Identification of new lead molecules against anticancer drug target TFIIH subunit P8 using biophysical and molecular docking studies.

The identification of molecules, which could modulate protein-protein interactions (PPIs), is of primary interest to medicinal chemists. Using biophysical methods during the current study, we have screened 76 compounds (grouped into 16 mixtures) against the p8 subunit of the general transcription factor (TFIIH), which has recently been validated as an anti-cancer drug target. 10% of the tested compounds showed interactions with p8 protein in STD-NMR experiments. These results were further validated by molecular docking studies where interactions between compounds and important amino acid residues were identified, including Lys20 in the hydrophobic core of p8, and Asp42 and 43 in the ß3 strand. Moreover, these compounds were able to destabilize the p8 protein by negatively shifting the Tm (≥2 °C) in thermal shift assay. Thus, this study has identified 8 compounds which are likely negative modulators of p8 protein stability, and could be further considered as potential anticancer agents.

Small molecule-based targeting of TTD-A dimerization to control TFIIH transcriptional activity represents a potential strategy for anticancer therapy.

The human transcription factor TFIIH is a large complex composed of 10 subunits that form an intricate network of protein-protein interactions critical for regulating its transcriptional and DNA repair activities. The trichothiodystrophy group A protein (TTD-A or p8) is the smallest TFIIH subunit, shuttling between a free and a TFIIH-bound state. Its dimerization properties allow it to shift from a homodimeric state, in the absence of a functional partner, to a heterodimeric structure, enabling dynamic binding to TFIIH. Recruitment of p8 at TFIIH stabilizes the overall architecture of the complex, whereas p8's absence reduces its cellular steady-state concentration and consequently decreases basal transcription, highlighting that p8 dimerization may be an attractive target for down-regulating transcription in cancer cells. Here, using a combination of molecular dynamics simulations to study p8 conformational stability and a >3000-member library of chemical fragments, we identified small-molecule compounds that bind to the dimerization interface of p8 and provoke its destabilization, as assessed by biophysical studies. Using quantitative imaging of TFIIH in living mouse cells, we found that these molecules reduce the intracellular concentration of TFIIH and its transcriptional activity to levels similar to that observed in individuals with trichothiodystrophy owing to mutated TTD-A Our results provide a proof of concept of fragment-based drug discovery, demonstrating the utility of small molecules for targeting p8 dimerization to modulate the transcriptional machinery, an approach that may help inform further development in anticancer therapies.

Quantity, quality, and functionality of peripheral blood cells derived from residual blood of different apheresis kits.

BACKGROUND: Research with primary human white blood cell (WBC) subpopulations requires high quantity, quality, and functionality of peripheral blood mononuclear cells (PBMCs) as a source to further characterize cellular subpopulations such as T and B lymphocytes, monocytes, or natural killer cells. Apart from buffy coats derived from whole blood, residual blood from preparative hemapheresis kits are used as a source for PBMCs, but knowledge on the yield and functionality of cells from different devices is limited. STUDY DESIGN AND METHODS: We evaluated quantity and quality of PBMCs isolated from apheresis kits of two apheresis devices (AMICUS, Fenwal; and Trima Accel, Terumo BCT), the latter being our standard source for many years. PBMCs derived from Trima or AMICUS were tested for yield and subtype composition by flow cytometry. Functionality was assessed by cytokine induction of CD4+ and CD8+ T cells and by degranulation. Moreover, cytotoxic activity of natural killer cells was quantified by a real-time killing assay. RESULTS: Mean numbers of isolated cells were 5.5 ± 2.4 × 108 for AMICUS, and 10.3 ± 6.4 × 108 for Trima Accel, respectively. The proportion of WBC subtypes corresponded to well-known numbers from whole blood, with minor differences between the two apheresis systems. Likewise, minor differences in cytokine induction were found in stimulated CD4+ or CD8+ T cells. Finally, PBMCs derived from the two systems showed comparable cytotoxic activity. CONCLUSION: PBMC derived from residual blood of the AMICUS and Trima Accel apheresis devices serve as an economic and easily accessible source for functional PBMCs with comparable quantity and quality to PBMCs derived from whole blood.

