Effect of Increased Lactate Dehydrogenase A Activity and Aerobic Glycolysis on the Proinflammatory Profile of Autoimmune CD8+ T Cells in Rheumatoid Arthritis.

OBJECTIVE: CD8+ T cells contribute to rheumatoid arthritis (RA) by releasing proinflammatory and cytolytic mediators, even in a challenging hypoxic and nutrient-poor microenvironment such as the synovial membrane. This study was undertaken to explore the mechanisms through which CD8+ T cells meet their metabolic demands in the blood and synovial membrane of patients with RA. METHODS: Purified blood CD8+ T cells from patients with RA, patients with psoriatic arthritis (PsA), and patients with spondyloarthritis (SpA), as well as healthy control subjects, and CD8+ T cells from RA synovial membrane were stimulated in medium containing 13 C-labeled metabolic substrates in the presence or absence of metabolic inhibitors, under conditions of normoxia or hypoxia. The production of metabolic intermediates was quantified by 1 H-nuclear magnetic resonance. The expression of metabolic enzymes, transcription factors, and immune effector molecules was assessed at both the messenger RNA (mRNA) and protein levels. CD8+ T cell functional studies were performed. RESULTS: RA blood CD8+ T cells met their metabolic demands through aerobic glycolysis, production of uniformly 13 C-enriched lactate in the RA blood (2.6 to 3.7-fold higher than in patients with SpA, patients with PsA, and healthy controls; P < 0.01), and induction of glutaminolysis. Overexpression of Warburg effect-linked enzymes in all RA CD8+ T cell subsets maintained this metabolic profile, conferring to the cells the capacity to proliferate under hypoxia and low-glucose conditions. In all RA CD8+ T cell subsets, lactate dehydrogenase A (LDHA) was overexpressed at the mRNA level (P < 0.03 versus controls; n = 6 per group) and protein level (P < 0.05 versus controls; n = 17 RA patients, n = 9 controls). In RA blood, inhibition of LDHA with FX11 led to reductions in lipogenesis, migration and proliferation of CD8+ T cells, and CD8+ T cell effector functions, while production of reactive oxygen species was increased by 1.5-fold (P < 0.03 versus controls). Following inhibition of LDHA with FX11, RA CD8+ T cells lost their capacity to induce healthy B cells to develop a proinflammatory phenotype. Similar metabolic alterations were observed in RA CD8+ T cells from the synovial membrane. CONCLUSION: Remodeling glucose and glutamine metabolism in RA CD8+ T cells by targeting LDHA activity can reduce the deleterious inflammatory and cytolytic contributions of these cells to the development of autoimmunity.

Multipotent mesenchymal stromal cells are sensitive to thermic stress - potential implications for therapeutic hyperthermia.

Purpose: Hyperthermia demonstrated clinical efficacy in multimodal cancer treatment. Multipotent mesenchymal stromal cells (MSCs) as part of the tumor-supporting stroma modulate tumor response and tissue regeneration after hyperthermia. We aimed to investigate the effects of hyperthermia on the survival, stem cell characteristics and heat shock expression of human MSCs.Materials and methods: Human MSCs and normal human dermal fibroblasts (NHDFs) were exposed to temperatures between 37 °C and 44 °C for 60 min, and hyperthermic sensitivity was examined by clonogenicity, proliferation and viability assays. The influence of 42 °C hyperthermia on the MSCs' adhesion potential, migratory capacity, surface marker expression and multi-lineage differentiation capability was investigated. Cell cycle distribution, apoptosis and senescence after 42 °C hyperthermia were determined by flow cytometry and ß-galactosidase staining. Heat shock protein expression was determined by Western Blots.Results: MSCs exhibited decreased clonogenic survival after 40 °C and 42 °C hyperthermia compared to NHDFs, while proliferative activity and viability were comparable after hyperthermia up to 44 °C. MSC adhesion was reduced after 42 °C hyperthermia, while the characteristic surface marker expression and the migratory ability remained unaffected in 42 °C hyperthermia-exposed MSCs. 42 °C hyperthermia diminished the adipogenic differential potential of all tested MSC samples. A pronounced G2/M arrest was found after 42 °C hyperthermia and was associated with increased apoptosis and senescence levels in MSCs. MSCs exhibited slightly lower heat shock protein levels compared to NHDFs.Conclusion: Human MSCs exhibit a thermosensitive phenotype which reduced the multipotent cells' regenerative abilities, resulting in impaired tissue regeneration after hyperthermia treatment or thermal injuries. On the other hand, tumor-associated MSCs may be efficiently targeted by hyperthermia treatment.

