Short-term glacier area changes, glacier geometry dependence, and regional climatic variations forcing, King George Island, Antarctica.

This study investigates the transient snowline (TSL) altitude for summer 2020, as well as glacial area loss in King George Island Icefields since 1988 using Sentinel-1 and 2 and Landsat Thematic Mapper (TM) imagery. Trends and anomalies in atmospheric temperature, U-wind, and V-wind were examined using ERA5 solutions. Results show the wet-snow zone corresponds to values of ≤ -13dB, and 44.3% of the glacial area is located above the TSL (≥ 300 m). Glacial area for 2020 is 999.95 km², and losses in the period represent 104.9 km² (error <1%) - a retreat of 3.17 km² / year. Glaciers in Keller Peninsula and Bellingshausen Dome lost the most area (28% and 17%, respectively) and did not have a TSL in 2020; followed by Warszawa (15%), Kraków (13%), and Eastern (10%), where the TSL was verified. Percentage area loss values increased with decreases in dimensions, area above TSL, and maximum elevation. Calving glaciers with ice-flow toward deeper and steeper submarine sectors (Bransfield Strait) exhibited greater glacier variations. The trend in warming atmospheric temperature was greater in the Bransfield Strait than in the Drake Passage. TSL and retreat difference between glaciers were influenced by climatic and ocean input, as well as multiple environmental factors.

Glacier fluctuations and a proglacial evolution in King George Bay (King George Island), Antarctica, since 1980 decade.

This study aims to investigate the glacier shrinkage and recent proglacial environment in King George Bay, Antarctica, since 1988 in response to climate change. Remote sensing data (SPOT, Sentinel, Landsat and Planet Scope images) were applied to glacial landforms and ice-marginal fluctuations mapping. Annual mean near-surface air temperature reanalysis solutions from ERA-Interim were analyzed. Moraines and glaciofluvial landforms were identified. The Ana Northern Glacier has the highest retreat value (3.64 km) (and area loss of 31%) in response to higher depth in frontal ice-margin and reveal ocean-glacier linkages. The Ana South Glacier changed from a tidewater glacier to land-terminating after 1995, and had an outline minimum elevation variation of 89 meters, a shrinkage of 0.63 km, and a new proglacial subaerial sector. The Ana South Glacier foreland had recessional moraines (probably formed between 1995 and 2022), lagoons, and lakes. There are many flutings in low-relief environments. The 1980-1989, 1990-1999, 2000-2009, 2010-2019 anomaly plots concerning to the 1980-2019 average for atmospheric temperature, are shown to be a driver of the local glacial trends.

Deciphering sources of PET signals in the tumor microenvironment of glioblastoma at cellular resolution.

Various cellular sources hamper interpretation of positron emission tomography (PET) biomarkers in the tumor microenvironment (TME). We developed an approach of immunomagnetic cell sorting after in vivo radiotracer injection (scRadiotracing) with three-dimensional (3D) histology to dissect the cellular allocation of PET signals in the TME. In mice with implanted glioblastoma, translocator protein (TSPO) radiotracer uptake per tumor cell was higher compared to tumor-associated microglia/macrophages (TAMs), validated by protein levels. Translation of in vitro scRadiotracing to patients with glioma immediately after tumor resection confirmed higher single-cell TSPO tracer uptake of tumor cells compared to immune cells. Across species, cellular radiotracer uptake explained the heterogeneity of individual TSPO-PET signals. In consideration of cellular tracer uptake and cell type abundance, tumor cells were the main contributor to TSPO enrichment in glioblastoma; however, proteomics identified potential PET targets highly specific for TAMs. Combining cellular tracer uptake measures with 3D histology facilitates precise allocation of PET signals and serves to validate emerging novel TAM-specific radioligands.

Translocator protein (18kDA) (TSPO) marks mesenchymal glioblastoma cell populations characterized by elevated numbers of tumor-associated macrophages.

