex disease as evidenced by a higher SYNTAX-Score (17.4 ± 8.5 vs. 14.5 ± 8.3; p<0.01). Restricting the survival analysis to patients with stable coronary artery disease, a significant prognostic advantage was found for patients received at least one DES compared to the BMS group (hazard ratio 0.58, 95% confidence interval 0.34-0.99) in the multivariate cox-regression-analysis., Conclusion: The introduction of DES leads to extension of treatment to more complex patients. The use of DES is associated with improved survival in stable patients undergoing MV-PCI., (Copyright © 2012 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

The human hair cycle is a complex, dynamic organ-transformation process during which the hair follicle repetitively progresses from a growth phase (anagen) to a rapid apoptosis-driven involution (catagen) and finally a relative quiescent phase (telogen) before returning to anagen. At present no theory satisfactorily explains the origin of the hair cycle rhythm. Based on experimental evidence we propose a prototypic model that focuses on the dynamics of hair matrix keratinocytes. We argue that a plausible feedback-control structure between two key compartments (matrix keratinocytes and dermal papilla) leads to dynamic instabilities in the population dynamics resulting in rhythmic hair growth. The underlying oscillation consists of an autonomous switching between two quasi-steady states. Additional features of the model, namely bistability and excitability, lead to new hypotheses about the impact of interventions on hair growth. We show how in silico testing may facilitate testing of candidate hair growth modulatory agents in human HF organ culture or in clinical trials., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Survival in patients with laryngeal cancer has not increased remarkably within the last years. It is presumed that a variety of factors act jointly in predicting survival after diagnosis: tumour stage, tumour site, treatment approaches, age and comorbidities. The aim of this German clinical multi-centre study is to present results from multivariate analysis. A retrospective cohort study was conducted in four hospitals in South-West Germany. Incident cases with laryngeal squamous cell carcinoma were included for the years 1998 to 2004, resulting in a population sample of 594 patients. Multivariate regression analysis was performed using the Cox proportional hazards model. Patients were followed up for 64.1months on average. Overall 5-year survival was 66% (95% confidence interval (CI): 62-70%). The strongest risk factors in multivariate analysis were age at first diagnosis (hazard ratio (HR): 1.5; 95% CI: 1.5-1.7 per each additional 10years), tumour stage, and the development of recurrences (HR 3.1; 95% CI: 2.3-4.2) or second primary carcinomas (HR 2.1; 95% CI: 1.4-3.1). A somewhat weaker effect was shown for patients with comorbidities (using Charlson's comorbidity index). The choice of treatment did not strongly affect survival when adjusting for other factors, possibly because the optimal treatment approach was applied for the specific constitution and requirements of each patient. For future research it would be desirable to study the effect of treatment on quality of life in multivariate analysis as well as other modifiable risk factors as smoking and drinking reduction or cessation after diagnosis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The Ca(2+) dependent transcription factor family known as nuclear factor of activated T cells (NFAT) has been shown to be important in T-cell immune responses. Because NFAT proteins have a weak DNA-binding capacity, they cooperate with other transcription factors at composite sites within the promoters of target genes. Recently, NFAT was shown to also be important for the induction of specific genetic programs that guide the differentiation and effector or regulatory activities of CD4(+) T helper subsets via the transcriptional regulation of their lineage-specific transcription factors, specifically T-bet (Th1), Gata3 (Th2), RORgammat (Th17), and Foxp3 (iTregs). In addition, the NFAT family governs the transcription of several signature cytokines, including their cytokine receptors. Subsequently, the integration of these complex intracellular signal transduction cascades is considered to critically determine the crosstalk between the T-cell receptor and receptors that are activated by both the adaptive and innate immune systems to determine pathways of T helper cell differentiation and function. Here, we carefully review the critical role of the established transcriptional partners and functional outcomes of these NFAT interactions in regard to the effector responses of these clinically relevant CD4(+) T helper subsets.

