Increased extracellular matrix and proangiogenic factor transcription in endothelial cells after cocultivation with primary human osteoblasts.

The most promising strategies in bone engineering have concentrated on providing sufficient vascularization to support the newly forming tissue. In this context, recent research in the field has focused on studying the complex interactions between bone forming and endothelial cells. Our previous work has demonstrated that direct contact cocultivation of human umbilical vein endothelial cells (HUVECs) with primary human osteoblasts (hOBs) induces the osteogenic phenotype and survival of hOBs. In order to investigate the mechanisms that lead to this effect, we performed microarray gene expression profiling on HUVECs following cocultivation with hOBs. Our data reveal profound transcriptomic changes that are dependent on direct cell contact between these cell populations. Pathway analysis using the MetaCore™ platform and literature research suggested a striking upregulation of transcripts related to extracellular matrix and cell-matrix interactions. Upregulation of a number of major angiogenetic factors confirms previous observations that HUVECs enter a proangiogenic state upon cocultivation with osteoblasts. Interestingly, the downregulated transcripts clustered predominantly around cell cycle-related processes. The microarray data were confirmed by quantitative real-time RT-PCR on selected genes. Taken together, this study provides a platform for further inquiries in complex interactions between endothelial cells and osteoblasts.

[Progress in the elimination of measles and rubella in the WHO European Region]. / Fortschritte bei der Eliminierung von Masern und Röteln in der Europäischen Region der WHO.

Substantial progress has been made in the World Health Organization (WHO) European Region toward reaching the goal of measles and rubella elimination. We analyzed the surveillance data of 2012 on measles and rubella for age-group, diagnosis confirmation status (clinical, laboratory-confirmed and epidemiologically linked), vaccination status, and measles-related deaths. For 2012, there were 23,871 measles cases and 29,361 rubella cases reported in the region, mostly among unvaccinated persons. Almost one in three patients with measles and one in five patients with rubella were aged 20 years and older. In a few countries, widespread outbreaks or indigenous transmission of measles persisted in 2012. While most countries in the region have controlled rubella, a small number still reported a high incidence and several outbreaks. Therefore, more efforts are required to achieve the goal of eliminating measles and rubella in the WHO European Region by 2015, particularly in high-incidence countries. The WHO measles and rubella elimination plan stipulates that all countries should achieve and maintain the required high vaccination coverage while conducting high-quality surveillance.

Potential hazards to embryo implantation: A human endometrial in vitro model to identify unwanted antigestagenic actions of chemicals.

Embryo implantation is a crucial step in human reproduction and depends on the timely development of a receptive endometrium. The human endometrium is unique among adult tissues due to its dynamic alterations during each menstrual cycle. It hosts the implantation process which is governed by progesterone, whereas 17ß-estradiol regulates the preceding proliferation of the endometrium. The receptors for both steroids are targets for drugs and endocrine disrupting chemicals. Chemicals with unwanted antigestagenic actions are potentially hazardous to embryo implantation since many pharmaceutical antiprogestins adversely affect endometrial receptivity. This risk can be addressed by human tissue-specific in vitro assays. As working basis we compiled data on chemicals interacting with the PR. In our experimental work, we developed a flexible in vitro model based on human endometrial Ishikawa cells. Effects of antiprogestin compounds on pre-selected target genes were characterized by sigmoidal concentration-response curves obtained by RT-qPCR. The estrogen sulfotransferase (SULT1E1) was identified as the most responsive target gene by microarray analysis. The agonistic effect of progesterone on SULT1E1 mRNA was concentration-dependently antagonized by RU486 (mifepristone) and ZK137316 and, with lower potency, by 4-nonylphenol, bisphenol A and apigenin. The negative control methyl acetoacetate showed no effect. The effects of progesterone and RU486 were confirmed on the protein level by Western blotting. We demonstrated proof of principle that our Ishikawa model is suitable to study quantitatively effects of antiprogestin-like chemicals on endometrial target genes in comparison to pharmaceutical reference compounds. This test is useful for hazard identification and may contribute to reduce animal studies.