TSPO is a promising novel tracer target for positron-emission tomography (PET) imaging of brain tumors. However, due to the heterogeneity of cell populations that contribute to the TSPO-PET signal, imaging interpretation may be challenging. We therefore evaluated TSPO enrichment/expression in connection with its underlying histopathological and molecular features in gliomas. We analyzed TSPO expression and its regulatory mechanisms in large in silico datasets and by performing direct bisulfite sequencing of the TSPO promotor. In glioblastoma tissue samples of our TSPO-PET imaging study cohort, we dissected the association of TSPO tracer enrichment and protein labeling with the expression of cell lineage markers by immunohistochemistry and fluorescence multiplex stains. Furthermore, we identified relevant TSPO-associated signaling pathways by RNA sequencing.We found that TSPO expression is associated with prognostically unfavorable glioma phenotypes and that TSPO promotor hypermethylation is linked to IDH mutation. Careful histological analysis revealed that TSPO immunohistochemistry correlates with the TSPO-PET signal and that TSPO is expressed by diverse cell populations. While tumor core areas are the major contributor to the overall TSPO signal, TSPO signals in the tumor rim are mainly driven by CD68-positive microglia/macrophages. Molecularly, high TSPO expression marks prognostically unfavorable glioblastoma cell subpopulations characterized by an enrichment of mesenchymal gene sets and higher amounts of tumor-associated macrophages.In conclusion, our study improves the understanding of TSPO as an imaging marker in gliomas by unveiling IDH-dependent differences in TSPO expression/regulation, regional heterogeneity of the TSPO PET signal and functional implications of TSPO in terms of tumor immune cell interactions.

Sclerotherapy of Venous Malformations Using Polidocanol: Effectiveness, Safety, and Predictors of Outcomes and Adverse Events.

PURPOSE: To assess the effectiveness, safety, and predictors of outcomes and adverse events for percutaneous sclerotherapy using polidocanol for the treatment of venous malformations (VMs). METHODS: A retrospective single-center analysis was performed, including patients with VMs who were treated with sclerotherapy using polidocanol between January 2011 and November 2021 at a tertiary center. Demographic characteristics, clinical data, and radiologic features were analyzed, and the influence of patient- and VM-related factors on the subjective clinical outcome and adverse events were investigated using a multivariate logistic regression analysis. RESULTS: In total, 167 patients who received 325 treatment sessions were included in this study. Overall symptom improvement was observed in 67.5%, stable symptoms were observed in 25.0%, and worsening was reported in 7.5% (clinical follow-up, 1.04 ± 1.67 years). The total adverse event rate was 10.2%, with an overall rate of 4.2% for permanent adverse events within the cohort. In multivariate analysis, the clinical outcome was worse in children (P = .01; 57.1% symptom improvement in children [age, <18 years] and 79.7% in adults), and adverse events were more frequently observed after the treatment of VMs located at the extremities (P < .01; 8.4% for VMs of the extremities and 1.2% for VMs in other locations). CONCLUSIONS: Sclerotherapy using polidocanol can be an effective treatment option for VMs with an acceptable safety profile. However, it can be less effective in children, and adverse events can be more frequently expected for VMs of the extremities.

Scaffold-Based (Matrigel™) 3D Culture Technique of Glioblastoma Recovers a Patient-like Immunosuppressive Phenotype.

Conventional 2D cultures are commonly used in cancer research though they come with limitations such as the lack of microenvironment or reduced cell heterogeneity. In this study, we investigated in what respect a scaffold-based (Matrigel™) 3D culture technique can ameliorate the limitations of 2D cultures. NGS-based bulk and single-cell sequencing of matched pairs of 2D and 3D models showed an altered transcription of key immune regulatory genes in around 36% of 3D models, indicating the reoccurrence of an immune suppressive phenotype. Changes included the presentation of different HLA surface molecules as well as cellular stressors. We also investigated the 3D tumor organoids in a co-culture setting with tumor-infiltrating lymphocytes (TILs). Of note, lymphocyte-mediated cell killing appeared less effective in clearing 3D models than their 2D counterparts. IFN-γ release, as well as live cell staining and proliferation analysis, pointed toward an elevated resistance of 3D models. In conclusion, we found that the scaffold-based (Matrigel™) 3D culture technique affects the transcriptional profile in a subset of GBM models. Thus, these models allow for depicting clinically relevant aspects of tumor-immune interaction, with the potential to explore immunotherapeutic approaches in an easily accessible in vitro system.