Human mesenchymal stem cells are resistant to UV-B irradiation.

Albeit being an effective therapy for various cutaneous conditions, UV-B irradiation can cause severe skin damage. While multipotent mesenchymal stem cells (MSCs) may aid the regeneration of UV-B-induced skin injuries, the influence of UV-B irradiation on MSCs remains widely unknown. Here, we show that human MSCs are relatively resistant to UV-B irradiation compared to dermal fibroblasts. MSCs exhibited higher clonogenic survival, proliferative activity and viability than dermal fibroblasts after exposure to UV-B irradiation. Cellular adhesion, morphology and expression of characteristic surface marker patterns remained largely unaffected in UV-irradiated MSCs. The differentiation ability along the adipogenic, osteogenic and chondrogenic lineages was preserved after UV-B treatment. However, UV-B radiation resulted in a reduced ability of MSCs and dermal fibroblasts to migrate. MSCs exhibited low apoptosis rates after UV-B irradiation and repaired UV-B-induced cyclobutane pyrimidine dimers more efficiently than dermal fibroblasts. UV-B irradiation led to prolonged p53 protein stability and increased p21 protein expression resulting in a prolonged G2 arrest and senescence induction in MSCs. The observed resistance may contribute to the ability of these multipotent cells to aid the regeneration of UV-B-induced skin injuries.

Mesenchymal stem cells preserve their stem cell traits after exposure to antimetabolite chemotherapy.

BACKGROUND: Mesenchymal stem cells (MSCs) participate in the regeneration of tissue lesions induced by antimetabolite chemotherapy; however, the influence of this class of anti-cancer compounds on the stem cells remains largely unknown. METHODS: The survival of MSCs after exposure to 5-fluorouracil (5-FU) and gemcitabine was measured by viability and clonogenic assays. MSC morphology, surface marker expression, adhesion potential, cellular velocity and differentiation potential were determined after antimetabolite treatment. Cell cycle distribution and apoptosis were assessed using flow cytometry, and senescence induction was evaluated by beta-galactosidase staining. Gene expression arrays were used to analyze the expression of enzymes involved in DNA metabolism and multidrug resistance. RESULTS: Here, we show that human primary bone marrow MSCs are relatively resistant to treatment with the widely used antimetabolite drugs 5-FU and gemcitabine. The stem cells were able to largely retain their functional abilities and defining stem cell traits after antimetabolite exposure. MSCs surface markers were found stably expressed, and the characteristic multi-lineage differentiation potential was maintained irrespective of 5-FU or gemcitabine treatment. High expression levels of enzymes involved in DNA metabolism and multidrug resistance transporters may contribute to the resistance to antimetabolite chemotherapy. DISCUSSION: The observed resistance and functional integrity may form the basis for further investigations of MSCs as a potential therapy for antimetabolite-induced tissue damage.

A Microcavity Array-Based 3D Model System of the Hematopoietic Stem Cell Niche.