Dual inhibition of PI3K and mTOR by VS-5584 suppresses thrombus formation.

VS-5584 is a highly selective dual kinase inhibitor which suppresses phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) activity. Because these kinases are crucially involved in primary hemostasis, we herein investigated the effect of this compound on thrombus formation in vitro and in vivo. Pretreatment of washed platelets (WP) or platelet-rich plasma (PRP) with VS-5584 inhibited the agonist-induced activation of surface glycoprotein complex (GP)IIb/IIIa and the upregulation of P-selectin. This was associated with a significantly reduced formation of platelet-leukocyte aggregates (PLA). VS-5584 further attenuated platelet aggregation and adhesion after agonist stimulation. In contrast, endothelial expression of intercellular adhesion molecule (ICAM)-1 and vascular cellular adhesion molecule (VCAM)-1 and secretion of von Willebrand Factor (vWF) were not affected by the dual kinase inhibitor. In vivo, VS-5584 inhibited photochemically induced thrombus formation as shown by a significantly prolonged time to complete vessel occlusion when compared to vehicle-treated controls. This was associated with an elevated tail vein bleeding time, indicating a potential hemorrhagic risk in VS-5584-treated mice. Taken together, these novel findings demonstrate that VS-5584 is a potent inhibitor of primary hemostasis targeting multiple platelet functions.

Deep characterization of blood cell miRNomes by NGS.

A systematic understanding of different factors influencing cell type specific microRNA profiles is essential for state-of-the art biomarker research. We carried out a comprehensive analysis of the biological variability and changes in cell type pattern over time for different cell types and different isolation approaches in technical replicates. All combinations of the parameters mentioned above have been measured, resulting in 108 miRNA profiles that were evaluated by next-generation-sequencing. The largest miRNA variability was due to inter-individual differences (34 %), followed by the cell types (23.4 %) and the isolation technique (17.2 %). The change over time in cell miRNA composition was moderate (<3 %) being close to the technical variations (<1 %). Largest variability (including technical and biological variance) was observed for CD8 cells while CD3 and CD4 cells showed significantly lower variations. ANOVA highlighted that 51.5 % of all miRNAs were significantly influenced by the purification technique. While CD4 cells were least affected, especially miRNA profiles of CD8 cells were fluctuating depending on the cell purification approach. To provide researchers access to the profiles and to allow further analyses of the tested conditions we implemented a dynamic web resource.

Tumor antigen-specific T cells for immune monitoring of dendritic cell-treated glioblastoma patients.

BACKGROUND AIMS: CD8(+) T cells are part of the adaptive immune system and, as such, are responsible for the elimination of tumor cells. Dendritic cells (DC) are professional antigen-presenting cells (APC) that activate CD8(+) T cells. Effector CD8(+) T cells in turn mediate the active immunotherapeutic response of DC vaccination against the aggressive glioblastoma (GBM). The lack of tumor response assays complicates the assessment of treatment success in GBM patients. METHODS: A novel assay to identify specific cytotoxicity of activated T cells by APC was evaluated. Tumor antigen-pulsed DCs from HLA-A*02-positive GBM patients were cultivated to stimulate autologous cytotoxic T lymphocytes (CTL) over a 12-day culture period. To directly correlate antigen specificity and cytotoxic capacity, intracellular interferon (IFN)-γ fluorescence flow cytometry-based measurements were combined with anti-GBM tumor peptide dextramer staining. IFN-γ response was quantified by real-time polymerase chain reaction (PCR), and selected GBM genes were compared with healthy human brain cDNA by single specific primer PCR characterization. RESULTS: Using CTL of GBM patients stimulated with GBM lysate-pulsed DCs increased IFN-γ messenger RNA levels, and intracellular IFN-γ protein expression was positively correlated with specificity against GBM antigens. Moreover, the GBM peptide-specific CD8(+) T-cell response correlated with specific GBM gene expression. Following DC vaccination, GBM patients showed 10-fold higher tumor-specific signals compared with unvaccinated GBM patients. DISCUSSION: These data indicate that GBM tumor peptide-dextramer staining of CTL in combination with intracellular IFN-γ staining may be a useful tool to acquire information on whether a specific tumor antigen has the potential to induce an immune response in vivo.