TSPO acts as an immune resistance gene involved in the T cell mediated immune control of glioblastoma.

Glioblastoma (GB) IDH-wildtype is the most malignant primary brain tumor. It is particularly resistant to current immunotherapies. Translocator protein 18 kDa (TSPO) is upregulated in GB and correlates with malignancy and poor prognosis, but also with increased immune infiltration. Here, we studied the role of TSPO in the regulation of immune resistance of human GB cells. The role of TSPO in tumor immune resistance was experimentally determined in primary brain tumor initiating cells (BTICs) and cell lines through genetic manipulation of TSPO expression and subsequent cocultures with antigen specific cytotoxic T cells and autologous tumor-infiltrating T cells. Death inducing intrinsic and extrinsic apoptotic pathways affected by TSPO were investigated. TSPO-regulated genes mediating apoptosis resistance in BTICs were identified through gene expression analysis and subsequent functional analyses. TSPO transcription in primary GB cells correlated with CD8+ T cell infiltration, cytotoxic activity of T cell infiltrate, expression of TNFR and IFNGR and with the activity of their downstream signalling pathways, as well as with the expression of TRAIL receptors. Coculture of BTICs with tumor reactive cytotoxic T cells or with T cell-derived factors induced TSPO up-regulation through T cell derived TNFα and IFNγ. Silencing of TSPO sensitized BTICs against T cell-mediated cytotoxicity. TSPO selectively protected BTICs against TRAIL-induced apoptosis by regulating apoptosis pathways. TSPO also regulated the expression of multiple genes associated with resistance against apoptosis. We conclude that TSPO expression in GB is induced through T cell-derived cytokines TNFα and IFNγ and that TSPO expression protects GB cells against cytotoxic T cell attack through TRAIL. Our data thereby provide an indication that therapeutic targeting of TSPO may be a suitable approach to sensitize GB to immune cell-mediated cytotoxicity by circumventing tumor intrinsic TRAIL resistance.

Update on quality assurance in neuropathology: Summary of the round robin trials on TERT promoter mutation, H3-3A mutation, 1p/19q codeletion, and KIAA1549::BRAF fusion testing in Germany in 2020 and 2021.

We previously reported on the first neuropathological round robin trials operated together with Quality in Pathology (QuIP) GmbH in 2018 and 2019 in Germany, i.e., the trials on IDH mutational testing and MGMT promoter methylation analysis [1]. For 2020 and 2021, the spectrum of round robin trials has been expanded to cover the most commonly used assays in neuropathological institutions. In addition to IDH mutation and MGMT promoter methylation testing, there is a long tradition for 1p/19q codeletion testing relevant in the context of the diagnosis of oligodendroglioma. With the 5th edition of the World Health Organization (WHO) classification of the central nervous system tumors, additional molecular markers came into focus: TERT promoter mutation is often assessed as a molecular diagnostic criterion for IDH-wildtype glioblastoma. Moreover, several molecular diagnostic markers have been introduced for pediatric brain tumors. Here, trials on KIAA1549::BRAF fusions (common in pilocytic astrocytomas) and H3-3A mutations (in diffuse midline gliomas, H3-K27-altered and diffuse hemispheric gliomas, H3-G34-mutant) were most desired by the neuropathological community. In this update, we report on these novel round robin trials. In summary, success rates in all four trials ranged from 75 to 96%, arguing for an overall high quality level in the field of molecular neuropathological diagnostics.

Evaluating the potential of urban areas for bat conservation with citizen science data.

