NUPR1 preserves insulin secretion of pancreatic ß-cells during inflammatory stress by multiple low-dose streptozotocin and high-fat diet.

Obesity is associated with dyslipidemia and subclinical inflammation that promotes metabolic disturbances including insulin resistance and pancreatic ß-cell dysfunction. The nuclear protein, transcriptional regulator 1 (NUPR1) responds to cellular stresses and features tissue protective properties. To characterize the role of NUPR1 in endocrine pancreatic islets during inflammatory stress, we generated transgenic mice with ß-cell-specific Nupr1 overexpression (ßNUPR1). Under normal conditions, ßNUPR1 mice did not differ from wild type (WT) littermates and display normal glucose homeostasis and ß-cell mass. For induction of inflammatory conditions, mice were treated with multiple low-dose streptozotocin (mld-STZ) and/or fed a high-fat diet (HFD). All treatments significantly worsened glycaemia in WT mice, while ßNUPR1 mice substantially preserved insulin secretion and glucose tolerance. HFD increased ß-cell mass in all animals, with ßNUPR1 mice tending to show higher values. The improved outcome of ßNUPR1 mice was accompanied by decreased NF-κB activation and lymphocyte infiltration in response to mld-STZ. In vitro, isolated ßNUPR1 islets preserved insulin secretion and content with insignificantly low apoptosis during culture stress and IL-1ß exposure. These findings suggest that NUPR1 plays a vital role in the protection of ß-cells from apoptosis, related degradation of insulin storages and subsequent secretion during inflammatory and obesity-related tissue stress.

Sustained release of rhBMP-2 from microporous tricalciumphosphate using hydrogels as a carrier.

BACKGROUND: Tissue engineering and bone substitutes are subjects of intensive ongoing research. If the healing of bone fractures is delayed, osteoinductive materials that induce mesenchymal stem cells (MSCs) to form bone are necessary. The use of Bone Morphogenetic Protein - 2 is a common means to enhance effectiveness and accelerate the healing process. A delivery system that maintains and releases BMP biological activity in controlled fashion at the surgical site while preventing systemic diffusion (and thereby the risk of undesirable effects by controlling the amount of protein implanted) is essential. In this study, we aimed to test a cylindrical TCP-scaffold (porosity ~ 40 %, mean pore size 5 µm, high interconnectivity) in comparison to BMP-2. Recombinant human BMP-2 was dissolved in different hydrogels as a carrier, namely gelatin and alginate cross-linked with CaCl2-solution, or a solution of GDL and CaCO3. FITC-labeled Protein A was used as a model substance for rhBMP-2 in the pre-trials. For loading, the samples were put in a flow chamber and sealed with silicone rings. Using a directional vacuum, the samples were loaded with the alginate-BMP-2-mixture and the loading success monitored by observing changes in a fluorescent dye (FITC labeled Protein A) under a fluorescence microscope. A fluorescence reader and ELISA were employed to measure the release. Efficacy was determined in cell culture experiments (MG63 cells) via Live-Dead-Assay, FACS, WST-1-Assay, pNPP alkaline phosphatase assay and confocal microscopy. For statistical analysis, we calculated the mean and standard deviation and carried out an analysis of variance. RESULTS: Directional vacuum makes it possible to load nearly 100 % of the interconnected micropores with alginate mixed with rhBMP-2. Using alginate hardened with CaCl2 as a carrier, BMP-2's release can be decelerated significantly longer than with other hydrogels - eg, for over 28 days. The effects on osteoblast-like cells were an increase of the growth rate and expression of alkaline phosphatase while triggering no toxic effect. CONCLUSION: The rhBMP-2-loaded microporous TCP scaffolds possess proliferative and osteoinductive potential. Alginate helps to lower the local growth factor dose below the cytotoxic limit, and allows the release period to be lengthened by at least 28 days.

Prospective clinical trial of patients who underwent ankle arthroscopy with articular diseases to match clinical and radiological scores with intra-articular cytokines.