The Marine-Derived Kinase Inhibitor Fascaplysin Exerts Anti-Thrombotic Activity.

BACKGROUND: The marine-derived kinase inhibitor fascaplysin down-regulates the PI3K pathway in cancer cells. Since this pathway also plays an essential role in platelet signaling, we herein investigated the effect of fascaplysin on thrombosis. METHODS: Fascaplysin effects on platelet activation, platelet aggregation and platelet-leukocyte aggregates (PLA) formation were analyzed by flow cytometry. Mouse dorsal skinfold chambers were used to determine in vivo the effect of fascaplysin on photochemically induced thrombus formation and tail-vein bleeding time. RESULTS: Pre-treatment of platelets with fascaplysin reduced the activation of glycoprotein (GP)IIb/IIIa after protease-activated receptor-1-activating peptide (PAR-1-AP), adenosine diphosphate (ADP) and phorbol-12-myristate-13-acetate (PMA) stimulation, but did not markedly affect the expression of P-selectin. This was associated with a decreased platelet aggregation. Fascaplysin also decreased PLA formation after PMA but not PAR-1-AP and ADP stimulation. This may be explained by an increased expression of CD11b on leukocytes in PAR-1-AP- and ADP-treated whole blood. In the dorsal skinfold chamber model of photochemically induced thrombus formation, fascaplysin-treated mice revealed a significantly extended complete vessel occlusion time when compared to controls. Furthermore, fascaplysin increased the tail-vein bleeding time. CONCLUSION: Fascaplysin exerts anti-thrombotic activity, which represents a novel mode of action in the pleiotropic activity spectrum of this compound.

Static image analysis as new approach for the characterization of tumor cell lysate used in dendritic cell vaccine preparation.

BACKGROUND: Safety is an important consideration for the clinical application of dendritic cells (DC) loaded with autologous tumor lysate (TL). Thus, avitalization of TL from living autologous tumor tissue has to be guaranteed. METHODS: Composition of TL was investigated by static image analysis (SIA) with the Morphologi G3 device, which simultaneously measures size and shape of up to 100,000 particles within one sample run. This approach was compared with sample characterization by high-resolution automated cell counting, trypan blue staining, and ATP quantification. RESULTS: Using SIA, we only detected fragmented, non-cellular structures in completely avitalized TL, indicating complete destruction of living cells. Analysis of particle size distribution by SIA as well as CASY cell counter showed that 95% of particles had a diameter of <10 µm as a sign of cell fragmentation. Complete avitalization of TL was confirmed with trypan blue staining and ATP analysis. CONCLUSION: Regarding generation of DC vaccines, the proof of avitality of TL from living tumor tissue can clearly be achieved by SIA alone or in combination with standard assays. Our data show that SIA is a highly precise method for TL characterization. The SIA device complies with FDA regulation and, therefore, might be suitable for characterization of cellular therapy medicinal products.

Bilberry-derived anthocyanins prevent IFN-γ-induced pro-inflammatory signalling and cytokine secretion in human THP-1 monocytic cells.