Global change, including urbanisation, threatens many of the >1400 bat species. Nevertheless, certain areas within highly urbanised cities may be suitable to harbour bat populations. Thus, managing urban habitats could contribute to bat conservation. Here, we wanted to establish evidence-based recommendations on how to improve urban spaces for the protection of bats. In a team effort with >200 citizen scientists, we recorded bat vocalisations up to six times over the course of 2 years at each of 600 predefined sites in the Berlin metropolitan area. For each species we identified the preferred and non-preferred landscape features. Our results show that artificial light at night (ALAN) had a negative impact on all species. For soprano pipistrelles and mouse-eared bats ALAN had the largest effect sizes among all environmental predictors. Canopy cover and open water were especially important for bat species that forage along vegetation edges and for trawling bats, respectively. Occurrence probability of species foraging in open space decreased with increasing distance to water bodies. On a larger scale, impervious surfaces tended to have positive effects on some species that are specialised on foraging along edge structures. Our study constitutes an important contribution to the growing body of literature showing that despite the many negative impacts of urbanisation on wildlife, urban environments can harbour bat populations if certain conditions are met, such as access to vegetation and water bodies and low levels of ALAN. Our findings are of high relevance for urban planners and conservationists, as they allow inferences on how to manage urban spaces in a bat-friendly way. We recommend limiting ALAN to the minimum necessary and maintaining and creating uninterrupted vegetated corridors between areas with high levels of canopy cover and water bodies, in which ALAN should be entirely avoided.

A comprehensive DNA panel next generation sequencing approach supporting diagnostics and therapy prediction in neurooncology.

Recent updates in the classification of central nervous system (CNS) tumors have increased the need for molecular testing. Assessment of multiple alterations in parallel, complex combinations of gene sequence and chromosomal changes, as well as therapy prediction by identification of actionable mutations are the major challenges. We here report on a customized next generation sequencing (NGS)-based DNA panel assay that combines diagnostic and predictive testing and -as a comprehensive approach- allows for simultaneous single nucleotide variant (SNP) / small insertion/deletion (InDel), copy number variation (CNV) and loss of heterozygosity (LOH) detection. We analyzed formalin-fixed and paraffin-embedded (FFPE) DNA from a total of 104 patients with CNS tumors. After amplicon capture-based library preparation, sequencing was performed on the relatively cost-efficient Illiumina MiniSeq platform and evaluated with freely available bioinformatical tools. 57 genes for exonic SNP/InDel calling (19 of those in intronic regions for CNV analysis), 3 chromosomal arms and 4 entire chromosomes for CNV and LOH analysis were covered. Results were extensively validated. Our approach yielded high accuracy, sensitivity and specificity. It led to refined diagnoses in a relevant number of analyzed cases, reliably enabled complex subclassifications (e.g. for medulloblastomas) and identified actionable targets for clinical use. Thus, our single-platform approach is an efficient and powerful tool to comprehensively support molecular testing in neurooncology. Future functionality is guaranteed as novel upcoming biomarkers can be easily incorporated in a modular panel design.

Quality assurance in neuropathology: Experiences from the round robin trials on IDH mutation and MGMT promoter methylation testing launched by the Quality Assurance Initiative Pathology (QuIP) in 2018 and 2019.

We here report on the first neuropathological round robin trials initiated by the Quality Assurance Initiative Pathology (QuIP) in Germany in the years 2018 and 2019. Testing services as external laboratory controls were offered for IDH1-R132H immunohistochemistry in 2018 followed by a molecular trial for IDH1 and IDH2 mutations in 2019 including the rare mutational variants. Also in 2019, a trial on MGMT promoter methylation testing was offered. On a national scale, trial offers were well received with around 40 participating institutions. The international announcement of the molecular IDH1/IDH2 mutational trial achieved only moderate European outspread. Success rates in all three trials were excellent (IDH1-R132H immunohistochemistry 2018: 94%, 18 out of 20 possible points required; IDH1/IDH2 mutational status 2019: 100%, 19 out of 20 possible points required; MGMT promoter methylation 2019: 94%, 19 out of 20 possible points required) indicating that quality standards are high in the broad majority of the institutions. Trial participation also involved filling in a questionnaire asking for background information on local testing procedures. We here present a first assessment of the information collected providing unique insights in the landscape of molecular testing in neuropathology. Derived from this information we identify future challenges and provide an outlook on the development of quality assurance in the field of neuropathology.

Deciphering source and sink responses of potato plants (Solanum tuberosum L.) to elevated temperatures.