OBJECTIVE: There is still a lack of reliable data on cytokine concentrations in the ankle and their value for prognosis. METHODS: In a prospective clinical trial, lavage fluids were collected from 49 patients with an arthroscopy of the ankle. The fluids were investigated by ELISA for cytokine levels. Clinical scores (FFI, AOFAS) were evaluated both pre-operatively and then again 12 months after surgery (n = 43, 88%). Radiological changes were noted with the Kellgren-Lawrence-Score (KLS) and the Ankle Osteoarthritis Scoring System (AOSS). Based on the difference between the pre- and postoperative clinical scores, two groups were defined according to whether they had benefited from the surgical therapy (Δ score ≥ 10) or not (Δ score < 10). RESULTS: The average clinical scores had improved to a statistically significant extent in the one-year follow-up (p < 0.01). BMP-2 (p = 0.02), IGF-1 (p = 0.04), BMP-7 (p = 0.01) and aggrecan (p = 0.04) showed significant correlations with pre-operative clinical and radiological scores (p = 0.02, p = 0.01, p = 0.01, p = 0.01). Furthermore, BMP-2 (p = 0.01), IGF-1/TPC (p = 0.03) and aggrecan (p = 0.01) correlated with scores after one year (p = 0.02, p = 0.01). High aggrecan concentrations were associated with a low clinical and a high radiological score at both time points, both indicating progress of cartilage degeneration in contrast to BMP-2 or IGF-1. Furthermore, MMP-13 concentrations were significantly higher in the non-benefit group (p = 0.02). CONCLUSION: BMP-2, IGF-1, aggrecan and MMP-13 seem to be involved in the degenerative process of cartilage in the ankle joint. Additionally, high synovial MMP-13 concentrations indicate a worse clinical outcome.

Association between intraarticular cytokine levels and clinical parameters of osteochondritis dissecans in the ankle.

BACKGROUND: Reliable data about in vivo regulation of cytokines in osteochondritis dissecans (OCD) of the ankle are still missing. Disease-specific regulation patterns were hypothesized. METHODS: 28 patients with a mean age of 30.7 ± 14.8 years undergoing an arthroscopy of the ankle because of OCD were prospectively included in a clinical trial. Lavage fluids were analyzed by ELISA for levels of aggrecan, BMP-2, BMP-7, IGF-1, IGF-1R, bFGF, endoglin, MMP-13, and IL-1ß. Additionally, clinical parameters and scores (FFI, CFSS, AOFAS) were evaluated and supplemented by the Kellgren Lawrence Score (KLS) for conventional X-rays and the Ankle Osteoarthritis Scoring System (AOSS) for MRI. RESULTS: Grading of OCD lesions statistically significant increased with age and was higher in case of previously performed operations (p<0.03). A worse clinical function reflected by low AOFAS and CFSS scores or high FFI was associated with high grading of cartilage damage or OCD (p<0.03). Similarly, high radiological scores (KLS and AOSS) indicating progress of OA positively correlated with grading of cartilage damage and OCD. The concordance between the MRI and arthroscopic classification was overall moderate (κ=0.52). Biochemically, only IGF/IGF-1R levels were consistently negatively associated with OCD grading, ICRS score, FFI and KLS (p<0.05). Correlation data is supported by post hoc statistics. CONCLUSIONS: Radiological and clinical parameters in association with synovial IGF-1/IGF-1R levels indicated an increasing joint degeneration with rising OCD stage. TRIAL REGISTRATION: German Clinical Trials Register DRKS00000365, 11/03/2008.

Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1.

Gene transfer into hematopoietic stem cells has been used successfully for correcting lymphoid but not myeloid immunodeficiencies. Here we report on two adults who received gene therapy after nonmyeloablative bone marrow conditioning for the treatment of X-linked chronic granulomatous disease (X-CGD), a primary immunodeficiency caused by a defect in the oxidative antimicrobial activity of phagocytes resulting from mutations in gp91(phox). We detected substantial gene transfer in both individuals' neutrophils that lead to a large number of functionally corrected phagocytes and notable clinical improvement. Large-scale retroviral integration site-distribution analysis showed activating insertions in MDS1-EVI1, PRDM16 or SETBP1 that had influenced regulation of long-term hematopoiesis by expanding gene-corrected myelopoiesis three- to four-fold in both individuals. Although insertional influences have probably reinforced the therapeutic efficacy in this trial, our results suggest that gene therapy in combination with bone marrow conditioning can be successfully used to treat inherited diseases affecting the myeloid compartment such as CGD.