Leukemia-targeting ligands isolated from phage-display peptide libraries.

Ligands specifically binding to leukemia cells may be used for drug targeting, resulting in more effective treatment with less side effects. Little is known about receptors specifically expressed on acute myeloid leukemia (AML) cells or ligands thereof. We selected random phage display peptide libraries on Kasumi-1 AML cells. A peptide with the sequence CPLDIDFYC was enriched. Phage displaying this peptide strongly bound to Kasumi-1 and SKNO-1 cells and binding could be inhibited by the cognate peptide. Both, Kasumi-1 and SKNO-1 cells carry the chromosomal translocation t(8;21), leading to aberrant expression of the fusion protein AML1/ETO. CPLDIDFYC also strongly and specifically bound primary AML1/ETO-positive AML blasts as well as U-937 cells with forced AML1/ETO expression, suggesting that the CPLDIDFYC receptor may be upregulated upon AML1/ETO expression. Gene expression profiling comparing a panel of CPLDIDFYC-binding and CPLDIDFYC-nonbinding cell lines identified a set of potential receptors for the CPLDIDFYC peptide. Further analysis suggested that alpha4beta1 integrin (VLA-4) is the CPLDIDFYC receptor. Finally, we showed that the CPLDIDFYC-phage is internalized upon receptor binding, suggesting that the CPLDIDFYC-receptor-ligand interaction may be exploitable for targeting drugs or gene therapy vectors to leukemia cells carrying the suitable receptor.

Whole blood miRNA expression analysis reveals miR-3613-3p as a potential biomarker for dedifferentiated liposarcoma.

BACKGROUND: Liposarcoma constitute about 13% of all soft tissue sarcoma and are associated with a high risk of metastases. As the preoperative differentiation between benign and malign lipomatous tumors is restricted to magnetic resonance imaging, computed tomography and biopsy, we performed a miRNA array to distinguish dedifferentiated liposarcoma patients from healthy controls and lipoma patients. METHODS: Blood samples of patients with dedifferentiated liposarcoma, healthy controls and lipoma patients were collected. Whole blood RNA was extracted and samples of patients with dedifferentiated liposarcoma (n= 6) and of healthy donors (n= 4) were analyzed using an Affymetrix GeneChip miRNA Array v. 4.0. qRT-PCR was carried out to confirm the most differentially expressed miRNA; being further analyzed in an independent cohort of healthy controls as well as in lipoma patients. RESULTS: As shown by the microarray, two miRNAs (miR-3613-3p, miR-4668-5p) were shown to be significantly upregulated (fold change: > 2.5; p< 0.05) in patients with dedifferentiated liposarcoma (n= 6) as compared to healthy controls (n= 4). miR-3613-3p was further validated by qRT-PCR to be significantly upregulated in dedifferentiated liposarcoma patients compared to an independent cohort of healthy controls (n= 3) and lipoma patients (n= 5). CONCLUSION: We identified a specific whole blood miRNA (miR-3613-3p) that may serve to distinguish between dedifferentiated liposarcoma patients and healthy controls, thus potentially serving as a specific biomarker for dedifferentiated liposarcoma.

The sequence complexity of exons trapped from the mouse genome.

BACKGROUND: A central issue in genome analysis is the identification and characterization of coding regions. Estimating the coding complexity of vertebrate genomes by measuring the kinetic complexity of mRNA populations and by sequence analysis of cDNAs is limited by the fact that any given source of mRNA represents a very biased sample of all genes. Exon trapping is a method that enables the identification of genes irrespective of their transcriptional status. RESULTS: Exons were trapped from the entire mouse genome, and the resulting fragments cloned. About 7% of a random sample of exons taken from this library have significant structural homology or sequence similarity to previously sequenced genes. Using cDNAs derived from several stages of mouse development, evidence for expression of about 62% of this sample of exons was found. These data suggest that the great majority of 'exons' in the library are derived from genes. We estimate that the fraction of the genome contained in trapped exons is 2.4%; this corresponds to a sequence complexity of about 72 megabases. CONCLUSIONS: The library of exons trapped from the entire mouse genome probably represents one of the least biased and most comprehensive libraries of mouse coding regions, and should therefore prove very useful for finding genes during genome mapping and sequencing.