Potato is an important staple food with increasing popularity worldwide. Elevated temperatures significantly impair tuber yield and quality. Breeding heat-tolerant cultivars is therefore an urgent need to ensure sustainable potato production in the future. An integrated approach combining physiology, biochemistry, and molecular biology was undertaken to contribute to a better understanding of heat effects on source- (leaves) and sink-organs (tubers) in a heat-susceptible cultivar. An experimental set-up was designed allowing tissue-specific heat application. Elevated day and night (29°C/27°C) temperatures impaired photosynthesis and assimilate production. Biomass allocation shifted away from tubers towards leaves indicating reduced sink strength of developing tubers. Reduced sink strength of tubers was paralleled by decreased sucrose synthase activity and expression under elevated temperatures. Heat-mediated inhibition of tuber growth coincided with a decreased expression of the phloem-mobile tuberization signal SP6A in leaves. SP6A expression and photosynthesis were also affected, when only the belowground space was heated, and leaves were kept under control conditions. By contrast, the negative effects on tuber metabolism were attenuated, when only the shoot was subjected to elevated temperatures. This, together with transcriptional changes discussed, indicated a bidirectional communication between leaves and tubers to adjust the source capacity and/or sink strength to environmental conditions.

Chronophin regulates active vitamin B6 levels and transcriptomic features of glioblastoma cell lines cultured under non-adherent, serum-free conditions.

BACKGROUND: The phosphatase chronophin (CIN/PDXP) has been shown to be an important regulator of glioma cell migration and invasion. It has two known substrates: p-Ser3-cofilin, the phosphorylated form of the actin binding protein cofilin, and pyridoxal 5'-phosphate, the active form of vitamin B6. Phosphoregulation of cofilin, among other functions, plays an important role in cell migration, whereas active vitamin B6 is a cofactor for more than one hundred enzymatic reactions. The role of CIN has yet only been examined in glioblastoma cell line models derived under serum culture conditions. RESULTS: We found that CIN is highly expressed in cells cultured under non-adherent, serum-free conditions that are thought to better mimic the in vivo situation. Furthermore, the substrates of CIN, p-Ser3-cofilin and active vitamin B6, were significantly reduced as compared to cell lines cultured in serum-containing medium. To further examine its molecular role we stably knocked down the CIN protein with two different shRNA hairpins in the glioblastoma cell lines NCH421k and NCH644. Both cell lines did not show any significant alterations in proliferation but expression of differentiation markers (such as GFAP or TUBB3) was increased in the knockdown cell lines. In addition, colony formation was significantly impaired in NCH644. Of note, in both cell lines CIN knockdown increased active vitamin B6 levels with vitamin B6 being known to be important for S-adenosylmethionine biosynthesis. Nevertheless, global histone and DNA methylation remained unaltered as was chemoresistance towards temozolomide. To further elucidate the role of phosphocofilin in glioblastoma cells we applied inhibitors for ROCK1/2 and LIMK1/2 to our model. LIMK- and ROCK-inhibitor treatment alone was not toxic for glioblastoma cells. However, it had profound, but antagonistic effects in NCH421k and NCH644 under chemotherapy. CONCLUSION: In non-adherent glioblastoma cell lines cultured in serum-free medium, chronophin knockdown induces phenotypic changes, e.g. in colony formation and transcription, but these are highly dependent on the cellular background. The same is true for phenotypes observed after treatment with inhibitors for kinases regulating cofilin phosphorylation (ROCKs and LIMKs). Targeting the cofilin phosphorylation pathway might therefore not be a straightforward therapeutic option in glioblastoma.

Attenuated lipotoxicity and apoptosis is linked to exogenous and endogenous augmenter of liver regeneration by different pathways.

