Resveratrol and ω-3 PUFAs Promote Human Macrophage Differentiation and Function.

Monocytes differentiate into M1 and M2 macrophages, which are classically activated by microbial products such as LPS or IFN-γ and interleukins (e.g., the anti-inflammatory and Th2 promoting IL-4), respectively. The contribution of nutrients or nutrient-based substances such as ω-3 polyunsaturated fatty acids (ω-3 PUFAs) and resveratrol (Res) on the differentiation and function of M1 and M2 macrophages was evaluated. THP-1 cells and peripheral blood mononuclear cells (PBMCs) were differentiated into M1 and M2 cells and activated with LPS/IFN-γ or IL-4/IL-13. Macrophage lineage specific surface determinants (e.g., CD11b, CD11c, CD14, CD206, CD209, CD274, HLA-DR, CCR7, CCR2) were analysed by cytofluorometry. Res and ω-3 PUFAs altered CD14, CD206, CD274 and HL-DR surface expression patterns in M1 and M2 macrophages differentiated from PBMC. LPS/IFN-γ or IL-14/IL-13 activated macrophages subpopulations, which secreted cytokines and chemokines as measured by multiplex ELISA. Res and ω-3 PUFA reduced IL-1ß, IL-6, TNF-α, CXCL10/IP-10, CCL13/MCP-4 and CCL20/MIP-3α in LPS/IFN-γ activated human leukaemia THP-1 cells, which is indicative of a dampening effect on M1 macrophages. However, Res increased M1 prototypic cytokines such as IL-1ß or IL-6 in macrophages derived from PBMCs and also modified the expression of IL-12p70. Collectively, Res and ω-3 PUFAs distinctly promoted the differentiation and function of M1 and M2 macrophages. We conclude that these substances strengthen the macrophage-mediated effects on the innate and adaptive immune response.

Toll-Like Receptors: Regulators of the Immune Response in the Human Gut.

Toll-like receptors (TLRs) are powerful molecular regulators by which the immune system may "sense" the environment and protect the host from pathogens or endogenous threats. In mammalian cells, several TLRs were identified with a tissue and cell type-specific distribution. Understanding the functions of specific TLRs is crucial for the development and discovery of compounds useful to maintaining or re-establishing homeostasis in the gastrointestinal tract (GIT). Due to their relevance in regulating the inflammatory response in the GIT, we will focus here on TLR2, TLR4, and TLR5. In particular, we describe (a) the molecular pathways activated by the stimulation of these receptors with their known bacterial ligands; (b) the non-bacterial ligands known to interact directly with TLR2 and TLR4 and their soluble forms. The scope of this minireview is to highlight the importance of bacterial and non-bacterial compounds in affecting the gut immune functions via the activation of the TLRs.

HAnd Suture Versus STApling for Closure of Loop Ileostomy (HASTA Trial): results of a multicenter randomized trial (DRKS00000040).

OBJECTIVES: The objective of the HASTA trial was to compare hand suture versus stapling loop ileostomy closure in a randomized controlled trial. BACKGROUND: Bowel obstruction is one of the main and the clinically and economically most relevant complication following closure of loop ileostomy after low anterior resection. The best surgical technique for closure of loop ileostomy has not been defined yet. METHODS: HASTA trial is a multicenter pragmatic randomized controlled surgical trial with 2 parallel groups to compare hand suture versus stapling for closure of loop ileostomy. The primary endpoint was the rate of bowel obstruction within 30 days after ileostomy closure. RESULTS: A total of 337 randomized patients undergoing closure of loop ileostomy after low anterior resection because of rectal cancer in 27 centers were included. The overall rate of postoperative ileus after ileostomy closure was 13.4%. Seventeen of 165 (10.3%) patients in the stapler group and 27 of 163 (16.6%) in the hand suture group developed bowel obstruction within 30 days postoperatively [odds ratio (OR) = 1.72; 95% confidence interval (CI): 0.89-3.31 = 0.10]. Duration of surgical intervention was significantly shorter in the stapler group (15 minutes; P < 0.001). Multivariable analysis of potential risk factors did not reveal any significant correlation with development of postoperative ileus. Rate of anastomotic leakage (stapler: 3.0%, hand suture: 1.8%, P = 0.48) did not differ significantly as well as all other secondary endpoints. CONCLUSIONS: Hand-sewn anastomosis versus stapler ileo-ileostomy for ileostomy closure are equally effective in terms of postoperative bowel obstruction, with stapler anastomosis leading to a shorter operation time. Postoperative ileus after ileostomy reversal remains a relevant complication.

