CAR virus receptor mediates erythroid differentiation and migration and is downregulated in MDS.

Myelodysplastic syndromes (MDS) are myeloid neoplasms presenting with dysplasia in the bone marrow (BM) and peripheral cytopenia. In most patients anemia develops. We screened for genes that are expressed abnormally in erythroid progenitor cells (EP) and contribute to the pathogenesis of MDS. We found that the Coxsackie-Adenovirus receptor (CAR = CXADR) is markedly downregulated in CD45low/CD105+ EP in MDS patients compared to control EP. Correspondingly, the erythroblast cell lines HEL, K562, and KU812 stained negative for CAR. Lentiviral transduction of the full-length CXADR gene into these cells resulted in an increased expression of early erythroid antigens, including CD36, CD71, and glycophorin A. In addition, CXADR-transduction resulted in an increased migration against a serum protein gradient, whereas truncated CXADR variants did not induce expression of erythroid antigens or migration. Furthermore, conditional knock-out of Cxadr in C57BL/6 mice resulted in anemia and erythroid dysplasia. Finally, decreased CAR expression on EP was found to correlate with high-risk MDS and decreased survival. Together, CAR is a functionally relevant marker that is down-regulated on EP in MDS and is of prognostic significance. Decreased CAR expression may contribute to the maturation defect and altered migration of EP and thus their pathologic accumulation in the BM in MDS.

Characterisation of Extracellular Vesicles from Equine Mesenchymal Stem Cells.

Extracellular vesicles (EVs) are nanosized lipid bilayer-encapsulated particles secreted by virtually all cell types. EVs play an essential role in cellular crosstalk in health and disease. The cellular origin of EVs determines their composition and potential therapeutic effect. Mesenchymal stem/stromal cell (MSC)-derived EVs have shown a comparable therapeutic potential to their donor cells, making them a promising tool for regenerative medicine. The therapeutic application of EVs circumvents some safety concerns associated with the transplantation of viable, replicating cells and facilitates the quality-controlled production as a ready-to-go, off-the-shelf biological therapy. Recently, the International Society for Extracellular Vesicles (ISEV) suggested a set of minimal biochemical, biophysical and functional standards to define extracellular vesicles and their functions to improve standardisation in EV research. However, nonstandardised EV isolation methods and the limited availability of cross-reacting markers for most animal species restrict the application of these standards in the veterinary field and, therefore, the species comparability and standardisation of animal experiments. In this study, EVs were isolated from equine bone-marrow-derived MSCs using two different isolation methods, stepwise ultracentrifugation and size exclusion chromatography, and minimal experimental requirements for equine EVs were established and validated. Equine EVs were characterised using a nanotracking analysis, fluorescence-triggered flow cytometry, Western blot and transelectron microscopy. Based on the ISEV standards, minimal criteria for defining equine EVs are suggested as a baseline to allow the comparison of EV preparations obtained by different laboratories.

Detection of synovial sepsis in horses using enzymes as biomarkers.