The basic mechanisms of serine/threonine protein kinase signaling networks have been elucidated in the past decade. Members of the protein kinase C (PKC) family are crucial in T cell signaling pathways. Particularly, PKC alpha, PKC beta, and PKC theta isotypes determine the nature of lymphocyte-specific in vivo effector responses. Therefore, PKC isotypes are validated drug targets in adaptive immunity. Selective PKC kinase inhibitors have been discovered and are currently in clinical development, where they may provide new therapeutic options for different immune disorders. Here we review the topic of PKC pathway activity in the regulation of T lymphocytes both in the cytokine response and adhesive capacity, and review recent results with PKC inhibitors in vitro and in vivo.

The antigen-specific interaction of a T cell with an antigen-presenting cell (APC) results in the formation of an immunologic synapse (IS) between the membranes of the 2 cells. beta(2) integrins on the T cell, namely, leukocyte function-associated antigen 1 (LFA-1) and its counter ligand, namely, immunoglobulin-like cell adhesion molecule 1 (ICAM-1) on the APC, critically stabilize this intercellular interaction. The small GTPase Rap1 controls T-cell adhesion through modulating the affinity and/or spatial organization of LFA-1; however, the upstream regulatory components triggered by the T-cell receptor (TCR) have not been resolved. In the present study, we identified a previously unknown function of a protein kinase C- theta (PKC-theta)/RapGEF2 complex in LFA-1 avidity regulation in T lymphocytes. After T-cell activation, the direct phosphorylation of RapGEF2 at Ser960 by PKC- theta regulates Rap1 activation as well as LFA-1 adhesiveness to ICAM-1. In OT-II TCR-transgenic CD4(+) T cells, clustering of LFA-1 after antigen activation was impaired in the absence of PKC- theta. These data define that, among other pathways acting on LFA-1 regulation, PKC- theta and its effector RapGEF2 are critical factors in TCR signaling to Rap1. Taken together, PKC- theta sets the threshold for T-cell activation by positively regulating both the cytokine responses and the adhesive capacities of T lymphocytes.

Objective: To introduce a sound synthesis tool for human EEG rhythms that is applicable in real time., Methods: We design an event-based sonification which suppresses irregular background and highlights normal and pathologic rhythmic activity., Results: We generated sound examples with rhythms from well-known epileptic disorders and find stereotyped rhythmic auditory objects in single channel and stereo display from generalized spike-wave runs. For interictal activity, we were able to separate focal rhythms from background activity and thus enable the listener to perceive its frequency, duration, and intensity while monitoring., Conclusions: The proposed event-based sonification allows quick detection and identification of different types of rhythmic EEE events in real time and can thus be used to complement visual displays in monitoring and EEG feedback tasks., Significance: The significance of the work lies in the fact that it can be implemented for on-line monitoring of clinical EEG and for EEG feedback applications where continuous screen watching can be substituted or improved by the auditory information stream.

We here investigate the crosstalk of PKC and PKA signaling during primary CD3(+) T-lymphocyte activation using pharmacologic inhibitors and activators in combination with our established panel of PKC isotype-deficient mouse T cells in vitro. PKCtheta and PKA inversely affect the CD3/CD28-induced IL-2 expression, whereas other PKC isotypes are dispensable in this signaling pathway. Gene ablation of PKCtheta selectively results in a profound reduction of IL-2 production; however, complete abrogation of IL-2 production in these PKCtheta(-/-) T cells was achieved only by simultaneous coactivation of the cAMP/PKA pathway in CD3(+) T cells. Conversely, the reduced IL-2 production in PKC inhibitor-treated T cells can be rescued by inhibition of the cAMP/PKA pathway in wild-type but not in PKCtheta(-/-) T cells. Mechanistically, the cAMP/PKA and PKCtheta pathways converge at the level of NF-AT, as shown by DNA binding analysis. The combined increase in PKA and decrease in PKCtheta activity leads to an enhanced inhibition of nuclear NF-AT translocation. This PKCtheta/PKA crosstalk significantly affects neither the NF-kappaB, the AP-1, nor the CREB pathways. Taken together, this opposite effect between the positive PKCtheta and the negative cAMP/PKA signaling pathways appears rate limiting for NF-AT transactivation and IL-2 secretion responses of CD3(+) T lymphocytes.

