Katre Maasalu

Treatment of children with Osteogenesis imperfecta in Estonia

Aim: To analyse the changes in fracture rate, bone density and histology in children with Osteogenesis imperfecta receiving treatment with alendronate (oral bisphosphonate) and calcitriol. Methods: Children treated at Tartu University Hospital from 1995 to 2001 were examined for Osteogenesis imperfecta. Radiographs and bone density measurements were obtained for all patients at the beginning of the study. Four patients also had bone biopsies prior to and one year after beginning treatment. The children were then given alendronate in weight‐dependent dosages and also calcitriol. The number of fractures during the treatment period was recorded and follow‐up bone density measurements were made. Results: Fifteen patients were treated during the 6‐y period; mean follow‐up approximately 3 y. It was found that the number of bone fractures had decreased significantly (p < 0.0001). Bone density improved in all 15 patients. Histologic studies revealed an increased number of osteoblasts and thickness of bone trabeculae as well as a more regular bone lamellar structure at the time of the second operation.

Elevated Serum IL-6, IL-8, MCP-1, CRP, and IFN-γ Levels in 10- to 11-Year-Old Boys with Increased BMI

Background/Aims: Many inflammation parameters are associated with obesity, but few comparable data are found in youth. This study aims to characterize the differences in serum levels of 13 biochemical inflammatory markers between boys with increased BMI and boys with normal BMI, and examine the relationships between inflammation markers, skinfold thicknesses, and body composition. Participants/Methods: The participants were 38 boys (BMI above 85th percentile) and 38 boys (normal BMI) at the age of 10–11 years. Measurements included BMI, 9 skinfold thicknesses, waist and hip circumferences, and total body and trunk fat mass and percentage as indices of obesity, fasting insulin, glucose, and serum concentrations of IL-2, IL-4, IL-6, IL-8, IL-10, VEGF, IFN-γ, TNF-α, IL-1α, IL-1β, monocyte chemoattractant protein-1 (MCP-1), epidermal growth factor, and CRP. Results: Overweight boys (OWB) were taller and more frequently in puberty than normal-weight boys (NWB). Skinfold thicknesses and body composition parameters were higher in OWB. They had significantly higher serum IL-6, IL-8, IFN-γ, MCP-1, and CRP values compared to NWB. Conclusions: Six of 13 measured biochemical markers were significantly increased in OWB, indicating that many low-grade inflammatory processes are already involved in the development of obesity in childhood.

Physical Activity and Bone Mineral Accrual in Boys with Different Body Mass Parameters during Puberty: A Longitudinal Study

The aim of our longitudinal study was to investigate the relationships between physical activity and bone mass in boys with different body mass status during the years surrounding pubertal growth spurt. Two hundred and six boys entering puberty took part in this study. The subjects were divided into underweight (), normal weight (), overweight () and obese () groups at baseline according to age related categories. Whole-body DXA scans were performed at baseline, after 12 and 24 months to assess body composition (lean body mass, fat mass), and total body (TB), lumbar spine (LS) and femoral neck (FN) bone mineral density (BMD) parameters. Physical activity was measured by 7- day accelerometry. For longitudinal analysis, multilevel fixed effects regression models were constructed. Biological age, height and lean body mass had an effect for explanation of TB BMD, FN BMD and LS BMD. Moderate to vigorous physical activity (MVPA), vigorous physical activity (VPA) and sedentary time (SED) had the significant effect only on FN BMD. Being an underweight boy at the baseline indicated greater chance (p<0.01) to have lower TB BMD in the future (2 years at follow up) development, compared to normal weight (estimates = −0.038), overweight (estimates = −0.061) and obese boys (estimates = −0.106).

Neonatal spinal muscular atrophy type 1 with bone fractures and heart defect.

The authors present the case of an infant girl with severe generalized weakness, multiple bone fractures, and heart defect. She needed mechanical ventilation from birth. Radiographs showed mid-diaphyseal fractures of both humeri and of the right femur as well as generalized osteopenia. Electroneuromyography showed spontaneous fibrillations at rest with no active movements. Motor response to a stimulus could not be registered. A systolic heart murmur was detected, and echocardiography showed a large atrial septal defect and an additional membrane in the left atrium. DNA analysis confirmed the diagnosis of spinal muscular atrophy on the third day of life. Histology of the muscle showed both hypertrophic and atrophic fibers. Degenerating swollen neurons were found in the ventral horns of the spinal cord and also in the mesencephalic red nucleus, which has not been described before. Humeral bone showed only partly formed cortical bone. The spectrum of spinal muscular atrophy is very diverse, and atypical clinical findings do not always rule out 5q spinal muscular atrophy. The SMN1 gene should still be investigated.