Expression of BMP-receptor type 1A correlates with progress of osteoarthritis in human knee joints with focal cartilage lesions.

BACKGROUND AIMS: Bone morphogenetic protein-2 (BMP-2) and its receptor type 1A (BMPR-1A) play significant roles in cartilage metabolism. The aim of this study was to evaluate a possible correlation between intra-articular expression of these proteins and the degree of osteoarthritis (OA) in human knees. METHODS: Biopsies of synovia and debrided cartilage were taken in 15 patients undergoing autologous chondrocyte implantation. Expression of BMP-2 and BMPR-1A was evaluated semi-quantitatively by immunohistologic staining. These data were complemented by grading of cartilage lesions according to International Cartilage Repair Society (ICRS), defect size, duration of complaints, knee osteoarthritis scoring system (KOSS) and Henderson and Kellgren-Lawrence scores. General histologic stainings were used to determine Mankin, Pritzker and Krenn scores. RESULTS: The expression of BMPR-1A but not of BMP-2 was significantly higher in cartilage biopsies taken in type 3 lesions with intact subchondral layer compared with type 4 defects (P < 0.05). In cartilage areas of bordering sectors, the intensity of immunohistologic staining of BMPR-1A was statistically significantly higher in mature cartilage compared with repair zones (P < 0.05). Expression of BMP-2 and its receptor 1A correlated in the cartilage biopsies (P < 0.02) but not in the synovia. The degree of OA was scored in all biopsies according to Mankin and Pritzker, and these scores correlated statistically significantly with BMPR-1A expression in the synovia (P < 0.05). In patients with an osteochondritis dissecans, the degree of OA was higher compared with other causes of chondromalacia, as evaluated by defect size, ICRS score, duration of complaints, Pritzker score and expression of BMPR-1A in cartilage (P < 0.05). CONCLUSIONS: These data support the role of BMPR-1A as an indicator of OA progression in human knees with circumscribed cartilage lesions.

Immunohistological localization of BMP-2, BMP-7, and their receptors in knee joints with focal cartilage lesions.

INTRODUCTION: Although it is well known that BMP-2 and BMP-7 play significant roles in cartilage metabolism, data about intra-articular expression and localization of these proteins and their receptors in humans are rare. METHODS: Biopsies of synovia and debrided cartilage were taken in patients undergoing autologous chondrocyte implantation. Expression of BMP-2, BMP-7, and their receptors BMPR-1A, BMPR-1B and BMPR-2 were semiquantitatively evaluated by immunohistological staining. RESULTS: BMP-7 was equally highly expressed in all cartilage and synovial biopsies. Increased levels of BMPR-1A, but not of BMPR-1B, and BMPR-2, were found in all synovial and 47% of all cartilage samples (P = 0.002). BMP-2 was positively scored in 47% of all cartilage and 40% of all synovial specimens. Defect size, KOSS, Henderson or Kellgren-Lawrence score did not statistically significant correlate with the expression of the analyzed proteins or Mankin and Pritzker scores. Duration of symptoms and localization of lesions were associated with KOSS (P < 0.02), but there was no influence of these parameters on protein expression. CONCLUSIONS: BMP-2, BMP-7, and BMPR-1A were expressed in cartilage and synovia of knees with focal cartilage lesions. Although defect localization and duration of symptoms decisively influence KOSS, there was no associated alteration of protein expression observed.

High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR).

