Novel Immortal Cell Lines Support Cellular Heterogeneity in the Human Annulus Fibrosus

Introduction

Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors.

Methods

Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor β (TGFβ).

Results

The AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFβ induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections.

Conclusion

We report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair.     

Reactive oxygen species and melanoma: an explanation for gender differences in survival?

Epidemiological research consistently shows a female advantage in melanoma survival. So far, no definite candidate for the explanation of this phenomenon has emerged. We propose that gender differences in oxidative stress caused by radical oxygen species (ROS) underlie these survival differences. It is known that males express lower amounts of anti-oxidant enzymes, resulting in more oxidative stress than females. The primary melanoma environment is characterized by high ROS levels, from exogenous sources as well as ROS production within melanoma cells themselves. ROS are known to be able to promote metastasis through a wide variety of mechanisms. We hypothesize that the higher levels of ROS in men enhance selection of ROS-resistance in melanoma cells. Subsequently, ROS can stimulate the metastatic potential of melanoma cells. In addition, due to the lower anti-oxidant defenses in men, ROS produced by melanoma cells cause more damage to healthy tissues surrounding the tumor, further stimulating metastasis. Therefore, ROS may explain the observed differences between males and females in melanoma survival.     

A MODEL APPROACH FOR SIZE-SELECTIVE COMPETITION OF MARINE PHYTOPLANKTON FOR FLUCTUATING NITRATE AND AMMONIUM1

Phytoplankton size-selective competition for fluctuating nutrients was studied with the use of a numerical model, which describes nitrate and ammonium uptake, nitrate reduction to ammonium, and growth as a function of cell she under fluctuating nitrogen limitation. The only size-dependent parameter in the model was the cell nutrient quota. Related to this, the cell surface area per biomass was negatively correlated to cell volume, and the vacuole volume per biomass ratio was positively correlated to cell volume. Simulations showed an inverse correlation between the maximum specific growth rate and cell size under steady-state conditions. With nitrate as the limiting nitrogen source, nitrogen quotas were always higher than with ammonium at the same specific growth rate. Net passive transport of ammonium due to unspecific diffusion of ammonia across the plasma membrane decreased the affinity for ammonium, whereas the affinity for nitrate was not influenced. Transient state-specific ammonium uptake was not dependent on cell size. However, small algae always have the highest specific growth rate in ammonium-controlled systems according to our model. Transient state nitrate uptake rate was positively correlated to cell size because larger algae have a higher vacuole volume per biomass, in which nitrate can be stored. Despite their lower maximum growth rate, larger algae became dominant during simulations under fluctuating nitrate supply when amplitude of and the period between nitrate pulses were high enough. Results from model simulations were qualitatively validated by earlier observations that large diatoms become dominant under fluctuating conditions when nitrate is the main nitrogen source.     

In Vivo Induction of Functionally Suppressive Induced Regulatory T Cells from CD4+CD25- T Cells Using an Hsp70 Peptide

Therapeutic peptides that target antigen-specific regulatory T cells (Tregs) can suppress experimental autoimmune diseases. The heat shock protein (Hsp) 70, with its expression elevated in inflamed tissue, is a suitable candidate antigen because administration of both bacterial and mouse Hsp70 peptides has been shown to induce strong immune responses and to reduce inflammation via the activation or induction of Hsp specific Tregs. Although two subsets of Tregs exist, little is known about which subset of Tregs are activated by Hsp70 epitopes. Therefore, we set out to determine whether natural nTregs (derived from the thymus), or induced iTregs (formed in the periphery from CD4⁺CD25^- naïve T cells) were targeted after Hsp70-peptide immunization. We immunized mice with the previously identified Hsp70 T cell epitope B29 and investigated the formation of functional iTregs by using an in vitro suppression assay and adoptive transfer therapy in mice with experimental arthritis. To study the in vivo induction of Tregs after peptide immunization, we depleted CD25⁺ cells prior to immunization, allowing the in vivo formation of Tregs from CD4⁺CD25^- precursors. This approach allowed us to study in vivo B29-induced Tregs and to compare these cells with Tregs from non-depleted immunized mice. Our results show that using this approach, immunization induced CD4⁺CD25⁺ T cells in the periphery, and that these cells were suppressive in vitro. Additionally, adoptive transfer of B29-specific iTregs suppressed disease in a mouse model of arthritis. This study shows that immunization of mice with Hsp70 epitope B29 induces functionally suppressive iTregs from CD4⁺CD25^- T cells.     