Blocking tumor-educated MSC paracrine activity halts osteosarcoma progression

Purpose: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets. Experimental Design: By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)–educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography. Results: We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFβ, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFβ-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFβ are increased in osteosarcoma patients. Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFβ-blocking agents as new therapeutic options for osteosarcoma patients. Clin Cancer Res; 23(14); 3721–33. ©2017 AACR.

Negative correlation between serum IL-6 level and cardiorespiratory fitness in 10- to 11-year-old boys with increased BMI

Abstract Background/Aims: Some markers of inflammation have been found to be associated with cardiorespiratory fitness levels, but only few studies have studied this in overweight children. The aim of this study was to investigate associations between markers of inflammation and the fitness levels measured by peak oxygen consumption (VO2peak and VO2peak/kg) in boys with increased body mass index (BMI) and with normal BMI. Participants/Methods: Subjects were 38 boys with BMI above 85th percentile (OWB) and 38 boys with normal BMI (NWB) at the age of 10 to 11 years. Serum concentrations of IL-2, IL-4, IL-6, IL-8, IL-10, VEGF, IFNγ, TNF-α, IL-1α, IL-1β, MCP-1, EGF, CRP and associations with measured cardiorespiratory fitness levels were studied. High-sensitive chips were used to measure 13 markers of inflammation. Results: Mean VO2peak was significantly higher (2.1±0.3 vs. 1.8±0.3 L/min; p<0.05) and mean VO2peak/kg significantly lower (33.7±4.7 vs. 48.9±6.4 mL/min/kg; p<0.05) in OWB than in NWB group. Out of 13 measured biochemical markers IL-6 correlated with VO2peak/kg (r=–0.37; p<0.05) and TNF-α with VO2peak (r=0.41; p<0.01) in OWB. BMI and IL-6 together explained 44.5% of the variability of VO2peak/kg in the OWB group. Conclusions: Overweight boys had lower cardiorespiratory fitness level measured by VO2peak/kg and this was negatively correlated with serum IL-6 level. Measurement of serum IL-6 level in overweight boys may help to identify subjects who need specific exercise formats to achieve maximal beneficial health effects and to reduce their risk for the development of type 2 diabetes and atherosclerosis later in life.

Whole exome sequencing of a single osteosarcoma case—integrative analysis with whole transcriptome RNA-seq data

BackgroundOsteosarcoma (OS) is a prevalent primary malignant bone tumour with unknown etiology. These highly metastasizing tumours are among the most frequent causes of cancer-related deaths. Thus, there is an urgent need for different markers, and with our study, we were aiming towards finding novel biomarkers for OS.MethodsFor that, we analysed the whole exome of the tumorous and non-tumour bone tissue from the same patient with OS applying next-generation sequencing. For data analysis, we used several softwares and combined the exome data with RNA-seq data from our previous study.ResultsIn the tumour exome, we found wide genomic rearrangements, which should qualify as chromotripsis—we detected almost 3,000 somatic single nucleotide variants (SNVs) and small indels and more than 2,000 copy number variants (CNVs) in different chromosomes. Furthermore, the somatic changes seem to be associated to bone tumours, whereas germline mutations to cancer in general. We confirmed the previous findings that the most significant pathway involved in OS pathogenesis is probably the WNT/β-catenin signalling pathway. Also, the IGF1/IGF2 and IGF1R homodimer signalling and TP53 (including downstream tumour suppressor gene EI24) pathways may have a role. Additionally, the mucin family genes, especially MUC4 and cell cycle controlling gene CDC27 may be considered as potential biomarkers for OS.ConclusionsThe genes, in which the mutations were detected, may be considered as targets for finding biomarkers for OS. As the study is based on a single case and only DNA and RNA analysis, further confirmative studies are required.

Osteocalcin is inversely associated with adiposity and leptin in adolescent boys.