BACKGROUND/AIMS: Anthocyanins are plant-derived dietary components that are highly abundant, for example, in bilberries. We have previously demonstrated that anthocyanins exert anti-inflammatory properties in mouse colitis models and ameliorate disease activity in ulcerative colitis patients. Here, we studied the molecular mechanisms through which anthocyanin-containing bilberry extract (BE) exerts anti-inflammatory effects in human monocytic THP-1 cells. METHODS: THP-1 cells were pre-incubated with BE 20 min prior to TNF-α or IFN-γ (100 ng/ml each) stimulation. Signalling protein activation was studied by Western blotting, mRNA expression by quantitative PCR and cytokine secretion by ELISA. RESULTS: IFN-γ-induced phosphorylation of STAT1 and STAT3 was significantly reduced by BE co-treatment. Consequently, levels of mRNA expression and/or cytokine secretion of MCP-1, IL-6, TNF-α, ICAM-1, and T-bet were lower with BE co-treatment. In contrast, BE enhanced TNF-α-mediated p65-NF-κB phosphorylation but reduced ERK1/2 phosphorylation. BE co-treatment further increased TNF-α-induced mRNA expression and secretion of NF-κB target genes, such as IL-6, IL-8, and MCP-1, while mRNA levels of ICAM-1 were reduced. CONCLUSIONS: BE co-treatment reduced IFN-γ-induced signal protein activation, pro-inflammatory gene expression, and cytokine secretion, whereas it enhanced TNF-α-induced responses. These findings suggest a distinct role for anthocyanins in modulating inflammatory responses that need to be further studied to fully understand anthocyanin-mediated effects.

Towards the classification of DYT6 dystonia mutants in the DNA-binding domain of THAP1.

The transcription factor THAP1 (THanatos Associated Protein 1) has emerged recently as the cause of DYT6 primary dystonia, a type of rare, familial and mostly early-onset syndrome that leads to involuntary muscle contractions. Many of the mutations described in the DYT6 patients fall within the sequence-specific DNA-binding domain (THAP domain) of THAP1 and are believed to negatively affect DNA binding. Here, we have used an integrated approach combining spectroscopic (NMR, fluorescence, DSF) and calorimetric (ITC) methods to evaluate the effect of missense mutations, within the THAP domain, on the structure, stability and DNA binding. Our study demonstrates that none of the mutations investigated failed to bind DNA and some of them even bind DNA stronger than the wild-type protein. However, some mutations could alter DNA-binding specificity. Furthermore, the most striking effect is the decrease of stability observed for mutations at positions affecting the zinc coordination, the hydrophobic core or the C-terminal AVPTIF motif, with unfolding temperatures ranging from 46°C for the wild-type to below 37°C for two mutations. These findings suggest that reduction in population of folded protein under physiological conditions could also account for the disease.

NMR studies of a new family of DNA binding proteins: the THAP proteins.

The THAP (THanatos-Associated Protein) domain is an evolutionary conserved C2CH zinc-coordinating domain shared with a large family of cellular factors (THAP proteins). Many members of the THAP family act as transcription factors that control cell proliferation, cell cycle progression, angiogenesis, apoptosis and epigenetic gene silencing. They recognize specific DNA sequences in the promoters of target genes and subsequently recruit effector proteins. Recent structural and functional studies have allowed getting better insight into the nuclear and cellular functions of some THAP members and the molecular mechanisms by which they recognize DNA. The present article reviews recent advances in the knowledge of the THAP domains structures and their interaction with DNA, with a particular focus on NMR. It provides the solution structure of the THAP domain of THAP11, a recently characterized human THAP protein with important functions in transcription and cell growth in colon cancer.

Low-dose antigen promotes induction of FOXP3 in human CD4+ T cells.

Low Ag dose promotes induction and persistence of regulatory T cells (Tregs) in mice, yet few studies have addressed the role of Ag dose in the induction of adaptive CD4(+)FOXP3(+) Tregs in humans. To this end, we examined the level of FOXP3 expression in human CD4(+)CD25(-) T cells upon activation with autologous APCs and varying doses of peptide. Ag-specific T cells expressing FOXP3 were identified by flow cytometry using MHC class II tetramer (Tmr). We found an inverse relationship between Ag dose and the frequency of FOXP3(+) cells for both foreign Ag-specific and self Ag-specific T cells. Through studies of FOXP3 locus demethylation and helios expression, we determined that variation in the frequency of Tmr(+)FOXP3(+) T cells was not due to expansion of natural Tregs, but instead, we found that induction, proliferation, and persistence of FOXP3(+) cells was similar in high- and low-dose cultures, whereas proliferation of FOXP3(-) T cells was favored in high Ag dose cultures. The frequency of FOXP3(+) cells positively correlated with suppressive function, indicative of adaptive Treg generation. The frequency of FOXP3(+) cells was maintained with IL-2, but not upon restimulation with Ag. Together, these data suggest that low Ag dose favors the transient generation of human Ag-specific adaptive Tregs over the proliferation of Ag-specific FOXP3(-) effector T cells. These adaptive Tregs could function to reduce ongoing inflammatory responses and promote low-dose tolerance in humans, especially when Ag exposure and tolerance is transient.