Despite huge advances in recent years, the interaction between hematopoietic stem and progenitor cells (HSPCs) and their niches in the bone marrow is still far from being fully understood. One reason is that hematopoiesis is a multi-step maturation process leading to HSPC heterogeneity. Subpopulations of HSPCs can be identified by clonogenic assays or in serial transplantation experiments in mice following sublethal irradiation, but it is very complex to reproduce or even maintain stem cell plasticity in vitro. Advanced model systems have been developed that allow to precisely control and analyze key components of the physiologic microenvironment for not only fundamental research purposes but, as a long-term goal, also for clinical applications. In this chapter, we describe our approach of building an artificial hematopoietic stem cell niche in the form of polymer film-based microcavities with a diameter of 300 µm and a depth of up to 300 µm and arranged in a 634-cavity array. The polymer films are provided with 3 µm pores and thus allow perfusion of the culture medium. The microcavity arrays can be inserted into a microbioreactor where a closed circulation loop can be tightly controlled with regard to medium flow and gas supply. The microcavity arrays were used for a three-dimensional (3D) co-culture of MSCs and HSPCs in a defined ratio over a time period of up to 21 days. With this setup, it could be demonstrated that the HSPCs maintained their stem cell characteristics more efficiently as compared to conventional monolayer co-culture controls.

The current understanding of mesenchymal stem cells as potential attenuators of chemotherapy-induced toxicity.

Chemotherapeutic agents are part of the standard treatment algorithms for many malignancies; however, their application and dosage are limited by their toxic effects to normal tissues. Chemotherapy-induced toxicities can be long-lasting and may be incompletely reversible; therefore, causative therapies for chemotherapy-dependent side effects are needed, especially considering the increasing survival rates of treated cancer patients. Mesenchymal stem cells (MSCs) have been shown to exhibit regenerative abilities for various forms of tissue damage. Preclinical data suggest that MSCs may also help to alleviate tissue lesions caused by chemotherapeutic agents, mainly by establishing a protective microenvironment for functional cells. Due to the systemic administration of most anticancer agents, the effects of these drugs on the MSCs themselves are of crucial importance to use stem cell-based approaches for the treatment of chemotherapy-induced tissue toxicities. Here, we present a concise review of the published data regarding the influence of various classes of chemotherapeutic agents on the survival, stem cell characteristics and physiological functions of MSCs. Molecular mechanisms underlying the effects are outlined, and resulting challenges of MSC-based treatments for chemotherapy-induced tissue injuries are discussed.

Author Correction: Dynamic cellular phenotyping defines specific mobilization mechanisms of human hematopoietic stem and progenitor cells induced by SDF1α versus synthetic agents.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

The Radiation Resistance of Human Multipotent Mesenchymal Stromal Cells Is Independent of Their Tissue of Origin.

PURPOSE: Human mesenchymal stromal cells (MSCs) may aid the regeneration of ionizing radiation (IR)-induced tissue damage. They can be harvested from different tissues for clinical purposes; however, the role of the tissue source on the radiation response of human MSCs remains unknown. METHODS AND MATERIALS: Human MSCs were isolated from adipose tissue, bone marrow, and umbilical cord, and cellular survival, proliferation, and apoptosis were measured after irradiation. The influence of IR on the defining functions of MSCs was assessed, and cell morphology, surface marker expression, and the differentiation potential were examined. Western blot analyses were performed to assess the activation of DNA damage signaling and repair pathways. RESULTS: MSCs from adipose tissue, bone marrow, and umbilical cord exhibited a relative radioresistance independent of their tissue of origin. Defining properties including cellular adhesion and surface marker expression were preserved, and irradiated MSCs maintained their potential for multilineage differentiation irrespective of their tissue source. Analysis of activated DNA damage recognition and repair pathways demonstrated an efficient repair of IR-induced DNA double-strand breaks in MSCs from different tissues, thereby influencing the induction of apoptosis. CONCLUSIONS: These data show for the first time that MSCs are resistant to IR and largely preserve their defining functions after irradiation irrespective of their tissue of origin. Efficient repair of IR-induced DNA double-strand breaks and consecutive reduction of apoptosis induction may contribute to the tissue-independent radiation resistance of MSCs.