BACKGROUND: Synovial sepsis is a commonly occurring, potentially career-ending or even life-threatening orthopaedic emergency. Diagnosis of synovial sepsis is currently primarily based on synovial fluid analysis, which often leaves diagnostic ambiguity due to overlap of clinicopathological parameters between septic and aseptic inflammatory synovitis. OBJECTIVES: To evaluate the reliability of lysozyme (LYS), myeloperoxidase (MPO) and elastase (ELT) as biomarkers for synovial sepsis in horses using a photometric assay to measure increased enzyme activity. STUDY DESIGN: Prospective, single-blinded, analytical, clinical study. METHODS: Equine synovial samples were assigned to one of three groups: (1) healthy controls (n = 10), (2) aseptic (n = 27) and (3) septic synovitis (n = 30). The enzyme activity assays (LYS, MPO and ELT) were compared with standard synovial fluid parameters and broad-range bacterial 16S rDNA PCR. RESULTS: LYS and MPO activities were significantly different between septic synovial samples, and both aseptic and control samples (P < .001, LYS: confidence interval [CI]: 2.25-3.41, resp., 2.21-3.8, MPO: CI 0.752-1.6, resp., 0.639-1.81). LYS achieved a 100% sensitivity and 100% specificity in differentiating between septic and aseptic (cut-off value 751.4) or control (cut-off: 484.6) samples (P < .001). MPO reached 93.33% sensitivity, 100% specificity for distinguishing septic from control (cut-off value: 0.1254) synovial samples and 93.33% sensitivity, 81.48% specificity for discriminating between septic and aseptic (cut-off value: 0.1305) synovial samples (P < .001). ELT activity could not be measured in any synovial sample. Both the LYS and the MPO measurements showed a highly significant correlation with PCR (LYS r = .79, MPO r = .69), synovial leukocyte count (LYS r = .752, MPO r = .571), % neutrophils (LYS r = .751, MPO r = 0.663) and each other (r = .744, all P < .001). MAIN LIMITATIONS: Variation in horses' signalment, affected synovial structures and synovial fluid freezing times may have affected the discriminative power of this study. CONCLUSIONS: Increased MPO and LYS activities allow reliable, rapid diagnosis of synovial sepsis with high sensitivity and specificity.

Molecular Mechanisms of Fetal Tendon Regeneration Versus Adult Fibrous Repair.

Tendinopathies are painful, disabling conditions that afflict 25% of the adult human population. Filling an unmet need for realistic large-animal models, we here present an ovine model of tendon injury for the comparative study of adult scarring repair and fetal regeneration. Complete regeneration of the fetal tendon within 28 days is demonstrated, while adult tendon defects remained macroscopically and histologically evident five months post-injury. In addition to a comprehensive histological assessment, proteome analyses of secretomes were performed. Confirming histological data, a specific and pronounced inflammation accompanied by activation of neutrophils in adult tendon defects was observed, corroborated by the significant up-regulation of pro-inflammatory factors, neutrophil attracting chemokines, the release of potentially tissue-damaging antimicrobial and extracellular matrix-degrading enzymes, and a response to oxidative stress. In contrast, secreted proteins of injured fetal tendons included proteins initiating the resolution of inflammation or promoting functional extracellular matrix production. These results demonstrate the power and relevance of our novel ovine fetal tendon regeneration model, which thus promises to accelerate research in the field. First insights from the model already support our molecular understanding of successful fetal tendon healing processes and may guide improved therapeutic strategies.

Fetal articular cartilage regeneration versus adult fibrocartilaginous repair: secretome proteomics unravels molecular mechanisms in an ovine model.

Osteoarthritis (OA), a degenerative joint disease characterized by progressive cartilage degeneration, is one of the leading causes of disability worldwide owing to the limited regenerative capacity of adult articular cartilage. Currently, there are no disease-modifying pharmacological or surgical therapies for OA. Fetal mammals, in contrast to adults, are capable of regenerating injured cartilage in the first two trimesters of gestation. A deeper understanding of the properties intrinsic to the response of fetal tissue to injury would allow us to modulate the way in which adult tissue responds to injury. In this study, we employed secretome proteomics to compare fetal and adult protein regulation in response to cartilage injury using an ovine cartilage defect model. The most relevant events comprised proteins associated with the immune response and inflammation, proteins specific for cartilage tissue and cartilage development, and proteins involved in cell growth and proliferation. Alarmins S100A8, S100A9 and S100A12 and coiled-coil domain containing 88A (CCDC88A), which are associated with inflammatory processes, were found to be significantly upregulated following injury in adult, but not in fetal animals. By contrast, cartilage-specific proteins like proteoglycan 4 were upregulated in response to injury only in fetal sheep postinjury. Our results demonstrate the power and relevance of the ovine fetal cartilage regeneration model presented here for the first time. The identification of previously unrecognized modulatory proteins that plausibly affect the healing process holds great promise for potential therapeutic interventions.