Integrating vector systems used in clinical gene therapy have proven their therapeutic potential in the long-term correction of immunodeficiencies. The integration loci of such vectors in the cellular genome represent a molecular marker unique for each transduced cell and its clonal progeny. To gain insight into the physiology of gene-modified hematopoietic repopulation and vector-related influences on clonal contributions, we have previously introduced a technology--linear amplification-mediated (LAM) PCR--for detecting and sequencing unknown DNA flanking sequences down to the single cell level (Supplementary Note online). LAM-PCR analyses have enabled qualitative and quantitative measurements of the clonal kinetics of hematopoietic regeneration in gene transfer studies, and uncovered the clonal derivation of non-leukemogenic and leukemogenic insertional side effects in preclinical and clinical gene therapy studies. The reliability and robustness of this method results from the initial preamplification of the vector-genome junctions preceding nontarget DNA removal via magnetic selection. Subsequent steps are carried out on a semisolid streptavidin phase, including synthesis of double complementary strands, restriction digest, ligation of a linker cassette onto the genomic end of the fragment and exponential PCR(s) with vector- and linker cassette-specific primers. LAM-PCR can be adjusted to all unknown DNA sequences adjacent to a known DNA sequence. Here we describe the use of LAM-PCR analyses to identify 5' long terminal repeat (LTR) retroviral vector adjacent genomic sequences.

NF-E2 overexpression delays erythroid maturation and increases erythrocyte production.

The transcription factor Nuclear Factor-Erythroid 2 (NF-E2) is overexpressed in the vast majority of patients with polycythaemia vera (PV). In murine models, NF-E2 overexpression increases proliferation and promotes cellular viability in the absence of erythropoietin (EPO). EPO-independent growth is a hallmark of PV. We therefore hypothesized that NF-E2 overexpression contributes to erythrocytosis, the pathognomonic feature of PV. Consequently, we investigated the effect of NF-E2 overexpression in healthy CD34+ cells. NF-E2 overexpression led to a delay in erythroid maturation, manifested by a belated appearance of glycophorin A-positive erythroid precursors. Maturation delay was similarly observed in primary PV patient erythroid cultures compared to healthy controls. Protracted maturation led to a significant increase in the accumulated number of erythroid cells both in PV cultures and in CD34+ cells overexpressing NF-E2. Similarly, NF-E2 overexpression altered erythroid colony formation, leading to an increase in erythroid burst-forming unit formation. These data indicate that NF-E2 overexpression delays the early phase of erythroid maturation, resulting in an expansion of erythroid progenitors, thereby increasing the number of erythrocytes derived from one CD34+ cell. These data propose a role for NF-E2 in mediating the erythrocytosis of PV.

Bone marrow-derived cells contribute to infarct remodelling.

OBJECTIVE: The paradigm that cardiac myocytes are non-proliferating and terminally differentiated cells has recently been challenged by several studies reporting the ability of bone marrow-derived cells (BMC) to transdifferentiate into cardiomyocytes. However, these results are controversial and could not be reproduced by others. Therefore, we studied the contribution and potential transdifferentiation of BMC into different cell types during the remodelling process in mouse hearts with experimental myocardial infarction. METHODS: Mice (C57BL/6J) were sublethally irradiated, and BM from enhanced green fluorescent protein (eGFP)-transgenic mice was transplanted. Coronary artery ligation was performed 3 months later. The hearts were studied 7 days (n=13) and 21 days (n=12) after infarction. Immunohistochemical staining was performed using antibodies against titin, connexin 43, vimentin, SMemb alpha-smooth muscle actin, CD45, CD34, F4/80, BS-1, CD31, and eGFP. Sections were analyzed using fluorescence and confocal laser microscopy. RESULTS: Success of BM transplantation was confirmed by FACS analysis. Occlusion of the coronary artery resulted in infarct sizes of 41+/-6% of the left ventricle. CD45+/eGFP+ inflammatory cells were found frequently after 7 days and to a lesser degree after 21 days. In 25 examined hearts, only 3 eGFP-positive cardiomyocytes were found. However, numerous BMC-derived fibroblasts and myofibroblasts were found in the infarct area. BMC contributed to scar tissue neoangiogenesis but not to angiogenesis in the periinfarct and remote zones. CONCLUSION: Transdifferentiation of BMC into viable cardiomyocytes is a negligible event in normal repair processes after myocardial damage. BMC-derived fibroblasts and myofibroblasts as well as neoangiogenesis significantly contribute to post-infarction scar formation and might be important in scar tissue remodelling.