Abstract Background/Aims: Recently, osteocalcin (OC), an osteoblast-derived hormone, has been found to correlate with adiposity, adipocytokines and insulin resistance in adults, but few studies have investigated this in children. The aim of this study was to investigate these associations in adolescent boys, for whom it is a time of significant bone mineral accrual, taking into account possible confounders related to adipose and bone tissues. Participants/Methods: Participants were 141 adolescent boys (mean age 13.9±0.7 years), who were divided into tertiles according to OC levels. Across these groups, differences in total body fat mass (FM), body fat distribution, adiponectin, leptin and insulin resistance values were examined with relation to age, pubertal stage, daily energy and calcium intakes, and physical activity. Results: Mean body mass index (BMI), FM, body fat% and leptin differed significantly between subjects in the three OC tertiles after adjustment for age, pubertal stage, energy and calcium intakes, and physical activity. There were no differences in fat free mass (FFM), bone mineral content, energy and calcium intakes, physical activity, adiponectin and insulin resistance values between study groups. For the entire cohort, mean serum OC was 130.2±45.2 ng/mL and was related to body mass, BMI, FM, body fat distribution and leptin. Circulating OC was not associated with FFM, daily energy and calcium intakes, physical activity, adiponectin or insulin resistance (insulin, glucose, homeostasis model assessment-insulin resistance) values. Conclusions: In male adolescents, OC is inversely related to body adiposity and leptin values, even after consideration of several factors that may affect bone and adipose tissues.

Adipocytokine and Ghrelin Levels in Relation to Body Composition in Rhythmic Gymnasts Entering into Puberty: A Three-Year Follow-Up Study

The aim of this study was to describe longitudinal changes in body composition, leptin, adiponectin, and ghrelin over a 36-month period in prepubertal rhythmic gymnasts (RG) and their age-matched untrained controls (UC) entering into puberty. Thirty-five RG (8.0 ± 0.6 yrs) and 33 UC (8.2 ± 0.6 yrs) were followed at 12-month intervals for the next 3 years. Height, weight, pubertal stage, body composition, leptin, adiponectin, and ghrelin were measured at each time points. The pubertal development over the next 36 months was slower in the RG compared with UC. Leptin was increased in UC and remained unchanged in RG over 3-year study period (3.7 ± 3.6 vs. 0.2 ± 1.1 ng/ml; p < .05). In RG, baseline leptin was negatively correlated with the change in body fat percentage over a 36-month period (r = -0.34; p < .05). The change in adiponectin over the study period was negatively correlated with the change in BMI (r = -0.43; p < .05). RG had relative leptin deficiency per body fat mass. In conclusion, relatively high leptin concentration at the beginning of puberty may predict those girls who do not increase their body fat percentage through coming years and therefore may have increased risk for delayed puberty.

Transcriptome analysis of osteosarcoma identifies suppression of wnt pathway and up-regulation of adiponectin as potential biomarker

Osteosarcoma (OS) is primary malignant bone tumour with complicated early diagnosis. There are no specific markers currently available for predicting the prognosis and chemosensitivity of OS. In present study we performed transcriptome profiling of single patient tumour tissue with RNA-seq technology. We analysed surgically removed sarcoma sample from single 16 years old male patient. Transciptome analysis was done with RNA-seq technology, bioinformatics with Lifescope and R Bioconductor. Validation experiments were done with quantitative real-time PCR (QRTPCR). After quality and coverage filtering, RNA-seq experiment resulted 29,311,899 mapped reads for sarcoma and 22,099,159 mapped reads for normal bone tissue. 65 genes were differentially expressed with FDR corrected statistical significance below 0.05. Seven genes were down-regulated and 58 genes were up-regulated in sarcoma. The most highly up-regulated gene in sarcoma was adiponectin, ADIPOQ (with adjusted p-value 5.5E-07, log2 fold change was 7.9). Many of the genes we found are related to the adipose tissue metabolism (ADIPOQ, PLIN1, FABP4) and to the Wnt signalling suppression (WIF1, SOST). We also found novel fusion transcript between the genes LMTK2 and ZSWIM5. LMTK2 is lemur tyrosine kinase 2, and it has been shown to be involved in NGF-TrkA signalling. Interestingly, studies support the involvement of LMTK2 in development of prostate cancer. ZSWIM5 is zinc finger SWIM domain protein 5 and its function is not known. Immunohistochemical analysis confirmed positive staining for adiponectin in osteosarcoma. This paper is a good illustration how transcriptome analysis can find new biomarkers and targets for complex diseases.