MicroRNA-146a reduces MHC-II expression via targeting JAK/STAT signaling in dendritic cells after stem cell transplantation.

Acute Graft-versus-host disease (GVHD) is a major immunological complication after allogeneic hematopoietic cell transplantation and a better understanding of the molecular regulation of the disease could help to develop novel targeted therapies. Here we found that a G/C polymorphism within the human microRNA-146a (miR-146a) gene of transplant recipients, which causes reduced miR-146a levels, was strongly associated with the risk of developing severe acute GVHD (n=289). In mice, deficiency of miR-146a in the hematopoietic system or transfer of recipient-type miR-146a-/- dendritic cells (DCs) enhanced GVHD, while miR-146a mimic-transfected DCs ameliorated disease. Mechanistically, lack of miR-146a enhanced JAK2-STAT1 pathway activity, which led to higher expression of class II-transactivator (CIITA) and consecutively increased MHCII-levels on DCs. Inhibition of JAK1/2 or CIITA knockdown in DCs prevented miR-146a-/- DC-induced GVHD exacerbation. Consistent with our findings in mice, patients with the miR-146a polymorphism rs2910164 in hematopoietic cells displayed higher MHCII levels on monocytes, which could be targeted by JAK1/2 inhibition. Our findings indicate that the miR-146a polymorphism rs2910164 identifies patients at high risk for GVHD before allo-HCT. Functionally we show that miR-146a acts as a central regulator of recipient-type DC activation during GVHD by dampening the pro-inflammatory JAK-STAT/CIITA/MHCII axis, which provides a scientific rationale for early JAK1/2 inhibition in selected patients.

Exon amplification from complete libraries of genomic DNA using a novel phage vector with automatic plasmid excision facility: application to the mouse neurofibromatosis-1 locus.

The identification of transcription units in the vicinity of chromosomal lesions found in tumours is an essential step in the identification of new oncogenes. Here, we describe a lambda phage vector system for genomic exon-trapping (lambda GET), which dramatically simplifies the task of exon amplification from genomic DNA. The vector accommodates about 6.5 to 19 kb of DNA and allows inserts to be automatically subcloned as multi-copy plasmids containing splice donor and acceptor sites positioned flanking the inserted genomic DNA. RNA transcripts derived from such plasmids are processed in vivo and exons contained within the inserted genomic fragments become flanked by known sequences in the resulting mRNAs. RNA-based PCR can then be used for subsequent cloning and sequence analysis of trapped exons. We have exploited the large cloning capacity of lambda GET to construct highly redundant complete genomic libraries from Sau3AI partially digested vertebrae DNAs. Using this system, we have analysed a region of about 1 MB around the mouse neurofibromatosis-1 locus and have identified novel transcription units flanking the Nf-1 gene.

Protein fingerprinting: a novel virus identification system.

Viral proteins separated by one-dimensional SDS-PAGE produce protein binding patterns (fingerprints) which are unique for different viruses. We have applied this concept successfully for the development of a practical and objective virus identification system which is applicable to most viruses. The method is simple, specific, and, unlike the currently available methods, free from all virus-specific reagents. Interference by host protein bands in SDS-PAGE preparations of virus-infected cell lysates was eliminated consistently by treating virus infected cell cultures with optimum concentration of NaCl for selective inhibition of host protein synthesis. The method utilizes the comparison of protein fingerprints of 'unknown' viruses with protein fingerprints of reference viruses stored in a computer data base, using pattern recognition software. All 113 'unknown' virus strains were correctly identified to the genus level by the protein fingerprint method, when compared with the conventional virus identification methods.

