Primary head and neck cancer cell cultures are susceptible to proliferation of Epstein-Barr virus infected lymphocytes.

BACKGROUND: New concepts for a more effective anti-cancer therapy are urgently needed. Experimental flaws represent a major counter player of this development and lead to inaccurate and unreproducible data as well as unsuccessful translation of research approaches into clinics. In a previous study we have created epithelial cell cultures from head and neck squamous cell carcinoma (HNSCC) tissue. METHODS: We characterize primary cell populations isolated from human papillomavirus positive HNSCC tissue for their marker expression by RT-qPCR, flow cytometry, and immunofluorescence staining. Their sensitivity to MDM2-inhibition was measured using cell viability assays. RESULTS: Primary HNSCC cell cultures showed the delayed formation of spheroids at higher passages. These spheroids mimicked the morphology and growth characteristics of other established HNSCC spheroid models. However, expression of epithelial and mesenchymal markers could not be detected in these cells despite the presence of the HNSCC stem cell marker aldehyde dehydrogenase 1 family member A1. Instead, strong expression of B- and T-lymphocytes markers was observed. Flow cytometry analysis revealed a heterogeneous mixture of CD3 + /CD25 + T-lymphocytes and CD19 + B-lymphocytes at a ratio of 4:1 at passage 5 and transformed lymphocytes at late passages (≥ passage 12) with CD45 + CD19 + CD20 + , of which around 10 to 20% were CD3 + CD25 + CD56 + . Interestingly, the whole population was FOXP3-positive indicative of regulatory B-cells (Bregs). Expression of transcripts specific for the Epstein-Barr-virus (EBV) was detected to increase in these spheroid cells along late passages, and this population was vulnerable to MDM2 inhibition. HPV + HNSCC cells but not EBV + lymphocytes were detected to engraft into immunodeficient mice. CONCLUSIONS: In this study we present a primary cell culture of EBV-infected tumor-infiltrating B-lymphocytes, which could be used to study the role of these cells in tumor biology in future research projects. Moreover, by describing the detailed characteristics of these cells, we aim to caution other researchers in the HNSCC field to test for EBV-infected lymphocyte contaminations in primary cell cultures ahead of further experiments. Especially researchers who are interested in TIL-based adopted immunotherapy should exclude these cells in their primary tumor models, e.g. by MDM2-inhibitor treatment. BI-12-derived xenograft tumors represent a suitable model for in vivo targeting studies.

Classification of three corynebacterial strains isolated from a small paddock in North Rhine-Westphalia: proposal of Corynebacterium kalinowskii sp. nov., Corynebacterium comes sp. nov. and Corynebacterium occultum sp. nov.

Three novel corynebacterial species were isolated from soil sampled at a paddock in Vilsendorf, North Rhine-Westphalia, Germany. The strains were coccoid or irregular rod-shaped, catalase-positive and pale white to yellow-orange in colour. By whole genome sequencing and comparison of the 16S rRNA genes as well as the whole genome structure, it was shown that all three strains represent novel species of the family Corynebacteriaceae, order Corynebacteriales, class Actinobacteria. This project describes the isolation, identification, sequencing, and phenotypic characterization of the three novel Corynebacterium species. We propose the names Corynebacterium kalinowskii sp. nov. (DSM 110639T=LMG 31801T), Corynebacterium comes sp. nov. (DSM 110640T=LMG 31802T), and Corynebacterium occultum sp. nov. (DSM 110642T=LMG 31803T).

A targeted transcriptomics approach for the determination of mixture effects of pesticides.

While real-life exposure occurs to complex chemical mixtures, toxicological risk assessment mostly focuses on individual compounds. There is an increasing demand for in vitro tools and strategies for mixture toxicity analysis. Based on a previously established set of hepatotoxicity marker genes, we analyzed mixture effects of non-cytotoxic concentrations of different pesticides in exposure-relevant binary mixtures in human HepaRG hepatocarcinoma cells using targeted transcriptomics. An approach for mixture analysis at the level of a complex endpoint such as a transcript pattern is presented, including mixture design based on relative transcriptomic potencies and similarities. From a mechanistic point of view, goal of the study was to evaluate combinations of chemicals with varying degrees of similarity in order to determine whether differences in mechanisms of action lead to different mixtures effects. Using a model deviation ratio-based approach for assessing mixture effects, it was revealed that most data points are consistent with the assumption of dose addition. A tendency for synergistic effects was only observed at high concentrations of some combinations of the test compounds azoxystrobin, cyproconazole, difenoconazole, propiconazole and thiacloprid, which may not be representative of human real-life exposure. In summary, the findings of our study suggest that, for the pesticide mixtures investigated, risk assessment based on the general assumption of dose addition can be considered sufficiently protective for consumers. The way of data analysis presented in this paper can pave the way for a more comprehensive use of multi-gene expression data in experimental studies related to mixture toxicity.