Image-based ex-vivo drug screening for patients with aggressive haematological malignancies: interim results from a single-arm, open-label, pilot study.

BACKGROUND: Patients with refractory or relapsed haematological malignancies have few treatment options and short survival times. Identification of effective therapies with genomic-based precision medicine is hampered by intratumour heterogeneity and incomplete understanding of the contribution of various mutations within specific cancer phenotypes. Ex-vivo drug-response profiling in patient biopsies might aid effective treatment identification; however, proof of its clinical utility is limited. METHODS: We investigated the feasibility and clinical impact of multiparametric, single-cell, drug-response profiling in patient biopsies by immunofluorescence, automated microscopy, and image analysis, an approach we call pharmacoscopy. First, the ability of pharmacoscopy to separate responders from non-responders was evaluated retrospectively for a cohort of 20 newly diagnosed and previously untreated patients with acute myeloid leukaemia. Next, 48 patients with aggressive haematological malignancies were prospectively evaluated for pharmacoscopy-guided treatment, of whom 17 could receive the treatment. The primary endpoint was progression-free survival in pharmacoscopy-treated patients, as compared with their own progression-free survival for the most recent regimen on which they had progressive disease. This trial is ongoing and registered with ClinicalTrials.gov, number NCT03096821. FINDINGS: Pharmacoscopy retrospectively predicted the clinical response of 20 acute myeloid leukaemia patients to initial therapy with 88·1% accuracy. In this interim analysis, 15 (88%) of 17 patients receiving pharmacoscopy-guided treatment had an overall response compared with four (24%) of 17 patients with their most recent regimen (odds ratio 24·38 [95% CI 3·99-125·4], p=0·0013). 12 (71%) of 17 patients had a progression-free survival ratio of 1·3 or higher, and median progression-free survival increased by four times, from 5·7 (95% CI 4·1-12·1) weeks to 22·6 (7·4-34·0) weeks (hazard ratio 3·14 [95% CI 1·37-7·22], p=0·0075). INTERPRETATION: Routine clinical integration of pharmacoscopy for treatment selection is technically feasible, and led to improved treatment of patients with aggressive refractory haematological malignancies in an initial patient cohort, warranting further investigation. FUNDING: Austrian Academy of Sciences; European Research Council; Austrian Science Fund; Austrian Federal Ministry of Science, Research and Economy; National Foundation for Research, Technology and Development; Anniversary Fund of the Austrian National Bank; MPN Research Foundation; European Molecular Biology Organization; and Swiss National Science Foundation.

First-in-human response of BCL-2 inhibitor venetoclax in T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive T-lymphoid malignancy usually refractory to current treatment strategies and associated with short overall survival. By applying next-generation functional testing of primary patient-derived lymphoma cells using a library of 106 US Food and Drug Administration (FDA)-approved anticancer drugs or compounds currently in clinical development, we set out to identify novel effective treatments for T-PLL patients. We found that the B-cell lymphoma 2 (BCL-2) inhibitor venetoclax (ABT-199) demonstrated the strongest T-PLL-specific response when comparing individual ex vivo drug response in 86 patients with refractory hematologic malignancies. Mechanistically, responses to venetoclax correlated with protein expression of BCL-2 but not with expression of the BCL-2 family members myeloid cell leukemia 1 (MCL-1) and BCL-XL in lymphoma cells. BCL-2 expression was inversely correlated with the expression of MCL-1. Based on the ex vivo responses, venetoclax treatment was commenced in 2 late-stage refractory T-PLL patients resulting in clinical responses. Our findings demonstrate first evidence of single-agent activity of venetoclax both ex vivo and in humans, offering a novel agent in T-PLL.

A narrowing range of bone scan in newly diagnosed prostate cancer patients: A retrospective comparative study.