Development and retranslational validation of an in vitro model to characterize acute infections in large human joints.

Bacterial infections can destroy cartilage integrity, resulting in osteoarthritis. Goal was to develop an in vitro model with in vivo validation of acute joint inflammation. Inflammation in cocultivated human synovial fibroblasts (SFB), chondrocytes (CHDR), and mononuclear cells (MNC) was successively relieved for 10 days. Articular effusions from patients with (n = 7) and without (n = 5) postoperative joint infection in healthy patients (ASA 1-2) were used as model validation. Inflammation in vitro resulted in an enormous increase in IL-1 and a successive reduction in SFB numbers. CHDR however, maintained metabolic activity and proteoglycan synthesis. While concentrations of bFGF in vivo and in vitro rose consistently, the mRNA increase was only moderate. Concurring with our in vivo data, cartilage-specific IGF-1 steadily increased, while IGF-1 mRNA in the CHDR and SFB did not correlate with protein levels. Similarly, aggrecan (ACAN) protein concentrations increased in vivo and failed to correlate in vitro with gene expression in either the CHDR or the SFB, indicating extracellular matrix breakdown. Anabolic cartilage-specific BMP-7 with highly significant intra-articular levels was significantly elevated in vitro on day 10 following maximum inflammation. Our in vitro model enables us to validate early inflammation of in vivo cell- and cytokine-specific regulatory patterns. This trial is registered with MISSinG, DRKS 00003536.

Mutation in the platelet-derived growth factor receptor alpha inhibits adeno-associated virus type 5 transduction.

Due to its non-pathogenic lifecycle, little is known about the cellular determinants of infection by adeno-associated virus (AAV). To identify these critical cellular factors, we took advantage of the gene transfer abilities of AAV in combination with a forward genetic selection to identify proteins critical for transduction by this virus. AAV serotype 5 (AAV5) vectors encoding the furin gene were used to transduce furin-deficient cells followed by selection with furin-dependent toxins. A population of cells specifically resistant to AAV5 transduction was identified and sequence analysis suggested all had a single amino acid mutation in the leader sequence of the platelet-derived growth factor receptor alpha (PDGFRα) gene. Characterization of this mutation suggested it inhibited PDGFRα trafficking resulting in limited expression on the plasma membrane. Mutagenesis and transfection experiments confirmed the effect of this mutation on PDGFRα trafficking, and the AAV5 resistant phenotype could be rescued by transfection with wild type PDGFRα.

Protein phosphatase 1 (PP-1)-dependent inhibition of insulin secretion by leptin in INS-1 pancreatic ß-cells and human pancreatic islets.

Leptin inhibits insulin secretion from pancreatic ß-cells, and in turn, insulin stimulates leptin biosynthesis and secretion from adipose tissue. Dysfunction of this adipoinsular feedback loop has been proposed to be involved in the development of hyperinsulinemia and type 2 diabetes mellitus. At the molecular level, leptin acts through various pathways, which in combination confer inhibitory effects on insulin biosynthesis and secretion. The aim of this study was to identify molecular mechanisms of leptin action on insulin secretion in pancreatic ß-cells. To identify novel leptin-regulated genes, we performed subtraction PCR in INS-1 ß-cells. Regulated expression of identified genes was confirmed by RT-PCR and Northern and Western blotting. Furthermore, functional impact on ß-cell function was characterized by insulin-secretion assays, intracellular Ca²(+) concentration measurements, and enzyme activity assays. PP-1α, the catalytic subunit of protein phosphatase 1 (PP-1), was identified as a novel gene down-regulated by leptin in INS-1 pancreatic ß-cells. Expression of PP-1α was verified in human pancreatic sections. PP-1α mRNA and protein expression is down-regulated by leptin, which culminates in reduction of PP-1 enzyme activity in ß-cells. In addition, glucose-induced insulin secretion was inhibited by nuclear inhibitor of PP-1 and calyculin A, which was in part mediated by a reduction of PP-1-dependent calcium influx into INS-1 ß-cells. These results identify a novel molecular pathway by which leptin confers inhibitory action on insulin secretion, and impaired PP-1 inhibition by leptin may be involved in dysfunction of the adipoinsular axis during the development of hyperinsulinemia and type 2 diabetes mellitus.