Dynamic cellular phynotyping defines specific mobilization mechanisms of human hematopoietic stem and progenitor cells induced by SDF1α versus synthetic agents.

Efficient mobilization of hematopoietic stem and progenitor cells (HSPC) is one of the most crucial issues for harvesting an adequate amount of peripheral HSPC for successful clinical transplantation. Applying well-defined surrogate models for the bone marrow niche, live cell imaging techniques, and novel tools in statistical physics, we have quantified the functionality of two mobilization agents that have been applied in the clinic, NOX-A12 and AMD3100 (plerixafor), as compared to a naturally occurring chemokine in the bone marrow, SDF1α. We found that NOX-A12, an L-enantiomeric RNA oligonucleotide to SDF1, significantly reduced the adhesion of HSPC to the niche surface mediated via the CXCR4-SDF1α axis, and stretched the migration trajectories of the HSPC. We found that the stretching of trajectories by NOX-A12 was more prominent than that by SDF1α. In contrast, plerixafor exhibited no detectable interference with adhesion and migration. We also found that the deformation of HSPC induced by SDF1α or plerixafor was also drastically suppressed in the presence of NOX-A12. This novel technology of quantitative assessment of "dynamic phenotypes" by physical tools has therefore enabled us to define different mechanisms of function for various extrinsic factors compared to naturally occurring chemokines.

Human mesenchymal stem cells lose their functional properties after paclitaxel treatment.

Mesenchymal stem cells (MSCs) are an integral part of the bone marrow niche and aid in the protection, regeneration and proliferation of hematopoietic stem cells after exposure to myelotoxic taxane anti-cancer agents, but the influence of taxane compounds on MSCs themselves remains incompletely understood. Here, we show that bone marrow-derived MSCs are highly sensitive even to low concentrations of the prototypical taxane compound paclitaxel. While MSCs remained metabolically viable, they were strongly impaired regarding both their proliferation and their functional capabilities after exposure to paclitaxel. Paclitaxel treatment resulted in reduced cell migration, delays in cellular adhesion and significant dose-dependent inhibition of the stem cells' characteristic multi-lineage differentiation potential. Cellular morphology and expression of the defining surface markers remained largely unaltered. Paclitaxel only marginally increased apoptosis in MSCs, but strongly induced premature senescence in these stem cells, thereby explaining the preservation of the metabolic activity of functionally inactivated MSCs. The reported sensitivity of MSC function to paclitaxel treatment may help to explain the severe bone marrow toxicities commonly caused by taxane-based anti-cancer treatments.

Cisplatin radiosensitizes radioresistant human mesenchymal stem cells.

Cisplatin-based chemo-radiotherapy is widely used to treat cancers with often severe therapy-associated late toxicities. While mesenchymal stem cells (MSCs) were shown to aid regeneration of cisplatin- or radiation-induced tissue lesions, the effect of the combined treatment on the stem cells remains unknown. Here we demonstrate that cisplatin treatment radiosensitized human bone marrow-derived MSCs in a dose-dependent manner and increased levels of radiation-induced apoptosis. However, the defining stem cell properties of MSCs remained largely intact after cisplatin-based chemo-radiation, and stem cell motility, adhesion, surface marker expression and the characteristic differentiation potential were not significantly influenced. The increased cisplatin-mediated radiosensitivity was associated with a cell cycle shift of MSCs towards the radiosensitive G2/M phase and increased residual DNA double-strand breaks. These data demonstrate for the first time a dose-dependent radiosensitization effect of MSCs by cisplatin. Clinically, the observed increase in radiation sensitivity and subsequent loss of regenerative MSCs may contribute to the often severe late toxicities observed after cisplatin-based chemo-radiotherapy in cancer patients.

Effects of CTGF Blockade on Attenuation and Reversal of Radiation-Induced Pulmonary Fibrosis.