Nonalcoholic fatty liver disease (NAFLD) covers a spectrum from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. Free fatty acids (FFA) induce steatosis and lipo-toxicity and correlate with severity of NAFLD. In this study we aimed to investigate the role of exogenous and endogenous ALR (augmenter of liver regeneration) for FFA induced ER (endoplasmatic reticulum) -stress and lipoapoptosis. Primary human hepatocytes or hepatoma cells either treated with recombinant human ALR (rhALR, 15kDa) or expressing short form ALR (sfALR, 15kDa) were incubated with palmitic acid (PA) and analyzed for lipo-toxicity, -apoptosis, activation of ER-stress response pathways, triacylglycerides (TAG), mRNA and protein expression of lipid metabolizing genes. Both, exogenous rhALR and cytosolic sfALR reduced PA induced caspase 3 activity and Bax protein expression and therefore lipotoxicity. Endogenous sfALR but not rhALR treatment lowered TAG levels, diminished activation of ER-stress mediators C-Jun N-terminal kinase (JNK), X-box binding protein-1 (XBP1) and proapoptotic transcription factor C/EBP-homologous protein (CHOP), and reduced death receptor 5 protein expression. Cellular ALR exerts its lipid lowering and anti-apoptotic actions by enhancing FABP1, which binds toxic FFA, increasing mitochondrial ß-oxidation by elevating the mitochondrial FFA transporter CPT1α, and decreasing ELOVL6, which delivers toxic FFA metabolites. We found reduced hepatic mRNA levels of ALR in a high fat diet mouse model, and of ALR and FOXA2, a transcription factor inducing ALR expression, in human steatotic as well as NASH liver samples, which may explain increased lipid deposition and reduced ß-oxidation in NASH patients. Present study shows that exogenous and endogenous ALR reduce PA induced lipoapoptosis. Furthermore, cytosolic sfALR changes mRNA and protein expression of genes regulating lipid metabolism, reduces ER-stress finally impeding progression of NASH.

Genome-wide analysis of starch metabolism genes in potato (Solanum tuberosum L.).

BACKGROUND: Starch is the principle constituent of potato tubers and is of considerable importance for food and non-food applications. Its metabolism has been subject of extensive research over the past decades. Despite its importance, a description of the complete inventory of genes involved in starch metabolism and their genome organization in potato plants is still missing. Moreover, mechanisms regulating the expression of starch genes in leaves and tubers remain elusive with regard to differences between transitory and storage starch metabolism, respectively. This study aimed at identifying and mapping the complete set of potato starch genes, and to study their expression pattern in leaves and tubers using different sets of transcriptome data. Moreover, we wanted to uncover transcription factors co-regulated with starch accumulation in tubers in order to get insight into the regulation of starch metabolism. RESULTS: We identified 77 genomic loci encoding enzymes involved in starch metabolism. Novel isoforms of many enzymes were found. Their analysis will help to elucidate mechanisms of starch biosynthesis and degradation. Expression analysis of starch genes led to the identification of tissue-specific isoenzymes suggesting differences in the transcriptional regulation of starch metabolism between potato leaf and tuber tissues. Selection of genes predominantly expressed in developing potato tubers and exhibiting an expression pattern indicative for a role in starch biosynthesis enabled the identification of possible transcriptional regulators of tuber starch biosynthesis by co-expression analysis. CONCLUSIONS: This study provides the annotation of the complete set of starch metabolic genes in potato plants and their genomic localizations. Novel, so far undescribed, enzyme isoforms were revealed. Comparative transcriptome analysis enabled the identification of tuber- and leaf-specific isoforms of starch genes. This finding suggests distinct regulatory mechanisms in transitory and storage starch metabolism. Putative regulatory proteins of starch biosynthesis in potato tubers have been identified by co-expression and their expression was verified by quantitative RT-PCR.

α-MSH modulates cell adhesion and inflammatory responses of synovial fibroblasts from osteoarthritis patients.