Signal transduction of pregnenolone sulfate in insulinoma cells: activation of Egr-1 expression involving TRPM3, voltage-gated calcium channels, ERK, and ternary complex factors.

The neurosteroid pregnenolone sulfate acts on the nervous system by modifying neurotransmission and receptor functions, thus influencing synaptic strength, neuronal survival, and neurogenesis. Here we show that pregnenolone sulfate induces a signaling cascade in insulinoma cells leading to enhanced expression of the zinc finger transcription factor Egr-1 and Egr-1-responsive target genes. Pharmacological and genetic experiments revealed that influx of Ca(2+) ions via transient receptor potential M3 and voltage-gated Ca(2+) channels, elevation of the cytosolic Ca(2+) level, and activation of ERK are essential for connecting pregnenolone sulfate stimulation with enhanced Egr-1 biosynthesis. Expression of a dominant-negative mutant of Elk-1, a key regulator of gene transcription driven by a serum response element, attenuated Egr-1 expression following stimulation, indicating that Elk-1 or related ternary complex factors connect the transcription of the Egr-1 gene with the pregnenolone sulfate-induced intracellular signaling cascade elicited by the initial influx of Ca(2+). The newly synthesized Egr-1 was biologically active and bound under physiological conditions to the regulatory regions of the Pdx-1, Synapsin I, and Chromogranin B genes. Pdx-1 is a major regulator of insulin gene transcription. Accordingly, elevated insulin promoter activity and increased mRNA levels of insulin could be detected in pregnenolone sulfate-stimulated insulinoma cells. Likewise, the biosynthesis of synapsin I, a synaptic vesicle protein that is found at secretory granules in insulinoma cells, was stimulated in pregnenolone sulfate-treated INS-1 cells. Together, these data show that pregnenolone sulfate induces a signaling cascade in insulinoma cells that is very similar to the signaling cascade induced by glucose in ß-cells.

Pregnenolone sulfate activates basic region leucine zipper transcription factors in insulinoma cells: role of voltage-gated Ca2+ channels and transient receptor potential melastatin 3 channels.

The neurosteroid pregnenolone sulfate activates a signaling cascade in insulinoma cells involving activation of extracellular signal-regulated protein kinase and enhanced expression of the transcription factor Egr-1. Here, we show that pregnenolone sulfate stimulation leads to a significant elevation of activator protein-1 (AP-1) activity in insulinoma cells. Expression of the basic region leucine zipper (bZIP) transcription factors c-Jun and c-Fos is up-regulated in insulinoma cells and pancreatic ß-cells in primary culture after pregnenolone sulfate stimulation. Up-regulation of a chromatin-embedded c-Jun promoter/luciferase reporter gene transcription in pregnenolone sulfate-stimulated insulinoma cells was impaired when the AP-1 binding sites were mutated, indicating that these motifs function as pregnenolone sulfate response elements. In addition, phosphorylation of cAMP response element (CRE)-binding protein is induced and transcription of a CRE-controlled reporter gene is stimulated after pregnenolone sulfate treatment, indicating that the CRE functions as a pregnenolone sulfate response element as well. Pharmacological and genetic experiments revealed that both L-type Ca(2+) channels and transient receptor potential melastatin 3 (TRPM3) channels are essential for connecting pregnenolone sulfate stimulation with enhanced AP-1 activity and bZIP-mediated transcription in insulinoma cells. In contrast, pregnenolone sulfate stimulation did not enhance AP-1 activity or c-Jun and c-Fos expression in pituitary corticotrophs that express functional L-type Ca(2+) channels but only trace amounts of TRPM3. We conclude that expression of L-type Ca(2+) channels is not sufficient to activate bZIP-mediated gene transcription by pregnenolone sulfate. Rather, additional expression of TRPM3 or depolarization of the cells is required to connect pregnenolone sulfate stimulation with enhanced gene transcription.