The natural compound ascorbigen modulates NADPH-quinone oxidoreductase (NQO1) mRNA and enzyme activity levels in cultured liver cells and in laboratory rats.

Ascorbigen (ABG) is a natural compound that represents a breakdown product of the glucosinolates that are present in Brassica vegetables. It is postulated that ABG may have anticarcinogenic activity; however, the underlying molecular and cellular mechanisms are largely unknown. In the present study we investigated the effect of ABG on the mRNA and enzyme activity levels of NADPH-quinone oxidoreductase (NQO1), which is centrally involved in the detoxification of xenobiotics, in cultured liver cells and in rats. The mRNA levels of NQO1 showed an increase of up to 100% in cultured liver cells (HepG2) following incubation with different concentrations of ABG (3-100 micromol/l) compared to control cells. Furthermore, NQO1 activity was elevated (up to 20%) by ABG treatment. The in vitro results were confirmed in rats who received either 5 mg/day ABG or vehicle for 7 days. Significantly higher mRNA (a 90% increase) and enzyme activity levels (a 40% increase) of NQO1 were detected in the liver of ABG-treated rats as compared to control animals. Current data indicate that ABG is a moderate inducer of the phase II enzyme NQO1, both in cultured hepatocytes and in vivo.

Sulfonylureas and glinides exhibit peroxisome proliferator-activated receptor gamma activity: a combined virtual screening and biological assay approach.

Most drugs currently employed in the treatment of type 2 diabetes either target the sulfonylurea receptor stimulating insulin release (sulfonylureas, glinides), or target the peroxisome proliferator-activated receptor (PPARgamma) improving insulin resistance (thiazolidinediones). Our work shows that sulfonylureas and glinides additionally bind to PPARgamma and exhibit PPARgamma agonistic activity. This activity was predicted in silico by virtual screening and confirmed in vitro in a binding assay, a transactivation assay, and by measuring the expression of PPARgamma target genes. Among the measured compounds, gliquidone and glipizide (two sulfonylureas), as well as nateglinide (a glinide), exhibit PPARgamma agonistic activity at concentrations comparable with those reached under pharmacological treatment. The most active of these compounds, gliquidone, is shown to be as potent as pioglitazone at inducing PPARgamma target gene expression. This dual mode of action of sulfonylureas and glinides may open new perspectives for the molecular pharmacology of antidiabetic drugs, because it provides evidence that drugs can be designed that target both the sulfonylurea receptor and PPARgamma. Targeting both receptors could increase pancreatic insulin secretion and improve insulin resistance. Glinides, sulfonylureas, and other acidified sulfonamides may be promising leads in the development of new PPARgamma agonists. In addition, we provide a unified concept of the PPARgamma binding ability of seemingly disparate compound classes.

Methods and biomarkers for the diagnosis and prognosis of cancer and other diseases: towards personalized medicine.