Sudden death from trophoblastic embolism in pregnancy.

A case of a 24-year-old multigravida, with dry cough, dyspnea, fatigue, and weight loss with normal foetal growth rate is reviewed. Upon admission the patient suddenly became tachycardic, tachypnoic, cyanotic, followed by a non-palpable peripheral pulse, and asystole unresponsive to resuscitation. The autopsy revealed massive pulmonary trophoblastic embolism, bilateral pregnancy luteoma, and accelerated placental maturation. Trophoblastic embolism should be taken into consideration whenever cardiorespiratory emergency develops during pregnancy.

Ponderal index and disproportionate fetal growth in IDDM pregnancies.

Disproportionate macrosomia refers to excessive weight characterized by a high weight/length ratio. Disproportionate macrosomia is associated with an increased likelihood of neonatal complications. The aim of the study was to investigate incidence of ponderal indexes and disproportionate fetal growth rate in newborns originating from IDDM and healthy pregnancies. 144 IDDM pregnancies and 432 uneventful pregnancies with normal findings of oral glucose tolerance test were studied, and matched 1:3 for gestational age, sex of newborn, mothers's parity and year of delivery. The pregnancies selected terminated between 30-40th gestational week and resulted with live birth. Mean birth weight (+/- SD) in IDDM group was 3558 +/- 817.6 compared to 3132.4 +/- 534.4 grams of control group (F = 51.49; p < 0.001), mean birth length was 49.8 +/- 3.5 vrs 49.1 +/- 2.5 (F = 8.55; p < 0.005), mean gestation age by examination for both study groups 37.9 +/- 1.9, mean ponderal index of IDDM group was 2.82 +/- 0.28 vrs. 2.63 +/- 0.24 (F = 64.52; p < 0.001) of control group, rate of Apgar score < 7 was 21.14% vrs. 5.08% (chi 2 = 30.30; p < 0.001). 53.4% of IDDMs had macrosomia compared with 8.33% of control infants (chi 2 = 140.25; p < 0.001), and 35.24% of IDDMs had disproportionate macrosomia compared with 5.79% of control infants. Significantly higher rate of both proportionally and disproportionally grown infants with macrosomia was found among IDDMs than among control infants. The rate of disproportionate macrosomic infants significantly differ among study group.

The effect of coagulation parameters on the placental respiratory and nutritive function in women having chronic hypertension with superimposed preeclampsia.

The aim of this study was to explore the relationship between coagulation and fibrinolytic system parameters with nutritive and respiratory placental function. We have analysed 79 pregnant women, of which 39 with severe preeclampsia (index group) and 41 healthy pregnant women. When comparing the study groups in third trimester, significantly lower platelet counts, fibrinogen values and antithrombin III values have been found in the index group compared to the control group. Factor VII levels were not found to be significantly different. The control group revealed significantly higher levels of coagulation factors II, V and VIII, compared to the index group. The increase of FDP, reduction of fibrinogen and increase of fibrinolysis in index group, when compared to the control group of healthy pregnant women, are the reflection of the intravascular fibrin deposition that leads to the described coagulation changes and consecutively to the foetal growth retardation. Indirect evidence are the correlation between newborns' weight and fibrinogen levels/fibrinolytic activity.

Effects of preeclampsia and eclampsia on cord blood coagulation tests.

Blood coagulation tests were determined in fifty-three paired umbilical cord blood and maternal venous blood samples originating from term singleton vaginal cephalic deliveries. The index group comprised seventeen deliveries complicated by preeclampsia or eclampsia, and the control group comprised thirty-six healthy women with uneventful pregnancies and deliveries. Mean values obtained from the coagulation and fibrinolytic assays did not significantly differ between study groups, except for antithrombin III levels in index group of neonates, which were significantly lower. Comparison of coagulation and fibrinolytic characteristics between mothers and their neonates produced expected level of difference due to immaturity of their haemostatic mechanisms. We found alterations in maternal blood coagulation and fibrinolysis and evidence of increased intravascular coagulation with severe preeclampsia and IUGR.