CK2 phosphorylation of Pdx-1 regulates its transcription factor activity.

The duodenal homeobox-1 protein Pdx-1 is one of the regulators for the transcription of the insulin gene. Pdx-1 is a phosphoprotein, and there is increasing evidence for the regulation of some of its functions by phosphorylation. Here, we asked whether protein kinase CK2 might phosphorylate Pdx-1 and how this phosphorylation could be implicated in the functional regulation of Pdx-1. We used fragments of Pdx-1 as well as phosphorylation mutants for experiments with protein kinase CK2. Transactivation was measured by reporter assays using the insulin promoter. Our data showed that Pdx-1 is phosphorylated by protein kinase CK2 at amino acids thr(231) and ser(232), and this phosphorylation was implicated in the regulation of the transcription factor activity of Pdx-1. Furthermore, inhibition of protein kinase CK2 by specific inhibitors led to an elevated release of insulin from pancreatic beta-cells. Thus, these findings identify CK2 as a novel mediator of the insulin metabolism.

Longitudinal Analyses of the Reciprocity of Depression and Anxiety after Traumatic Brain Injury and Its Clinical Implications.

Depression and anxiety are common following traumatic brain injury (TBI). Understanding their prevalence and interplay within the first year after TBI with differing severities may improve patients' outcomes after TBI. Individuals with a clinical diagnosis of TBI recruited for the large European collaborative longitudinal study CENTER-TBI were screened for patient-reported major depression (MD) and generalized anxiety disorder (GAD) at three, six, and twelve months post-injury (N = 1683). Data were analyzed using autoregressive cross-lagged models. Sociodemographic, premorbid and injury-related factors were examined as risk factors. 14.1-15.5% of TBI patients reported moderate to severe MD at three to twelve months after TBI, 7.9-9.5% reported GAD. Depression and anxiety after TBI presented high within-domain persistency and cross-domain concurrent associations. MD at three months post-TBI had a significant impact on GAD at six months post-TBI, while both acted bidirectionally at six to twelve months post-TBI. Being more severely disabled, having experienced major extracranial injuries, an intensive care unit stay, and being female were risk factors for more severe MD and GAD. Major trauma and the level of consciousness after TBI were additionally associated with more severe MD, whereas being younger was related to more severe GAD. Individuals after TBI should be screened and treated for MD and GAD early on, as both psychiatric disturbances are highly persistent and bi-directional in their impact. More severely disabled patients are particularly vulnerable, and thus warrant timely screening and intensive follow-up treatment.

Cholinergic Signaling Attenuates Pro-Inflammatory Interleukin-8 Response in Colonic Epithelial Cells.