An eight-compound mixture but not corresponding concentrations of individual chemicals induces triglyceride accumulation in human liver cells.

In real life, organisms are exposed to complex mixtures of chemicals at low concentration levels, whereas research on toxicological effects is mostly focused on single compounds at comparably high doses. Mixture effects deviating from the assumption of additivity, especially synergistic effects, are of concern. In an adverse outcome pathway (AOP)-guided manner, we analyzed the accumulation of triglycerides in human HepaRG liver cells by a mixture of eight steatotic chemicals (amiodarone, benzoic acid, cyproconazole, flusilazole, imazalil, prochloraz, propiconazole and tebuconazole), each present below its individual effect concentration at 1-3 µM. Pronounced and significantly enhanced triglyceride accumulation was observed with the mixture, and similar effects were seen at the level of pregnane-X-receptor activation, a molecular initiating event leading to hepatic steatosis. Transcript pattern analysis indicated subtle pro-steatotic changes at low compound concentrations, which did not exert measurable effects on cellular triglycerides. Mathematical modeling of mixture effects indicated potentially more than additive behavior using a model for compounds with similar modes of action. The present data underline the usefulness of AOP-guided in vitro testing for the identification of mixture effects and highlight the need for further research on chemical mixtures and harmonization of data interpretation of mixture effects.

Pesticide mixture effects on liver protein abundance in HepaRG cells.

Toxicological effects of chemicals are mostly tested individually. However, consumers encounter exposure to complex mixtures, for example multiple pesticide residues, by consuming food such as crops, fruits or vegetables. Currently, more than 450 active substances are approved in the European Union, and there is little data on effects after combined exposure to several pesticides. Toxicological animal studies would increase enormously, if pesticide combinations had to be analyzed in vivo. Therefore, in vitro methods addressing this issue are needed. We have developed 32 immunoaffinity-based mass spectrometry assays to investigate the impact of hepatotoxic active substances on liver proteins in human HepaRG cells. Five compounds were selected based on their (dis)similar capability to modulate protein levels, and on their combined use in commercially available formulations. Four binary mixtures were prepared from these five substances and tested in different concentrations over three time points. We applied a novel statistical method to describe deviations from additivity and to detect antagonistic and synergistic effects. The results regarding the abundance of hepatotoxicity-related proteins showed additive behavior for 1323 out of 1427 endpoints tested, while 104 combinatorial effects deviating from additivity, such as antagonism or synergism were observed.

Transcript and protein marker patterns for the identification of steatotic compounds in human HepaRG cells.

Non-alcoholic fatty liver disease is a major health concern especially in Western countries. Animal studies suggest that certain chemicals may contribute to hepatocellular triglyceride accumulation, among them a number of hepatotoxic pesticidal active compounds. In order to improve the identification of potential liver steatosis inducers in vitro in a human cell culture system, HepaRG cells were treated with a selection of 30 steatotic or non-steatotic pesticides. Induction of triglyceride accumulation was monitored, and changes in the expression of hepatotoxicity marker genes were measured at the mRNA and protein levels. Based on these data, transcript and protein marker signatures predictive of triglyceride accumulation in HepaRG cells were derived. The predictive transcript set consisted of POR, ANXA10, ARG1, CCL20, FASN, INSIG1, SREBF1, CD36, CYP2D6, and SLCO1B1. The predictive protein set consisted of NCPR (POR), CYP2E1, CYP1A1, ALDH3A1, UGT2B7, UGT2B15, S100P, LMNA, and PRKDC. In conclusion, the present study presents for the first time transcript and protein marker patterns to separate steatotic from non-steatotic compounds in a human liver cell line.

RNA-protein correlation of liver toxicity markers in HepaRG cells.