Influence of hyperglycemia on early embryonal growth in IDDM pregnant women.

The association between maternal diabetes mellitus and congenital anomalies is well established. Congenital malformations in the offspring of diabetic mothers account for approximately forty percent of perinatal deaths. The aim of the study was to identify incidence of early embryonal delay in diabetic and normal pregnancies, and to examine relationship between the HbA1c values and early embryonal growth delay. One hundred twenty IDDM and fifty and four healthy women enrolled into the study. Pregnancy duration was confirmed by beta-HCG measurements within a fortnight from the missed menstrual period. No statistical difference was detected between the studied groups for gestational age, prepregnancy weight, newborns' birthweight and sex. The risk of spontaneous abortion in IDDM pregnancy with delayed embryonal growth was eight times higher than in IDDM pregnancies with normal growth pattern. No fetal malformations were determined in fetuses or newborns of either groups. The mean value and standard deviation of HbA1c in the IDDM patients with normal embryonic growth was 7.3 +/- 1.5%, and in the group of early embryonic growth, delay 9.39 +/- 2.37% respectively (F = 7.79; p = 0.006). This study confirmed the relationship between embryonal growth, spontaneous abortions and abnormal metabolic control of IDDM pregnancies.

Glycosylated hemoglobin and fetal growth in normal, gestational and insulin dependent diabetes mellitus pregnancies.

Aims of the study were: evaluation of HbA1c levels in the peripheral blood of pregnant women with insulin dependent diabetes, gestational diabetes, glucose intolerance, and healthy pregnant controls; implications of HbA1c concentration on detection and the control of women with impaired carbohydrate metabolism in pregnancy; comparison of HbA1c levels with appearance of miscarriages, and premature deliveries; comparison of weight gain during pregnancy to HbA1c levels; comparison of difference from ideal body weight with HbA1c in diabetic pregnant women; comparison of neonatal birth weight and HbA1c levels. 290 pregnant women were enrolled to the study. The highest value of HbA1c was in the group IDDM pregnant women (7.7% +/- 1.8%), and the lowest value of HbA1c was in the control group (4.1% +/- 0.5%). Statistically significant coefficients were found between HbA1c and weight gain during pregnancy, between weight deviation from ideal body weight and HbA1c (r = 0.54 and r = 0.48 respectively); and between newborns weight and HbA1c (r = 0.51). Well regulated glycemia and intensive pregnancy follow-up of diabetic women reduces stillbirths, neonatal complications and neonatal macrosomia incidence.

Successful treatment of alloimmune thrombocytopenia using corticosteroid therapy in a woman with two consecutive neonatal deaths--case report.

Alloimmune thrombocytopenia is a serious fetal disorder resulting from platelet-antigen incompatibility between the mother and the fetus. In mild cases, the diagnosis is usually made upon detection of neonatal thrombocytopenia, but serious consequences such as fetal intracranial hemorrhage and/or unexplained fetal death may complicate the disorder. Various treatment modalities are suggested in the management of alloimmune thrombocytopenia, however, none has yet been confirmed as obviously superior. We report on the successful use of corticosteroids during pregnancy in a woman with a history of two consecutive neonatal deaths due to severe thrombocytopenia and HPA 5b platelet-specific antigen incompatibility.

[Fetal growth in pregnant women with chronic hypertension]. / Rast fetusa u trudnica s kronicnom hipertenzijom.

