Comparative analysis of whole plant, flower and root extracts of Chamomilla recutita L. and characteristic pure compounds reveals differential anti-inflammatory effects on human T cells.

Introduction: Chronic inflammation is a hallmark of chronic wounds and inflammatory skin diseases. Due to a hyperactive and prolonged inflammation triggered by proinflammatory immune cells, transitioning to the repair and healing phase is halted. T cells may exacerbate the proinflammatory milieu by secreting proinflammatory cytokines. Chamomilla recutita L. (chamomile) has been suggested for use in several inflammatory diseases, implying a capability to modulate T cells. Here, we have characterized and compared the effects of differently prepared chamomile extracts and characteristic pure compounds on the T cell redox milieu as well as on the migration, activation, proliferation, and cytokine production of primary human T cells. Methods: Phytochemical analysis of the extracts was carried out by LC-MS/MS. Primary human T cells from peripheral blood (PBTs) were pretreated with aqueous or hydroethanolic chamomile extracts or pure compounds. Subsequently, the effects on intracellular ROS levels, SDF-1α induced T cell migration, T cell activation, proliferation, and cytokine production after TCR/CD3 and CD28 costimulation were determined. Gene expression profiling was performed using nCounter analysis, followed by ingenuity pathway analysis, and validation at protein levels. Results: The tested chamomile extracts and pure compounds differentially affected intracellular ROS levels, migration, and activation of T cells. Three out of five differently prepared extracts and two out of three pure compounds diminished T cell proliferation. In line with these findings, LC-MS/MS analysis revealed high heterogeneity of phytochemicals among the different extracts. nCounter based gene expression profiling identified several genes related to T cell functions associated with activation and differentiation to be downregulated. Most prominently, apigenin significantly reduced granzyme B induction and cytotoxic T cell activity. Conclusion: Our results demonstrate an anti-inflammatory effect of chamomile- derived products on primary human T cells. These findings provide molecular explanations for the observed anti-inflammatory action of chamomile and imply a broader use of chamomile extracts in T cell driven chronic inflammatory diseases such as chronic wounds and inflammatory skin diseases. Importantly, the mode of extract preparation needs to be considered as the resulting different phytochemicals can result in differential effects on T cells.

Monitoring methylation changes in cancer.

Methylation of cytosines at their carbon-5 position plays an important role both during development and in tumorgenesis. The methylation occurs almost exclusively in CpG dinucleotides. While the bulk of human genomic DNA is depleted in CpG sites, there are CpG-rich stretches, so-called CpG islands, which are located in promoter regions of more than 70% of all known human genes. In normal cells, CpG islands are unmethylated, reflecting an transcriptionally active state of the respective gene. Epigenetic silencing of tumor suppressor genes by hypermethylation of CpG islands is a very early and stable characteristic of tumorigenesis. The detection of DNA methylation is based on a treatment of genomic DNA with sodium bisulfite, which converts only unmethylated cytosines to uracil, while methylated cytosines stay unaltered. This sequence conversion can be detected in the same way as a single nucleotide polymorphism. Even though different approaches have been established for analysing DNA methylation, so far detection methods that are capable of surveying the methylation status of multiple gene promoters have been restricted to a limited number of cytosines. The use of oligonucleotide microarrays permits the parallel analysis of the methylation status of individual cytosines on a genome-wide and gene-specific level. On the one hand, a hybridization-based setup is described employing microarrays that contain oligonucleotide probes of 17-25 bases in length reflecting the methylated as well as the unmethylated status of each CpG site. After hybridization of sodium bisulfite treated and fluorescently labeled targets, methylation status of individual CpG dinucleotides can be computed based on resulting signal intensities. Secondly, a microarray-based approach for detecting methylation-specific sequence polymorphisms via an on-chip enzymatic primer extension is described.

Array-based analysis of genomic DNA methylation patterns of the tumour suppressor gene p16INK4A promoter in colon carcinoma cell lines.

Aberrant DNA methylation at CpG dinucleotides can result in epigenetic silencing of tumour suppressor genes and represents one of the earliest events in tumourigenesis. To date, however, high-throughput tools that are capable of surveying the methylation status of multiple gene promoters have been restricted to a limited number of cytosines. Here, we present an oligonucleotide microarray that permits the parallel analysis of the methylation status of individual cytosines, thus combining high throughput and high resolution. The approach was used to study the CpG island in the promoter region of the tumour suppressor gene p16(INK4A). In total, 876 oligonucleotide probes of 21 nt in length were used to inspect the methylation status of 53 CpG dinucleotides, producing correct signals in colorectal cancer cell lines as well as control samples with a defined methylation status. The information was validated by established alternative methods. The overall methylation pattern was consistent for each cell line, while different between them. At the level of individual cytosines, however, significant variations between individual cells of the same type were found, but also consistencies across the panel of cancer cell lines were observed.

Fluorescent sample labeling for DNA microarray analyses.

Three fluorophor-labeling methods for gene expression profiling on deoxyribonucleic acid (DNA) microarrays are described. All three techniques start from total ribonucleic acid (RNA) samples. Two procedures are based on first-strand complementary DNA synthesis by reverse transcription. Label is introduced either by direct incorporation of fluorescently labeled nucleotides or indirectly by incorporation of aminoally-dUTP and subsequent coupling of fluorescent dyes. The third method is based on an amplification of antisense RNA by in vitro transcription subsequent to first- and second-strand complementary DNA synthesis. While the first two methods are applied mainly in analyses on microarrays made from spotted polymerase chain reaction products or long oligonucleotides, the last procedure is mostly used for experiments on in situ synthesized oligonucleotide arrays.