The liver is a main target organ for the toxicity of many different compounds. While in general, in vivo testing is still routinely used for assessing the hepatotoxic potential of test chemicals, the use of in vitro models offers advantages with regard to throughput, consumption of resources, and animal welfare aspects. Using the human hepatoma cell line HepaRG, we performed a comparative evaluation of a panel of hepatotoxicity marker mRNAs and proteins after exposure of the cells to 30 different pesticidal active compounds comprising herbizides, fungicides, insecticides, and others. The panel of hepatotoxicity markers included nuclear receptor target genes, key players of fatty acid and bile acid metabolism-related pathways, as well as recently identified biomarkers of drug-induced liver injury. Moreover, marker genes and proteins were identified, for example, S100P, ANXA10, CYP1A1, and CYP7A1. These markers respond with high sensitivity to stimulation with chemically diverse test compounds already at non-cytotoxic concentrations. The potency of the test compounds, determined as an overall parameter of their ability to deregulate marker expression in vitro, was very similar between the mRNA and protein levels. Thus, this study does not only characterize the response of human liver cells to 30 different pesticides but also demonstrates that hepatotoxicity testing in human HepaRG cells yields well comparable results at the mRNA and protein levels. Furthermore, robust hepatotoxicity marker genes and proteins were identified in HepaRG cells.

Assessment of mixture toxicity of (tri)azoles and their hepatotoxic effects in vitro by means of omics technologies.

Consumers are constantly exposed to chemical mixtures such as multiple residues of different pesticides via the diet. This raises questions concerning potential combination effects, especially because these substances are tested for regulatory purposes on an individual basis. With approximately 500 active substances approved as pesticides, there are too many possible combinations to be tested in standard animal experiments generally requested for regulatory purposes. Therefore, the development of in vitro tools and alternative testing strategies for the assessment of mixture effects is extremely important. As a first step in the development of such in vitro tools, we used (tri)azoles as model substances in a set of different cell lines derived from the primary target organ of these substances, the liver (human: HepaRG, rat: H4IIE). Concentrations were reconciled with measured tissue concentrations obtained from in vivo experiments to ensure comparable effect levels. The effects of the substances were subsequently analyzed by transcriptomics and metabolomics techniques and compared to data from corresponding in vivo studies. The results show that similar toxicity pathways are affected by substances and combinations, thus indicating a similar mode of action and additive effects. Two biomarkers obtained by the approach, CAR and Cyp1A1, were used for mixture toxicity modeling and confirmed the concentration-additive effects, thus supporting the selected testing strategy and raising hope for the development of in vitro methods suitable to detect combination effects and prioritize mixtures of concern for further testing.

Genome wide transcription start sites analysis of Xanthomonas campestris pv. campestris B100 with insights into the gum gene cluster directing the biosynthesis of the exopolysaccharide xanthan.

Xanthomonas campestris pv. campestris (Xcc) is the major producer of the exopolysaccharide xanthan, the commercially most important natural polysaccharide of microbial origin. The current work provides deeper insights into the yet uncharacterized transcriptomic features of the xanthan producing strain Xcc-B100. Towards this goal, RNA sequencing of a library based on the selective enrichment of the 5' ends of native transcripts was performed. This approach resulted in the genome wide identification of 3067 transcription start sites (TSSs) that were further classified based on their genomic positions. Among them, 1545 mapped upstream of an actively transcribed CDS and 1363 were classified as novel TSSs representing antisense, internal, and TSSs belonging to previously unidentified genomic features. Analyzing the transcriptional strength of primary and antisense TSSs revealed that in some instances antisense transcription seemed to be initiated at a higher level than its sense counterpart. Mapping the exact positions of TSSs aided in the identification of promoter consensus motifs, ribosomal binding sites, and enhanced the genome annotation of 159 in silico predicted translational start (TLS) sites. The global view on length distribution of the 5' untranslated regions (5'-UTRs) deduced from the data pointed to the occurrence of leaderless transcripts and transcripts with unusually long 5'-UTRs, in addition to identifying seven putative riboswitch elements for Xcc-B100. Concerning the biosynthesis of xanthan, we focused on the transcriptional organization of the gum gene cluster. Under the conditions tested, we present evidence for a complex transcription pattern of the gum genes with multiple TSSs and an obvious considerable role of antisense transcription. The gene gumB, encoding an outer membrane xanthan exporter, is presented here as an example for genes that possessed a strong antisense TSS.

Transcriptome sequencing revealed the transcriptional organization at ribosome-mediated attenuation sites in Corynebacterium glutamicum and identified a novel attenuator involved in aromatic amino acid biosynthesis.