INTRODUCTION: The synovium is a target for neuropeptides. Melanocortins have attained particular attention as they elicit antiinflammatory effects. Although synovial fluid from patients with rheumatic diseases contains α-melanocyte-stimulating hormone (α-MSH) it is unknown whether synovial fibroblasts generate α-MSH and respond to melanocortins. METHODS: Synovial tissue was obtained from osteoarthritis (OA) patients. Cells were isolated and prepared either as primary mixed synoviocytes or propagated as synovial fibroblasts (OASFs). Melanocortin receptor (MC) and proopiomelanocortin (POMC) expression were investigated by endpoint RT-PCR, immunofluorescence and Western immunoblotting. Functional coupling of MC1 was assessed by cAMP and Ca(2+) assays. Cell adhesion was monitored by the xCELLigence system. Secretion of α-MSH, tumour necrosis factor (TNF), interleukin (IL)-6 and IL-8 was determined by ELISA. RESULTS: OASFs in vitro expressed MC1. MC1 transcripts were present in synovial tissue and appropriate immunoreactivity was detected in synovial fibroblasts in situ. OASFs contained truncated POMC transcripts but neither full-length POMC mRNA, POMC protein nor α-MSH were detectable. In accordance with this only truncated POMC transcripts were present in synovial tissue. α-MSH increased cAMP dose-dependently but did not alter calcium in OASFs. α-MSH also enhanced adhesion of OASFs to fibronectin and reduced TNF, IL-6 and IL-8 secretion in primary mixed synoviocyte cultures. In OASFs, α-MSH modulated basal and TNF/IL-1ß-mediated secretion of IL-6 and IL-8. CONCLUSION: Synovial fibroblasts express MC1in vitro and in situ. α-MSH elicits biological effects in these cells suggesting an endogenous immunomodulatory role of melanocortins within the synovium. Our results encourage in vivo studies with melanocortins in OA models.

Tissue-engineering strategies to repair chondral and osteochondral tissue in osteoarthritis: use of mesenchymal stem cells.

Focal chondral or osteochondral lesions can be painful and disabling because they have insufficient intrinsic repair potential, and constitute one of the major extrinsic risk factors for osteoarthritis (OA). Attention has, therefore, been paid to regenerative therapeutic procedures for the early treatment of cartilaginous defects. Current treatments for OA are not regenerative and have little effect on the progressive degeneration of joint tissue. One major reason for this underrepresentation of regenerative therapy is that approaches to treating OA with cell-based strategies have to take into consideration the larger sizes of the defects, as compared with isolated focal articular-cartilage defects, and the underlying disease process. Here, we review current treatment strategies using mesenchymal stem cells (MSCs) for chondral and osteochondral tissue repair in trauma and OA-affected joints. We discuss tissue-engineering approaches, in preclinical large-animal models and clinical studies in humans, which use crude bone-marrow aspirates and MSCs from different tissue sources in combination with bioactive agents and materials.

Melanocortin 1 receptor-signaling deficiency results in an articular cartilage phenotype and accelerates pathogenesis of surgically induced murine osteoarthritis.

Proopiomelanocortin-derived peptides exert pleiotropic effects via binding to melanocortin receptors (MCR). MCR-subtypes have been detected in cartilage and bone and mediate an increasing number of effects in diathrodial joints. This study aims to determine the role of MC1-receptors (MC1) in joint physiology and pathogenesis of osteoarthritis (OA) using MC1-signaling deficient mice (Mc1re/e). OA was surgically induced in Mc1re/e and wild-type (WT) mice by transection of the medial meniscotibial ligament. Histomorphometry of Safranin O stained articular cartilage was performed with non-operated controls (11 weeks and 6 months) and 4/8 weeks past surgery. µCT-analysis for assessing epiphyseal bone architecture was performed as a longitudinal study at 4/8 weeks after OA-induction. Collagen II, ICAM-1 and MC1 expression was analysed by immunohistochemistry. Mc1re/e mice display less Safranin O and collagen II stained articular cartilage area compared to WT prior to OA-induction without signs of spontaneous cartilage surface erosion. This MC1-signaling deficiency related cartilage phenotype persisted in 6 month animals. At 4/8 weeks after OA-induction cartilage erosions were increased in Mc1re/e knees paralleled by weaker collagen II staining. Prior to OA-induction, Mc1re/e mice do not differ from WT with respect to bone parameters. During OA, Mc1re/e mice developed more osteophytes and had higher epiphyseal bone density and mass. Trabecular thickness was increased while concomitantly trabecular separation was decreased in Mc1re/e mice. Numbers of ICAM-positive chondrocytes were equal in non-operated 11 weeks Mc1re/e and WT whereas number of positive chondrocytes decreased during OA-progression. Unchallenged Mc1re/e mice display smaller articular cartilage covered area without OA-related surface erosions indicating that MC1-signaling is critical for proper cartilage matrix integrity and formation. When challenged with OA, Mc1re/e mice develop a more severe OA-pathology. Our data suggest that MC1-signaling protects against cartilage degradation and subchondral bone sclerosis in OA indicating a beneficial role of the POMC system in joint pathophysiology.