OBJECTIVES: The objective of the following study is to clarify a suitable group whereby a bone scan could be spared at the initial staging of prostate cancer, we wished to identify the possible relationship between bone metastasis and clinical and pathological parameters including serum total prostate specific antigen (PSA) concentration, alkaline phosphatase (ALP), biopsy Gleason Score (GS), and percentage of pathological cores. MATERIALS AND METHODS: We reviewed the results of 220 bone scintigraphies, which were done between January 1, 2011 and June 30, 2013 in patients with newly diagnosed prostate cancer. These parameters were evaluated together with standard clinicopathological data to determine the prediction ability of the bone scan by univariate and multivariate analyses. RESULTS: Bone metastases were seen in 44 patients of all 220 patients (20%, 95% confidence interval, 17-24%). In univariate analysis, PSA and biopsy GS were useful in predicting the bone scan result, but ALP and percentage of pathological cores was not. In multivariate analysis, the single most useful parameter in predicting the bone scan result was PSA (P < 0.001). CONCLUSIONS: A bone scan seems to be impractical in newly diagnosed prostate cancer patients with serum PSA level <20 ng/ml and GS up to seven and pre-treatment PSA is the best predictor of the need for the bone scan according to results of this study.

Stage-specific control of early B cell development by the transcription factor Ikaros.

The transcription factor Ikaros is an essential regulator of lymphopoiesis. Here we studied its B cell-specific function by conditional inactivation of the gene encoding Ikaros (Ikzf1) in pro-B cells. B cell development was arrested at an aberrant 'pro-B cell' stage characterized by increased cell adhesion and loss of signaling via the pre-B cell signaling complex (pre-BCR). Ikaros activated genes encoding signal transducers of the pre-BCR and repressed genes involved in the downregulation of pre-BCR signaling and upregulation of the integrin signaling pathway. Unexpectedly, derepression of expression of the transcription factor Aiolos did not compensate for the loss of Ikaros in pro-B cells. Ikaros induced or suppressed active chromatin at regulatory elements of activated or repressed target genes. Notably, binding of Ikaros and expression of its target genes were dynamically regulated at distinct stages of early B lymphopoiesis.

Solitary fibrous tumor of neck mimicking cold thyroid nodule in 99m tc thyroid scintigraphy.

A 68-year-old man had a rapidly growing, painless neck mass, thought to be nodular goiter. Ultrasonography showed a giant, heterogeneous mass occupying the middle and superior poles and protruding outside of the left thyroid lobe. The results of the thyroid function tests were normal. Thyroid scintigraphy revealed a large hypoactive nodule in the left thyroid lobe. Complete surgical removal of tumor was performed and macroscopically demonstrated a well-demarked lesion outside the thyroid gland. Microscopically, the lesion was composed of fibroblast-like spindle cells in a patternless architecture and extensive stromal hyalinization. Immunohistochemistry showed positive reaction for CD34 in spindle cells and diffuse bcl-2 staining. The pathology was confirmed as solitary fibrous tumor. In the follow-up period after surgery, thyroid scintigraphy showed normal left thyroid lobe. Solitary fibrous tumor originated from or associated with thyroid gland is extremely rare. According to our knowledge, this is the first reported solitary fibrous tumor presenting like a cold thyroid nodule. This pathology must be considered for differential diagnosis of neck masses in the thyroid region.

The effect of telomerase template antagonist GRN163L on bone-marrow-derived rat mesenchymal stem cells is reversible and associated with altered expression of cyclin d1, cdk4 and cdk6.