The Gram-positive bacterium Corynebacterium glutamicum belongs to the order Corynebacteriales and is used as a producer of amino acids at industrial scales. Due to its economic importance, gene expression and particularly the regulation of amino acid biosynthesis has been investigated extensively. Applying the high-resolution technique of transcriptome sequencing (RNA-seq), recently a vast amount of data has been generated that was used to comprehensively analyze the C. glutamicum transcriptome. By analyzing RNA-seq data from a small RNA cDNA library of C. glutamicum, short transcripts in the known transcriptional attenuators sites of the trp operon, the ilvBNC operon and the leuA gene were verified. Furthermore, whole transcriptome RNA-seq data were used to elucidate the transcriptional organization of these three amino acid biosynthesis operons. In addition, we discovered and analyzed the novel attenuator aroR, located upstream of the aroF gene (cg1129). The DAHP synthase encoded by aroF catalyzes the first step in aromatic amino acid synthesis. The AroR leader peptide contains the amino acid sequence motif F-Y-F, indicating a regulatory effect by phenylalanine and tyrosine. Analysis by real-time RT-PCR suggests that the attenuator regulates the transcription of aroF in dependence of the cellular amount of tRNA loaded with phenylalanine when comparing a phenylalanine-auxotrophic C. glutamicum mutant fed with limiting and excess amounts of a phenylalanine-containing dipeptide. Additionally, the very interesting finding was made that all analyzed attenuators are leaderless transcripts.

Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique.

BACKGROUND: The use of RNAseq to resolve the transcriptional organization of an organism was established in recent years and also showed the complexity and dynamics of bacterial transcriptomes. The aim of this study was to comprehensively investigate the transcriptome of the industrially relevant amino acid producer and model organism Corynebacterium glutamicum by RNAseq in order to improve its genome annotation and to describe important features for transcription and translation. RESULTS: RNAseq data sets were obtained by two methods, one that focuses on 5'-ends of primary transcripts and another that provides the overall transcriptome with an improved resolution of 3'-ends of transcripts. Subsequent data analysis led to the identification of more than 2,000 transcription start sites (TSSs), the definition of 5'-UTRs (untranslated regions) for annotated protein-coding genes, operon structures and many novel transcripts located between or in antisense orientation to protein-coding regions. Interestingly, a high number of mRNAs (33%) is transcribed as leaderless transcripts. From the data, consensus promoter and ribosome binding site (RBS) motifs were identified and it was shown that the majority of genes in C. glutamicum are transcribed monocistronically, but operons containing up to 16 genes are also present. CONCLUSIONS: The comprehensive transcriptome map of C. glutamicum established in this study represents a major step forward towards a complete definition of genetic elements (e.g. promoter regions, gene starts and stops, 5'-UTRs, RBSs, transcript starts and ends) and provides the ideal basis for further analyses on transcriptional regulatory networks in this organism. The methods developed are easily applicable for other bacteria and have the potential to be used also for quantification of transcriptomes, replacing microarrays in the near future.

Comprehensive discovery and characterization of small RNAs in Corynebacterium glutamicum ATCC 13032.

BACKGROUND: Recent discoveries on bacterial transcriptomes gave evidence that small RNAs (sRNAs) have important regulatory roles in prokaryotic cells. Modern high-throughput sequencing approaches (RNA-Seq) enable the most detailed view on transcriptomes offering an unmatched comprehensiveness and single-base resolution. Whole transcriptome data obtained by RNA-Seq can be used to detect and characterize all transcript species, including small RNAs. Here, we describe an RNA-Seq approach for comprehensive detection and characterization of small RNAs from Corynebacterium glutamicum, an actinobacterium of high industrial relevance and model organism for medically important Corynebacterianeae, such as C. diphtheriae and Mycobacterium tuberculosis. RESULTS: In our RNA-Seq approach, total RNA from C. glutamicum ATCC 13032 was prepared from cultures grown in minimal medium at exponential growth or challenged by physical (heat shock, cold shock) or by chemical stresses (diamide, H2O2, NaCl) at this time point. Total RNA samples were pooled and sequencing libraries were prepared from the isolated small RNA fraction. High throughput short read sequencing and mapping yielded over 800 sRNA genes. By determining their 5'- and 3'-ends and inspection of their locations, these potential sRNA genes were classified into UTRs of mRNAs (316), cis-antisense sRNAs (543), and trans-encoded sRNAs (262). For 77 of trans-encoded sRNAs significant sequence and secondary structure conservation was found by a computational approach using a whole genome alignment with the closely related species C. efficiens YS-314 and C. diphtheriae NCTC 13129. Three selected trans-encoded sRNAs were characterized by Northern blot analysis and stress-specific transcript patterns were found. CONCLUSIONS: The study showed comparable numbers of sRNAs known from genome-wide surveys in other bacteria. In detail, our results give deep insight into the comprehensive equipment of sRNAs in C. glutamicum and provide a sound basis for further studies concerning the functions of these sRNAs.