Experimental osteoarthritis models in mice.

Osteoarthritis (OA) is a slowly progressing, degenerative disorder of synovial joints culminating in the irreversible destruction of articular cartilage and subchondral bone. It affects almost everyone over the age of 65 and influences life quality of affected individuals with enormous costs to the health care system. Current therapeutic strategies seek to ameliorate pain and increase mobility; however, to date none of them halts disease progression or regenerates damaged cartilage or bone. Thus, there is an ultimate need for the development of new, noninvasive treatments that could substitute joint replacement for late- or end-stage patients. Therefore, osteoarthritis animal models for mimicking of all OA features are important. Mice develop an OA pathology that is comparable to humans, rapidly develop OA due to the short lifetime and show reproducible OA symptoms. They provide a versatile and widely used animal model for analyzing molecular mechanisms of OA pathology. One major advantage over large animal models is the availability of knockout or transgenic mice strains to examine genetic predispositions/contributions to OA.In this chapter, we describe three widely used instability-inducing murine osteoarthritis models. The most common two methods for surgical induction are: (1) destabilization of the medial meniscus (DMM) and (2) anterior cruciate ligament transection (ACLT). In the DMM model, the medial meniscotibial ligament is transected while in the ACLT model the anterior cruciate ligament is destroyed. In the third, chemical induced instability method, intraarticular collagenase is injected into the knee joint. Intraarticular collagenase weakens articular ligaments which cause instability of the joint, and full-blown OA develops within 6 weeks. For morphological evaluation, we correspond mainly to the recommendations of OARSI for histological assessment of osteoarthritis in mouse. For statistical evaluation summed or mean scores of all four knee areas (medial tibial plateau (MTP), medial tibial condyle (MFC), lateral tibial plateau (LTP) or lateral femoral condyle (LFC)), medial and/or lateral regions are used.In future, not only large animal models like guinea pigs, sheep, goats, or horses will be important for a better understanding of osteoarthritis, but especially the mouse model with its rapid development of osteoarthritis and its numerous advantages by providing knockout or transgenic strains will become more and more relevant for drug development and determination of genetic predispositions of osteoarthritis pathology.

Substrate specificity of the human renal sodium dicarboxylate cotransporter, hNaDC-3, under voltage-clamp conditions.

Proximal tubule cells extract dicarboxylates from filtrate and blood, using cotransporters located in the brush border [sodium dicarboxylate cotransporter (NaDC-1)] and basolateral cell membrane (NaDC-3). We expressed the human NaDC-3 (hNaDC-3) in Xenopus laevis oocytes and characterized it by the two-electrode voltage-clamp technique. At -60 mV, succinate (4 carbons) and glutarate (5 carbons) generated inward currents due to translocation of three sodium ions and one divalent dicarboxylate, whereas oxalate (2 carbons) and malonate (3 carbons) did not. The cis-dicarboxylate maleate produced currents smaller in magnitude, whereas the trans-dicarboxylate fumarate generated currents similar to succinate. The substituted succinate derivatives, malate, 2,2- and 2,3-dimethylsuccinate, and 2,3-dimercaptosuccinate elicited inward currents, whereas aspartate and guanidinosuccinate showed hardly detectable currents. The C-5 dicarboxylates glutarate and alpha-ketoglutarate produced larger currents than succinate; glutamate and folate failed to cause inward currents. Kinetic analysis revealed, at -60 mV, K(0.5) values of 25 +/- 12 microM for succinate and 45 +/- 13 microM for alpha-ketoglutarate, values close to the plasma concentration of these compounds. For both compounds, the K(0.5) was independent of voltage, whereas the maximal current increased with hyperpolarization. As opposed to the rat and flounder orthologs, hNaDC-3 was hardly inhibited by lithium concentrations up to 5 mM. In the absence of sodium, however, lithium can mediate succinate-dependent currents. The narrow substrate specificity prevents interaction of drugs with dicarboxylate-like structure with hNaDC-3 and ensures sufficient support of the proximal tubule cells with alpha-ketoglutarate for anion secretion via organic anion transporter 1 or 3.