The rapid development of new diagnostic procedures, the mapping of the human genome, progress in mapping genetic polymorphisms, and recent advances in nucleic acid- and protein chip technologies are driving the development of personalized therapies. This breakthrough in medicine is expected to be achieved largely due to the implementation of "lab-on-the-chip" technology capable of performing hundreds, even thousands of biochemical, cellular and genetic tests on a single sample of blood or other body fluid. Focusing on a few disease-specific examples, this review discusses selected technologies and their combinations likely to be incorporated in the "lab-on-the-chip" and to provide rapid and versatile information about specific diseases entities. Focusing on breast cancer and after an overview of single-nucleotide polymorphism (SNP)-screening methodologies, we discuss the diagnostic and prognostic importance of SNPs. Next, using Duchenne muscular dystrophy (DMD) as an example, we provide a brief overview of powerful and innovative integration of traditional immuno-histochemistry techniques with advanced biophysical methods such as NMR-spectroscopy or Fourier-transformed infrared (FT-IR) spectroscopy. A brief overview of the challenges and opportunities provided by protein and aptamer microarrays follows. We conclude by highlighting novel and promising biochemical markers for the development of personalized treatment of cancer and other diseases: serum cytochrome c, cytokeratin-18 and -19 and their proteolytic fragments for the detection and quantitation of malignant tumor mass, tumor cell turn-over, inflammatory processes during hepatitis and Epstein-Barr virus (EBV)-induced hemophagocytic lymphohistiocytosis and apoptotic/necrotic cancer cell death.

ADRIS - The Adverse Drug Reactions Information Scheme.

Under the Adverse Drug Reactions Information Scheme (ADRIS) data and knowledge relevant to the etiology of adverse drug reactions (ADRs) such as chemical structure of parent compounds, metabolites, covalent adducts, nucleic acid and protein sequences, protein structures, pharmaco-, toxico- and enzyme kinetics, pharmaco- and toxicodynamics, protein interactions, molecular pathways and complexes, as well as toxicological and clinical outcomes, are collected and logically and semantically related. ADRIS reflects the ontological prerequisite for the creation of databases and knowledge discovery systems for the abstraction and visualization of theragenomic concepts. A final outcome is the prediction of ADRs based on a profound knowledge of drug function and the molecular basics for personalized drug safety and eventually, personalized medicine. 2004

Advances in recombinant antibody microarrays.

Antibody microarrays, one emerging class of proteomic technologies, have broad applications in proteome analysis, disease diagnostics and quantitative analysis. Compared to DNA microarrays, protein targets have significantly more complex interactions with their ligands such as antibodies. To introduce antibody microarrays for clinical diagnostics and thus to complement or replace conventional immunoassays, several new developments are addressed. We discuss different microarray surfaces, immobilization techniques, detection systems and advantages and disadvantages of antibody microarrays compared to standard clinical techniques. Currently, the probes with highest specificity, well-characterized binding properties, and the possibility of large-scale production using display libraries are recombinant antibodies.

A streptavidin-biotin-based microarray platform for immunoassays.

The authors describe a microarray system for disease diagnosis based on antibody-antigen interactions. Either biotinylated antibodies or antigens are coupled via streptavidin linkers onto a gold surface. This platform has been used to establish recombinant antibody-antigen interactions and to detect specific IgM antibodies in sera of patients suspected of Lyme borreliosis. Therefore, this microarray system can be adapted for further applications.

ADRIS--The Adverse Drug Reactions Information Scheme.

Under the Adverse Drug Reactions Information Scheme (ADRIS) data and knowledge relevant to the etiology of adverse drug reactions (ADRs) such as chemical structure of parent compounds, metabolites, covalent adducts, nucleic acid and protein sequences, protein structures, pharmaco-, toxico- and enzyme kinetics, pharmaco- and toxicodynamics, protein interactions, molecular pathways and complexes, as well as toxicological and clinical outcomes, are collected and logically and semantically related. ADRIS reflects the ontological prerequisite for the creation of databases and knowledge discovery systems for the abstraction and visualization of theragenomic concepts. A final outcome is the prediction of ADRs based on a profound knowledge of drug function and the molecular basics for personalized drug safety and eventually, personalized medicine.

Measurement of the number of molecules of a single mRNA species in a complex mRNA preparation.