Of mice and men: human RNA polymerase III promoter U6 is more efficient than its murine homologue for shRNA expression from a lentiviral vector in both human and murine progenitor cells.

OBJECTIVE: RNA interference mediated by transcription of short hairpin RNAs (shRNAs) from lentiviral expression vectors has emerged as an efficient method to effectively and specifically silence gene expression in a vast variety of mammalian cells. shRNA expression is routinely driven by a RNA polymerase III promoter, most often by the U6 promoter. Here we demonstrate that U6 promoter activity-and consequently gene silencing success-differs significantly among species. MATERIALS AND METHODS: We have modified pLeGO-G, an HIV-based third-generation lentivector, to express a 19nt shRNA sequence against the human transcription factor nuclear factor erythroid 2 or against its murine homologue, as well as an shRNA against murine JAK2, from either the human or the murine U6 promoter. Gene silencing efficiency was analyzed in a human erythroleukemic cell line, in primary human CD34(+) cells, as well as in a murine erythroleukemic cell line and in primary murine bone marrow. RESULTS: ShRNA expression from the human U6 promoter resulted in a fourfold increase in knockdown efficiency compared to expression from the murine U6 promoter in both human and murine cells. CONCLUSIONS: The U6 promoter constitutes an important determinant for efficient gene silencing by shRNAs.

Stable differentiation and clonality of murine long-term hematopoiesis after extended reduced-intensity selection for MGMT P140K transgene expression.

Efficient in vivo selection increases survival of gene-corrected hematopoietic stem cells (HSCs) and protects hematopoiesis, even if initial gene transfer efficiency is low. Moreover, selection of a limited number of transduced HSCs lowers the number of cell clones at risk of gene activation by insertional mutagenesis. However, a limited clonal repertoire greatly increases the proliferation stress of each individual clone. Therefore, understanding the impact of in vivo selection on proliferation and lineage differentiation of stem-cell clones is essential for its clinical use. We established minimal cell and drug dosage requirements for selection of P140K mutant O6-methylguanine-DNA-methyltransferase (MGMT P140K)-expressing HSCs and monitored their differentiation potential and clonality under long-term selective stress. Up to 17 administrations of O6-benzylguanine (O6-BG) and 1,3-bis(2-chloroethyl)-1-nitroso-urea (BCNU) did not impair long-term differentiation and proliferation of MGMT P140K-expressing stem-cell clones in mice that underwent serial transplantation and did not lead to clonal exhaustion. Interestingly, not all gene-modified hematopoietic repopulating cell clones were efficiently selectable. Our studies demonstrate that the normal function of murine hematopoietic stem and progenitor cells is not compromised by reduced-intensity long-term in vivo selection, thus underscoring the potential value of MGMT P140K selection for clinical gene therapy.

Acute myeloid leukemia is associated with retroviral gene transfer to hematopoietic progenitor cells in a rhesus macaque.

We report, for the first time, a replication-defective retroviral vector-associated neoplasia in a nonhuman primate. Five years after transplantation with CD34+ cells transduced with a retroviral vector expressing enhanced green fluorescent protein (eGFP) and a drug-resistant variant of the dihydrofolate reductase gene (L22Y), a rhesus macaque developed a fatal myeloid sarcoma, a type of acute myeloid leukemia. Tumor cells contained 2 clonal vector insertions. One insertion was found in BCL2-A1, an antiapoptotic gene. This event suggests that currently available retroviral vectors may have long-term side effects, particularly in hematopoietic stem and progenitor cells.