This study represents a retrospective analysis of pregnancies with chronic arterial hypertension and their outcomes. The aim was to evaluate the influence of arterial hypertension on 101 essential and 109 cases of secondary hypertension in comparison to the control group consisting of 499 normotensive pregnancies. According to the obtained data, 27.7% of the women with chronic hypertension had proteinuria, 61% had bacteriuria and 58.6% had superimposed EPH gestosis. The occurrence of EPH gestosis among the controls was 5.6%, that is significantly less than in the experimental group (X2 = 282.8%; p < 0.001). The outcomes of pregnancies associated with chronic hypertension were: 19% preterm deliveries compared to the controls in which only 9.2% preterm deliveries occurred (X2 = 14.4; p < 0.001). Newborns from pregnancies with essential hypertension were significantly heavier, weighing 3177 +/- 734 g, than those from pregnancies with secondary hypertension, which weighted 2578 +/- 932 g. Perinatal mortality was higher in the study group and significantly higher in the pregnancies with associated secondary hypertension (30.3%) than in pregnancies associated with essential hypertension (15.8%).

The state of measles and rubella in the WHO European Region, 2013.

Measles and rubella persist in the World Health Organization European Region despite long-standing and widespread use of vaccines against them. Our aim was to review the epidemiology of measles and rubella in relation to the goal of eliminating these diseases from the Region by 2015. We report on the number of measles and rubella cases by country in 2012 and present an analysis of preliminary measles and rubella surveillance data for 2013. We analysed data of these diseases for 2013 by age group, diagnosis confirmation (clinical, laboratory-confirmed and epidemiologically linked), and vaccination, hospitalization and importation status. We also report on measles-related deaths. For 2012, there were 26,785 [corrected] measles cases and 29,601 rubella cases reported in the Region. For 2013, these figures were 31,520 and 39,367 respectively. Most measles cases in 2013 (96%; n = 30,178) were reported by nine countries: Georgia (7830), Germany (1773), Italy (2216), the Netherlands (2499), Romania (1074), the Russian Federation (2174), Turkey (7404), Ukraine (3308) and the United Kingdom (1900). In 2013, most measles cases were among unvaccinated persons and over one in three patients were aged 20 years and older. For 2013, almost all rubella cases were reported by Poland (n = 38,585; 98%). High population immunity and high-quality surveillance are the cornerstones to eliminate measles and rubella. Without sustained political commitment and accelerated action by Member States and partners, the elimination of measles and rubella in the WHO European Region may not be achieved.

Depletion of STAT5 blocks TEL-SYK-induced APMF-type leukemia with myelofibrosis and myelodysplasia in mice.

The spleen tyrosine kinase (SYK) was identified as an oncogenic driver in a broad spectrum of hematologic malignancies. The in vivo comparison of three SYK containing oncogenes, SYK(wt), TEL-SYK and IL-2-inducible T-cell kinase (ITK)-SYK revealed a general myeloexpansion and the establishment of three different hematologic (pre)diseases. SYK(wt) enhanced the myeloid and T-cell compartment, without leukemia/lymphoma development. ITK-SYK caused lethal T-cell lymphomas and the cytoplasmic TEL-SYK fusion induced an acute panmyelosis with myelofibrosis-type acute myeloid leukemia (AML) with up to 50% immature megakaryoblasts infiltrating bone marrow, spleen and liver, additional MPN features (myelofibrosis and granulocyte expansion) and MDS stigmata with megakaryocytic and erythroid dysplasia. LKS cells were reduced and all subsets (LT/ST/MPP) showed reduced proliferation rates. SYK inhibitor treatment (R788) of diseased TEL-SYK mice reduced leukocytosis, spleen and liver infiltration, enhanced the hematocrit and prolonged survival time, but could not significantly reduce myelofibrosis. Stat5 was identified as a major downstream mediator of TEL-SYK in vitro as well as in vivo. Consequently, targeted deletion of Stat5 in vivo completely abrogated TEL-SYK-induced AML and myelofibrosis development, proving Stat5 as a major driver of SYK-induced transformation. Our experiments highlight the important role of SYK in AML and myelofibrosis and prove SYK and STAT5 inhibitors as potent treatment options for those diseases.