FeEDDHA-facilitated Fe uptake in relation to the behaviour of FeEDDHA components in the soil-plant system as a function of time and dosage

FeEDDHA products are widely used to prevent and remedy Fe chlorosis in crops grown on calcareous soils. These products consist of a mixture of FeEDDHA components: racemic o,o-FeEDDHA, meso o,o-FeEDDHA, o,p-FeEDDHA and rest-FeEDDHA. The FeEDDHA components differ in physical and chemical properties, and as a consequence also in effectiveness as Fe fertilizer. In order to efficiently match dose, frequency and moment of FeEDDHA application with the Fe requirements of plants, it is important to understand the behaviour of the FeEDDHA components in the soil-plant system as a function of time and dosage, and to relate this behaviour to Fe uptake by plants. These issues have been examined in a pot trial study with soybean plants (Glycine max (L.) Merr. cv Mycogen 5072) grown on calcareous soil from Santomera, Spain. Four FeEDDHA treatments (two compositions, two dosages) were applied prior to the set in of chlorosis. Leaching of FeEDDHA components was prevented. Plant and soil were sampled every week, for six weeks. From one week onward the Fe concentration in the pore water was largely gouverned by racemic and meso o,o-FeEDDHA. The concentration behaviour of the o,o-FeEDDHA isomers underwent two stages: a strong decline within the first week resulting from linear adsorption, and a gradual decline from one week onward. For meso o,o-FeEDDHA, unlike racemic o,o-FeDDHA, the gradual decline could be mathematically well described with an exponential decay function. Soybean plants mainly took up Fe in the progressed vegetative stage (3rd and 4th week) and in the reproductive stage, when the pods were being filled with seeds (6th week). Fe uptake and removal of racemic o,o-FeEDDHA from the soil system display a similar time-trend, whereas the removal of meso o,o-FeEDDHA had a plant-independent character. This indicates the removal of racemic o,o-FeEDDHA is to a larger extent plant-related.     

Linking phylogenetic identities of bacteria to starch fermentation in an in vitro model of the large intestine by RNA-based stable isotope probing

Carbohydrates, including starches, are an important energy source for humans, and are known for their interactions with the microbiota in the digestive tract. Largely, those interactions are thought to promote human health. Using 16S ribosomal RNA (rRNA)-based stable isotope probing (SIP), we identified starch-fermenting bacteria under human colon-like conditions. To the microbiota of the TIM-2 in vitro model of the human colon 7.4 g l⁻¹ of [U-¹³C]-starch was added. RNA extracted from lumen samples after 0 (control), 2, 4 and 8 h was subjected to density-gradient ultracentrifugation. Terminal-restriction fragment length polymorphism (T-RFLP) fingerprinting and phylogenetic analyses of the labelled and unlabelled 16S rRNA suggested populations related to Ruminococcus bromii, Prevotella spp. and Eubacterium rectale to be involved in starch metabolism. Additionally, 16S rRNA related to that of Bifidobacterium adolescentis was abundant in all analysed fractions. While this might be due to the enrichment of high-GC RNA in high-density fractions, it could also indicate an active role in starch fermentation. Comparison of the T-RFLP fingerprints of experiments performed with labelled and unlabelled starch revealed Ruminococcus bromii as the primary degrader in starch fermentation in the studied model, as it was found to solely predominate in the labelled fractions. LC-MS analyses of the lumen and dialysate samples showed that, for both experiments, starch fermentation primarily yielded acetate, butyrate and propionate. Integration of molecular and metabolite data suggests metabolic cross-feeding in the system, where populations related to Ruminococcus bromii are the primary starch degrader, while those related to Prevotella spp., Bifidobacterium adolescentis and Eubacterium rectale might be further involved in the trophic chain.