The normalization of data obtained from hybridization experiments with DNA chips to determine mRNA expression and concentration (gene expression profiling) is an unsolved problem. Furthermore, slight changes in mRNA expression or small numbers of mRNA molecules which may be relevant to disease cannot be detected so far. We have designed a method to calculate the number of molecules of a single mRNA species in a complex mRNA preparation. The basic concept is the transformation of a quantitative problem into a qualitative problem. Individual molecules pertaining to the same molecular species (IMPSMS) are transformed to a mixture of new different molecular species (DMS) and amplified. We propose two implementations of the method. The first procedure is based on a method for cloning tagged nucleic acid molecules onto the surface of micro-beads. It should be possible to transform and determine up to 10(6) IMPSMS into new DMS. The second strategy uses multimeric linkers, a method frequently used in DNA computing to assemble random DNA. The second strategy should be easier to implement but is limited to a few hundred IMPSMS.

Anticancer drugs of tomorrow: apoptotic pathways as targets for drug design.

Apoptosis or programmed cell death is a set of ordered events that enables the selective removal of cells from tissue and is essential for homeostasis and proper function of multicellular organisms. Components of this signaling network, which include ligands, such as CD95, tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand, as well as downstream molecules, such as caspases, Bcl-2 family members, and inhibitor-of-apoptosis proteins, which trigger and regulate apoptosis, are crucial targets for conventional drug development and gene therapy of cancer and other diseases. Here, we focus on apoptotic pathways and propose new potential molecular targets that could prove effective in controlling cell death in the clinical setting.

Differential calcium response in HeLa and HeLa-Fas cells by cytotoxic T lymphocytes.

We constructed a CD95 overexpressing HeLa cell line which was extremely sensitive towards CD95 mediated apoptosis. In these CD95 overexpressing cells, CD95 blocks the nuclear calcium signal induced by perforin positive and CD95 ligand positive killer cells. This phenomenon is highly relevant in states of inflammatory syndromes such as systemic inflammatory response syndrome (SIRS) and sepsis which are associated with a high probability to reactivate latent viruses due to a functional deficiency of cytotoxic effectors.

HIPK2 associates with RanBPM.

Using the yeast two-hybrid system, we have identified the Ran-binding protein (RanBPM) as an interaction partner of homeodomain-interacting protein kinase 2 (HIPK2). RanBPM has been described as a centrosomal protein through which Ran regulates the centrosomal function. HIPK2 is mainly a nuclear protein, which among other functions represses transcription mediated by homeodomain containing transcription factors. Here, we show that overexpressed wildtype HIPK2 and a kinase defective mutant of HIPK2 directly interact with RanBPM in the nucleus of mammalian cells. Overexpressed wildtype RanBPM and a kinase defective mutant of HIPK2 co-localise with HIPK2 in defined nuclear structures. A carboxy- and an amino-terminal deletion of HIPK2 do not seem to be able to bind to RanBPM.

Caspases--their role in apoptosis and other physiological processes as revealed by knock-out studies.

Caspases are crucial mediators of apoptosis, a form of physiological cell death. Their activation is carefully controlled by a philogenetically conserved death program, which is indispensable for the homeostasis and development of higher organisms. Dysregulation of apoptosis contributes to the pathogenesis of many human diseases. As effectors of the apoptotic machinery, caspases are considered potential therapeutic targets. In vitro studies have demonstrated the requirement of caspase activity for both the triggering phase as well as the execution of apoptosis, thus providing a molecular base for the fine-tuning of this process by pharmacological agents. The precise roles of the individual caspases in vivo and their functional relation to each other have been best demonstrated in genetically modified animals. The generation of single caspase-deficient mice have confirmed most of the data obtained in vitro and exposed some new aspects previously undetected in the cell culture system. Interestingly, inactivation of many caspases revealed not only their expected participation in apoptotic events as well as in the maturation of cytokines, but also provided hints about the role of at least some caspases in cell differentiation and stimulatory responses. In this review we will discuss what these studies have unveiled about the role of individual caspases in development, apoptosis, and inflammation, with particular focus on their role beyond the apoptotic process.