Activated cell-mediated immunity is known to be accompanied by elevated concentrations of 7,8-dihydroneopterin which in high concentrations was found to interfere with the oxidant-antioxidant balance. In this study we investigated whether 7,8-dihydroneopterin mediates apoptosis of Jurkat T-lymphocytes via a CrmA- or Bcl-2-sensitive pathway. Transient transfection assays with CrmA and Bcl-2 expression constructs showed that apoptosis was not affected by CrmA whereas it was significantly decreased upon cotransfection with Bcl-2 constructs. Results suggest that 7,8-dihydroneopterin-induced apoptosis of T-lymphocytes is mediated by a Bcl-2-sensitive pathway.

Interferon-gamma is a cytokine released in large amounts during cell-mediated immune response. It induces the expression of proinflammatory cytokines and enhances macrophage capacity to secrete reactive oxygen intermediates and the pteridines neopterin and 7,8-dihydroneopterin. To assay the role of these pteridines in the immune system several studies were performed. Thereby, 7,8-dihydroneopterin was found to induce apoptosis in T lymphocytes. In this study we report that caspases are involved in 7,8-dihydroneopterin-mediated apoptosis in Jurkat T cells. In connection with this result we found that 7,8-dihydroneopterin can increase Fas ligand expression detected in Western blot analysis and promoter reporter assays. Antioxidants potently reduced the effect of 7,8-dihydroneopterin on Fas ligand promoter activation suggesting an involvement of oxidative stress. In further investigations, ESR-measurements were performed to evaluate the role of 7,8-dihydroneopterin in the formation of radicals. We found that the pteridine in combination with the spin trap DMPO induces the production of DMPO-OH spin adducts. This reaction was sensitive to the presence of chelated metal ions and could completely be blocked by the addition of superoxide dismutase. These data suggest that 7,8-dihydroneopterin in aqueous solution leads to the formation of .OH radicals via generation of superoxide anion. We hypothesize that an overproduction of radicals caused by high levels of 7,8-dihydroneopterin is likely to be responsible for the pro-apoptotic effects observed in cell cultures and possibly contributes to the pathogenesis of diseases involving immune activation and elevated concentrations of neopterin-derivatives.

Protein kinase C (PKC)-&theta;, a serine/threonine protein kinase and novel PKC subfamily member, has been recently identified as an essential component of the T cell synapse which activates the NF-kB signaling cascade leading to expression of the IL-2 gene during T cell activation. By RNA in situ hybridization to whole-body embryo sections it is shown that the murine PKC&theta; is specifically expressed in tissues with hematopoietic and lymphopoietic activity. Expression is also evident in skeletal muscle. A further highly specific expression was observed in the peripheral and central nervous system which is described in detail. Expression in the brain persists up to adult stages.

A biochemical oscillator involving feedback inhibition can be extended to generate a chaotic attractor. The simple chaotic attractor may be exploited to create signals of higher dimension by passive forcing of other reactions. Diffusively coupled oscillators with negative feedback exhibit regular and irregular patterns in time and space associated with periodic and hyperchaotic attractors, respectively.Copyright 1998 Academic Press