Telomerase activity is essential for the continued growth and survival of malignant cells, therefore inhibition of this activity presents an attractive target for anti-cancer therapy. The telomerase inhibitor GRN163L, was shown to inhibit the growth of cancer cells both in vitro and in vivo. Mesenchymal stem cells (MSCs) also show telomerase activity in maintaining their self-renewal; therefore the effects of telomerase inhibitors on MSCs may be an issue of concern. MSCs are multipotent cells and are important for the homeostasis of the organism. In this study, we sought to demonstrate in vitro effects of GRN163L on rat MSCs. When MSCs were treated with 1 microM GRN163L, their phenotype changed from spindle-shaped cells to rounded ones and detached from the plate surface, similar to cancer cells. Quantitative-RT-PCR and immunoblotting results revealed that GRN163L holds MSCs at the G1 state of the cell cycle, with a drastic decrease in mRNA and protein levels of cyclin D1 and its cdk counterparts, cdk4 and cdk6. This effect was not observed when MSCs were treated with a mismatch control oligonucleotide. One week after GRN163L was removed, mRNA and protein expressions of the genes, as well as the phenotype of MSCs returned to those of untreated cells. Therefore, we concluded that GRN163L does not interfere with the self-renewal and differentiation of MSCs under short term in vitro culture conditions. Our study provides additional support for treating cancers by administrating GRN163L without depleting the body's stem cell pools.

Radioiodine whole-body scans, thyroglobulin levels, 99mTc-MIBI scans and computed tomography: results in patients with lung metastases from differentiated thyroid cancer.

OBJECTIVES: The correlation between a 131I whole-body scan (WBS), a 99mTc sestamibi (99mTc-MIBI) WBS, a computed tomography (CT) scan and the value of routine follow-up for 131I WBS and thyroglobulin (Tg) levels in patients with lung metastases from differentiated thyroid cancer was assessed. METHOD: Pulmonary metastases were detected in 32 patients out of 583 with differentiated thyroid cancer (DTC) who were admitted to our clinic between 1985 and 2004 (age range, 22-79 years; mean, 58 +/- 19 years; 15 women and 17 men). Pulmonary metastases were diagnosed by considering the 131I WBS, increased Tg levels and/or other positive radiological findings. Papillary carcinoma was diagnosed in 15/32 patients and follicular carcinoma in 13/32. A mixed type found in 4/32 patients was classified histopathologically. A total of 3.7-53.65 GBq (100-1450 mCi) 131I was given to each patient. The duration of follow-up ranged from 36 to 240 months. A 131I WBS, the determination of Tg levels and/or a CT scan were carried out in the assessment of a diagnosis and follow-up of patients with lung metastases. A 99mTc-MIBI WBS was performed on 19 patients who were chosen at random from the 583. RESULTS: Nineteen of 32 patients had lung metastases before they received the first 131I treatment. Six of the 32 had distant-organ metastases other than in the lungs. Four of these six patients had only lung and bone metastases. Pulmonary metastases were observed on the 131I WBS patients 31/32 (96.8%) whereas no pulmonary metastases, were detected on the CT scans in 3/32 patients. The last diagnostic whole-body scan (DWBS) was normal in 13/32 patients. At the first examination, the Tg levels in 27/32 (84.4%) patients were below 30 ng . ml(-1). At the final examination, 20/32 (62.5%) patients had Tg levels higher than 30 ng . ml(-1), while Tg levels were lower than 30 ng . ml(-1) in 12/32 patients. Tg levels decreased in 21/32 and increased in 3/32 patients. The 131I WBS continued to be abnormal in 2/3 patients with increased Tg levels but became normal in one patient whose CT scan still showed macro-nodular lesions. Tg levels did not change significantly in 8/32 patients. The 131I WBS became normal in 5/8 patients, while the CT scans for 4/5 showed micro-nodules. Metastases were detected in 12/19 patients who underwent 99mTc-MIBI whole-body scanning: 18/19 showed metastases on the 131I WBSs and 17/19 on the CT scans. Of the seven patients without a sign of metastasis on the 99mTc-MIBI WBS, one was negative in terms of metastasis on the 131I WBS and one on the CT scan. Fibrosis was observed on the CT scans of 2/32 patients. One patient developed dedifferentiation, as determined by the negative 131I WBS and positive CT scan. CONCLUSION: 131I whole-body scanning and the determination of Tg levels are the most important procedures for the evaluation of lung metastases in differentiated thyroid cancer. Computed tomography is a useful addition to 131I whole-body scanning. MIBI imaging alone may not be enough to detect lung metastases from differentiated thyroid cancer.