Background: Radiotherapy is a mainstay for the treatment of lung cancer that can induce pneumonitis or pulmonary fibrosis. The matricellular protein connective tissue growth factor (CTGF) is a central mediator of tissue remodeling. Methods: A radiation-induced mouse model of pulmonary fibrosis was used to determine if transient administration of a human antibody to CTGF (FG-3019) started at different times before or after 20 Gy thoracic irradiation reduced acute and chronic radiation toxicity. Mice (25 mice/group; 10 mice/group in a confirmation study) were examined by computed tomography, histology, gene expression changes, and for survival. In vitro experiments were performed to directly study the interaction of CTGF blockade and radiation. All statistical tests were two-sided. Results: Administration of FG-3019 prevented (∼50%-80%) or reversed (∼50%) lung remodeling, improved lung function, improved mouse health, and rescued mice from lethal irradiation ( P < .01). Importantly, when antibody treatment was initiated at 16 weeks after thoracic irradiation, FG-3019 reversed established lung remodeling and restored lung function. CTGF blockade abrogated M2 polarized macrophage influx, normalized radiation-induced gene expression changes, and reduced myofibroblast abundance and Osteopontin expression. Conclusion: These results indicate that blocking CTGF attenuates radiation-induced pulmonary remodeling and can reverse the process after initiation. CTGF has a central role in radiation-induced fibrogenesis, and FG-3019 may benefit patients with radiation-induced pulmonary fibrosis or patients with other forms or origin of chronic fibrotic diseases.

Mesenchymal stem cells are sensitive to bleomycin treatment.

Mesenchymal stem cells (MSCs) have been shown to attenuate pulmonary damage induced by bleomycin-based anticancer treatments, but the influence of bleomycin on the stem cells themselves remains largely unknown. Here, we demonstrate that human bone marrow-derived MSCs are relatively sensitive to bleomycin exposure compared to adult fibroblasts. MSCs revealed increased levels of apoptosis after bleomycin treatment, while cellular morphology, stem cell surface marker expression and the ability for adhesion and migration remained unchanged. Bleomycin treatment also resulted in a reduced adipogenic differentiation potential of these stem cells. MSCs were found to efficiently repair DNA double strand breaks induced by bleomycin, mostly through non-homologous end joining repair. Low mRNA and protein expression levels of the inactivating enzyme bleomycin hydrolase were detected in MSCs that may contribute to the observed bleomycin-sensitive phenotype of these cells. The sensitivity of MSCs against bleomycin needs to be taken into consideration for ongoing and future treatment protocols investigating these stem cells as a potential treatment option for bleomycin-induced pulmonary damage in the clinic.

Evaluation of GMP-compliant culture media for in vitro expansion of human bone marrow mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) from human bone marrow serve as a resource for cell-based therapies in regenerative medicine. Clinical applications require standardized protocols according to good manufacturing practice (GMP) guidelines. Donor variability as well as the intrinsic heterogeneity of MSC populations must be taken into consideration. The composition of the culture medium is a key factor in successful MSC expansion. The aim of this study was to comparatively assess the efficiency of xeno-free human platelet lysate (HPL)-based cell expansion with two commercially available media-StemPro MSC SFM CTS (for human ex vivo tissue and cell culture processing applications) and MSCGM (non-GMP-compliant, for research only)-in an academic setting as the first optimization step toward GMP-compliant manufacturing. We report the feasibility of MSC expansion up to the yielded cell number with all three media. MSCs exhibited the typical fibroblastoid morphology, with distinct differences in cell size depending on the medium. The differentiation capacity and characteristic immunophenotype were confirmed for all MSC populations. Proliferation was highest using StemPro MSC SFM CTS, whereas HPL medium was more cost-effective and its composition could be adjusted individually according to the respective needs. In summary, we present a comprehensive evaluation of GMP-compatible culture media for MSC expansion. Both StemPro and HPL medium proved to be suitable for clinical application and allowed sufficient cell proliferation. Specific differences were observed and should be considered according to the intended use. This study provides a detailed cost analysis and tools that may be helpful for the establishment of GMP-compliant MSC expansion.