Lipoprotein(a) [Lp(a)], which has been shown to interact with fibrin(ogen) and other components of the blood clotting cascade, is a major independent risk factor for atherothrombotic disease in humans. The physiological function(s) of Lp(a), as well as the precise mechanism(s) by which high plasma levels of Lp(a) increase risk are unknown. Identification of further potential apo(a)-protein ligands may be crucial to illuminate apo(a)'s function(s) and pathophysiological properties. We used the repetitive apo(a) kringle IV type 2, which is variable in number in apo(a), to screen a human liver cDNA library by the yeast two-hybrid interaction trap system. Among 11 positive clones that emerged from the screen, eight clones were identified as beta-2 glycoprotein I and one as fibronectin. Coimmunoprecipitation experiments confirmed that beta-2 glycoprotein I and apo(a)/Lp(a) interact in human plasma and in cell culture supernatants of COS-1 cells, which ectopically expressed apo(a). The apo(a)-beta2-glycoprotein I interaction indicates new potential roles for Lp(a) in fibrinolysis and autoimmunity.

Crosslinking of the CD4 coreceptor can inhibit subsequent T-cell activation via the T-cell antigen receptor (TCR)/CD3 complex. The ability of the human immunodeficiency virus (HIV) envelope protein, gp 120, to cause similar inhibition has implicated this inhibitory signal in the induction of T-cell anergy and apoptosis observed in the acquired immunodeficiency syndrome (AIDS). In order to clarify the biochemical basis of this inhibition, we analyzed the effect of CD4 ligation on early signaling events induced by subsequent CD3xCD4 co-crosslinking. By comparison with CD3 crosslinking alone, CD3xCD4 co-crosslinking of a CD3+CD4+ human T-cell leukemia line (SupT1) resulted in an enhanced increase in free intracellular calcium concentration and tyrosine phosphorylation of several cellular substrates, including the prominent phosphorylation of an unidentified 120-kDa protein (p120). Prior CD4 ligation inhibited these responses. Similar results were obtained with A3.01, another CD3+CD4+ T leukemic line. However, P120 was only minor phosphorylated on tyrosine upon receptor crosslinking in A2.01/CD4(-cyt401), a derivative line expressing a truncated CD4 coreceptor lacking its cytoplasmic domain which binds the p56lck protein tyrosine kinase (PTK). Furthermore, prior CD4 ligation failed to inhibit in this line the increased tyrosine phosphorylation induced by subsequent CD3xCD4 co-crosslinking. Thus, prior CD4 crosslinking, or expression of truncated CD4, are both associated with reduced p120 phosphorylation. These results suggest that p120 is a p56lck substrate playing an important role during T-cell activation.

Two novel tyrosine kinase cDNAs were obtained from murine mast cells. These kinases, Emb and Emt, constitute a novel tyrosine kinase subfamily which may also include Tec, a kinase preferentially expressed in liver, and Dsrc28, a fruit fly kinase. Both lack hydrophobic stretches characteristic of the transmembrane domains found in growth factor receptor tyrosine kinases and carboxyl-terminal, negative regulatory tyrosine residue found in Src family kinases. In addition to the Src homology region 2 (SH2) and SH3 domains characteristic of the Src family kinases and other signaling molecules, Emb and Emt share a similar amino-terminal domain comprised mainly of two repeat segments. The emb 2.7-kb transcript was expressed in mast cells, myeloid cells and B lymphocytes while the emt 4.6-kb mRNA in mast cells, myeloid cells and T lymphocytes. The evidence for in vitro tyrosine kinase activity of Emb and Emt proteins is also provided.

A comparison of the efficiencies of hydrophobic interaction chromatography, ion-exchange chromatography, reversed-phase chromatography and gel permeation chromatography in the separation of tear proteins was made using a variety of different buffers. Separation of immunoglobulins, lactoferrin, albumin, PMFA (protein migrating faster than albumin) and lysozyme was accomplished by gel permeation chromatography in less than 30 min using a TSK-type SW3000 column equilibrated with ammonium acetate buffer (pH 4.1) with a high reproducibility. When gel permeation chromatography was used as a completely automated diagnostic method, only minute volumes (1.0 microliter) of tear samples were necessary for the quantitative analysis of proteins. The other three methods proved to be more suitable for the preparation of individual tear proteins but were less suitable for their quantitation.