Infants affected by Hirschsprung disease (HSCR), a neurodevelopmental congenital disorder, lack ganglia of the intrinsic enteric nervous system (aganglionosis) in a variable length of the colon, and are prone to developing severe Hirschsprung-associated enterocolitis (HAEC). HSCR patients typically show abnormal dense innervation of extrinsic cholinergic nerve fibers throughout the aganglionic rectosigmoid. Cholinergic signaling has been reported to reduce inflammatory response. Consequently, a sparse extrinsic cholinergic innervation in the mucosa of the rectosigmoid correlates with increased inflammatory immune cell frequencies and higher incidence of HAEC in HSCR patients. However, whether cholinergic signals influence the pro-inflammatory immune response of intestinal epithelial cells (IEC) is unknown. Here, we analyzed colonic IEC isolated from 43 HSCR patients with either a low or high mucosal cholinergic innervation density (fiber-low versus fiber-high) as well as from control tissue. Compared to fiber-high samples, IEC purified from fiber-low rectosigmoid expressed significantly higher levels of IL-8 but not TNF-α, IL-10, TGF-ß1, Muc-2 or tight junction proteins. IEC from fiber-low rectosigmoid showed higher IL-8 protein concentrations in cell lysates as well as prominent IL-8 immunoreactivity compared to IEC from fiber-high tissue. Using the human colonic IEC cell line SW480 we demonstrated that cholinergic signals suppress lipopolysaccharide-induced IL-8 secretion via the alpha 7 nicotinic acetylcholine receptor (a7nAChR). In conclusion, we showed for the first time that the presence of a dense mucosal cholinergic innervation is associated with decreased secretion of IEC-derived pro-inflammatory IL-8 in the rectosigmoid of HSCR patients likely dependent on a7nAChR activation. Owing to the association between IL-8 and enterocolitis-prone, fiber-low HSCR patients, targeted therapies against IL-8 might be a promising immunotherapy candidate for HAEC treatment.

Lack of Mucosal Cholinergic Innervation Is Associated With Increased Risk of Enterocolitis in Hirschsprung's Disease.

BACKGROUND & AIMS: Hirschsprung's disease (HSCR) is a congenital intestinal motility disorder defined by the absence of enteric neuronal cells (ganglia) in the distal gut. The development of HSCR-associated enterocolitis remains a life-threatening complication. Absence of enteric ganglia implicates innervation of acetylcholine-secreting (cholinergic) nerve fibers. Cholinergic signals have been reported to control excessive inflammation, but the impact on HSCR-associated enterocolitis is unknown. METHODS: We enrolled 44 HSCR patients in a prospective multicenter study and grouped them according to their degree of colonic mucosal acetylcholinesterase-positive innervation into low-fiber and high-fiber patient groups. The fiber phenotype was correlated with the tissue cytokine profile as well as immune cell frequencies using Luminex analysis and fluorescence-activated cell sorting analysis of colonic tissue and immune cells. Using confocal immunofluorescence microscopy, macrophages were identified in close proximity to nerve fibers and characterized by RNA-seq analysis. Microbial dysbiosis was analyzed in colonic tissue using 16S-rDNA gene sequencing. Finally, the fiber phenotype was correlated with postoperative enterocolitis manifestation. RESULTS: The presence of mucosal nerve fiber innervation correlated with reduced T-helper 17 cytokines and cell frequencies. In high-fiber tissue, macrophages co-localized with nerve fibers and expressed significantly less interleukin 23 than macrophages from low-fiber tissue. HSCR patients lacking mucosal nerve fibers showed microbial dysbiosis and had a higher incidence of postoperative enterocolitis. CONCLUSIONS: The mucosal fiber phenotype might serve as a prognostic marker for enterocolitis development in HSCR patients and may offer an approach to personalized patient care and new therapeutic options.

Regulation of AP-1 activity in glucose-stimulated insulinoma cells.

The transcriptional activity of AP-1 has been analyzed in glucose-stimulated INS-1 insulinoma cells using a chromosomally embedded AP-1-responsive reporter gene. We show that AP-1 activity was significantly elevated in glucose-treated INS-1 cells. Preincubation of the cells with nifedipine or expression of the Ca(2+) binding protein parvalbumin in the cytoplasm of INS-1 cells reduced AP-1 activity. Thus, activation of L-type Ca(2+) channels and an elevated cytoplasmic Ca(2+) concentration are crucial to connecting glucose stimulation with enhanced AP-1 activity. Expression of dominant negative forms of A-Raf, MKK4 or MKK6 and pharmacological inhibition of MEK and p38 revealed that extracellular signal-regulated protein kinase, p38 and c-Jun NH(2)-terminal protein kinase participate in the upregulation of AP-1 activity. Expression of dominant-negative mutants of c-Jun and Elk-1 reduced AP-1 transcriptional activity in INS-1 cells indicating that c-Jun and ternary complex factors are involved in the regulation of AP-1 activity in glucose-stimulated insulinoma cells.