Microcavity arrays as an in vitro model system of the bone marrow niche for hematopoietic stem cells.

In previous studies human mesenchymal stromal cells (MSCs) maintained the "stemness" of human hematopoietic progenitor cells (HPCs) through direct cell-cell contact in two-dimensional co-culture systems. We establish a three-dimensional (3D) co-culture system based on a custom-made chip, the 3(D)-KITChip, as an in vitro model system of the human hematopoietic stem cell niche. This array of up to 625 microcavities, with 300 µm size in each orientation, was inserted into a microfluidic bioreactor. The microcavities of the 3(D)-KITChip were inoculated with human bone marrow MSCs together with umbilical cord blood HPCs. MSCs used the microcavities as a scaffold to build a complex 3D mesh. HPCs were distributed three-dimensionally inside this MSC network and formed ß-catenin- and N-cadherin-based intercellular junctions to the surrounding MSCs. Using RT(2)-PCR and western blots, we demonstrate that a proportion of HPCs maintained the expression of CD34 throughout a culture period of 14 days. In colony-forming unit assays, the hematopoietic stem cell plasticity remained similar after 14 days of bioreactor co-culture, whereas monolayer co-cultures showed increasing signs of HPC differentiation and loss of stemness. These data support the notion that the 3D microenvironment created within the microcavity array preserves vital stem cell functions of HPCs more efficiently than conventional co-culture systems.

Mesenchymal stem cells exhibit resistance to topoisomerase inhibition.

BACKGROUND: Inhibition of cellular topoisomerases has been established as an effective way of treating certain cancers, albeit with often high levels of toxicity to the bone marrow. While the involvement of mesenchymal stem cells (MSCs) in bone marrow homeostasis and regeneration has been well established, the effects of topoisomerase-inhibiting anticancer agents remain largely unknown. MATERIALS AND METHODS: Human bone marrow MSCs were treated with topoisomerase I inhibitor irinotecan or topoisomerase II inhibitor etoposide, and survival and apoptosis levels were measured. The influence of topoisomerase inhibition on cellular morphology, adhesion and migration potential and the ability to differentiate was assessed. Additionally, the role of individual DNA double-strand break repair pathways in MSCs was investigated as a potential cellular mechanism of resistance to topoisomerase inhibitors. RESULTS: Human bone marrow MSCs were found relatively resistant to topoisomerase I and II inhibitors and show survival levels comparable to these of differentiated fibroblasts. Treatment with irinotecan or etoposide did not significantly influence cellular adhesion, migratory ability, surface marker expression or induction of apoptosis in human MSCs. The ability to differentiate was found preserved in MSCs after exposure to high doses of irinotecan or etoposide. MSCs were able to efficiently repair DNA double-strand breaks induced by topoisomerase inhibitors both by non-homologous end joining and homologous recombination pathways. CONCLUSION: Our data demonstrate a topoisomerase-resistant phenotype of human MSCs that may at least in part be due to the stem cells' ability to efficiently remove DNA damage caused by these anticancer agents. The observed resistance of MSCs warrants further investigation of these cells as a potential therapeutic option for treating topoisomerase inhibitor-induced bone marrow damage.

Mesenchymal stem cells maintain their defining stem cell characteristics after treatment with cisplatin.

Mesenchymal stem cells (MSCs) aid the regeneration of tissues damaged by treatment with cisplatin. However, the effects of this cytotoxic drug on the stem cells have been largely unknown. Here we demonstrate that human bone marrow-derived MSCs are relatively resistant to cisplatin treatment and show resistance levels comparable to these of differentiated fibroblasts. Cisplatin did not affect cellular morphology, adhesion or induction of apoptosis in MSCs. The potential for differentiation was preserved after exposure to cisplatin, and established MSC surface markers were observed to be stably expressed irrespective of cisplatin treatment. Cytoskeletal rearrangements and high expression levels of individual heat shock proteins were detected in MSCs and may be partly responsible for the observed cisplatin resistance. The cisplatin-resistant phenotype of human MSCs supports the concept of further investigating these stem cells as a potential treatment option for cisplatin-induced tissue damage.

Radio-resistant mesenchymal stem cells: mechanisms of resistance and potential implications for the clinic.

Mesenchymal stem cells (MSCs) comprise a heterogeneous population of multipotent stromal cells and can be isolated from various tissues and organs. Due to their regenerative potential, they have been subject to intense research efforts, and they may provide an efficient means for treating radiation-induced tissue damage. MSCs are relatively resistant to ionizing radiation and retain their stem cell characteristics even after high radiation doses. The underlying mechanisms for the observed MSC radioresistance have been extensively studied and may involve efficient DNA damage recognition, double strand break repair and evasion of apoptosis. Here, we present a concise review of the published scientific data on the radiobiological features of MSCs. The involvement of different DNA damage recognition and repair pathways in the creation of a radioresistant MSC phenotype is outlined, and the roles of apoptosis, senescence and autophagy regarding the reported radioresistance are summarized. Finally, potential influences of the radioresistant MSCs for the clinic are discussed with respect to the repair and radioprotection of irradiated tissues.

Quantifying adhesion mechanisms and dynamics of human hematopoietic stem and progenitor cells.

Using planar lipid membranes with precisely defined concentrations of specific ligands, we have determined the binding strength between human hematopoietic stem cells (HSC) and the bone marrow niche. The relative significance of HSC adhesion to the surrogate niche models via SDF1α-CXCR4 or N-cadherin axes was quantified by (a) the fraction of adherent cells, (b) the area of tight adhesion, and (c) the critical pressure for cell detachment. We have demonstrated that the binding of HSC to the niche model is a cooperative process, and the adhesion mediated by the CXCR4- SDF1α axis is stronger than that by homophilic N-cadherin binding. The statistical image analysis of stochastic morphological dynamics unraveled that HSC dissipated energy by undergoing oscillatory deformation. The combination of an in vitro niche model and novel physical tools has enabled us to quantitatively determine the relative significance of binding mechanisms between normal HSC versus leukemia blasts to the bone marrow niche.

Novel activating mutation of human calcium-sensing receptor in a family with autosomal dominant hypocalcaemia.

INTRODUCTION: Autosomal dominant hypocalcaemia (ADH) is caused by activating mutations in the calcium sensing receptor gene (CaR) and characterised by mostly asymptomatic mild to moderate hypocalcaemia with low, inappropriately serum concentration of PTH. OBJECTIVE: The purpose of the present study was to biochemically and functionally characterise a novel mutation of CaR. PATIENTS: A female proband presenting with hypocalcaemia was diagnosed to have "idiopathic hypoparathyroidism" at the age of 10 with a history of muscle pain and cramps. Further examinations demonstrated hypocalcaemia in nine additional family members, affecting three generations. MAIN OUTCOME MEASURE: P136L CaR mutation was predicted to cause gain of function of CaR. RESULTS: Affected family members showed relevant hypocalcaemia (mean ± SD; 1.9 ± 0.1 mmol/l). Patient history included mild seizures and recurrent nephrolithiasis. Genetic analysis confirmed that hypocalcaemia cosegregated with a heterozygous mutation at codon 136 (CCC → CTC/Pro → Leu) in exon 3 of CaR confirming the diagnosis of ADH. For in vitro studies P136L mutant CaR was generated by site-directed mutagenesis and examined in transiently transfected HEK293 cells. Extracellular calcium stimulation of transiently transfected HEK293 cells showed significantly increased intracellular Ca(2+) mobilisation and MAPK activity for mutant P136L CaR compared to wild type CaR. CONCLUSIONS: The present study gives insight about a novel activating mutation of CaR and confirms that the novel P136L-CaR mutation is responsible for ADH in this family.