Low occurrence of the HLA-C*04:09N allele in a large Hungarian cohort.

The presence of null alleles may affect the outcome of stem cell transplantation. HLA-C*04:09N was defined as 'common' with a frequency of 2-5/1000 in Caucasians, and its presence is routinely tested as part of haplotypes HLA-A*02:01/A*23:01-B*44:03-DRB1*07:01-DQB1*02:01. We aimed to investigate HLA-C*04:09N in a representative Hungarian cohort. HLA-typing data of 7345 unrelated persons were analyzed. The presence of HLA-C*04:09N was excluded in 157 chromosomes with either serology typing or with an allele-specific polymerase chain reaction for HLA-C*04:09N. HLA-C*04:09N was identified in a single chromosome with HLA-A*02, B*44, C*04, DRB1*07 resulting in a HLA-C*04:09N allele frequency of 0.0068% (1/14,690). This is approximately a 10- to 40-fold lower frequency compared with the previous data. Our results emphasize the need of precise local population-specific HLA-data, allowing appropriate modifications of local HLA-typing protocols.

HLA genetic diversity in Hungarians and Hungarian Gypsies: complementary differentiation patterns and demographic signals revealed by HLA-A, -B and -DRB1 in Central Europe.

Systematic analyses of human leukocyte antigen (HLA) profiles in different populations may increase the efficiency of bone marrow donor selection and help reconstructing human peopling history. We typed HLA-A, -B, and -DRB1 allele groups in two bone marrow donor cohorts of 2402 Hungarians and 186 Hungarian Gypsies and compared them with several Central-European, Spanish Gypsy, and Indian populations. Our results indicate that different European Gypsy populations share a common origin but diverged genetically as a consequence of founder effect and rapid genetic drift, whereas other European populations are related genetically in relation to geography. This study also suggests that while HLA-A accurately depicts the effects of genetic drift, HLA-B, and -DRB1 conserve more signatures of ancient population relationships, as a result of balancing selection.

Reduction of Bcr-Abl function leads to erythroid differentiation of K562 cells via downregulation of ERK.

The chimeric bcr-abl gene encodes a constitutively active tyrosine kinase that leads to abnormal transduction of growth and survival signals leading to chronic myeloid leukemia (CML). According to our previous observations, in vitro differentiation of several erythroid cell lines is accompanied by the downregulation of extracellular signal-regulated kinases (ERK)1/2 mitogen-activated protein kinase (MAPK) activities. In this work we investigated whether ERKs have a decisive role in either the erythroid differentiation process or apoptosis of bcr-abl+ K562 cells by means of direct (MEK1/2 inhibitor UO126) and indirect (reduced Bcr-Abl function) inhibition of their activities. We found that both Gleevec and UO126 induced hemoglobin expression. Gleevec treatment reduced the phosphorylation of Bcr-Abl, ERK and STAT-5 for up to 24 h, decreased Bcl-XL levels, and induced caspase-3-dependent apoptosis. In contrast, UO126 treatment resulted in only a transient decrease of ERK activity and did not induce cell death. For studying the effect of reduced Bcr-Abl function on erythroid differentiation at the level of the bcr-abl transcript, we applied the siRNA approach. Stable degradation of bcr-abl mRNA was achieved by using a retroviral vector with enhanced green fluorescent protein (EGFP) reporter. Despite a high (>90%) transduction efficiency we detected only a transient decrease in Bcr-Abl protein and in phosphorylated ERK1/2 levels. This transient change in Bcr-Abl signaling was sufficient to induce hemoglobin expression without significant cell death. These results suggest that by transiently reducing Bcr-Abl function it is possible to overcome the differentiation blockade without evoking apoptosis in CML cells and that reduced ERK activity may have a crucial role in this process.

The potential role of HLA-DRB1*11 in the development and outcome of haematopoietic stem cell transplantation-associated thrombotic microangiopathy.

Transplantation-associated thrombotic microangiopathy (TA-TMA) is a serious complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT) with high mortality rate. We retrospectively studied the frequency, clinical and genetic associations and prognostic effect of TA-TMA, in a total of 425 consecutive adult patients, who underwent allo-HSCT for a malignant haematological condition between 2007 and 2013 at our single centre. TA-TMA developed in 19% of the patients. Unrelated donor type (P<0.001), acute GvHD grades II-IV (P<0.001), myeloablative conditioning regimens (P=0.003), tacrolimus-based GvHD prophylaxis (P=0.003), CMV infection (P=0.003) and carriership for HLA-DRB1*11 (P=0.034) were associated with the development of TA-TMA. Survival was adversely affected by the presence of TA-TMA (P<0.001). Among patients with TA-TMA, the outcome of HLA-DRB1*11 carriers was significantly better compared with non-carriers (P=0.003). As a new finding, our observations suggest that the presence of HLA-DRB1*11 antigen contributes to the development of TA-TMA and affects the outcome.

Image watermarking using block site selection and DCT domain constraints.

In this paper we propose an image watermarking algorithm based on constraints in the Discrete Cosine Transform (DCT) domain. An image watermarking algorithm has two stages: signature casting (embedding) and signature detection. In the first stage it embeds an identifying label in the image. This is recognized in the second stage. The proposed algorithm has two processing steps. In the first step certain pixel blocks are selected using a set of parameters while in the second step a DCT coefficient constraint is embedded in the selected blocks. Two different constraint rules are suggested for the parametric modification of the DCT frequency coefficients. The first one embeds a linear constraint among certain selected DCT coefficients and the second defines circular detection regions according to the given parameters. The watermarks cast by the proposed algorithm are resistant to JPEG compression and filtering.

Optical flow estimation and moving object segmentation based on median radial basis function network.

Various approaches have been proposed for simultaneous optical flow estimation and segmentation in image sequences. In this study, the moving scene is decomposed into different regions with respect to their motion, by means of a pattern recognition scheme. The inputs of the proposed scheme are the feature vectors representing still image and motion information. Each class corresponds to a moving object. The classifier employed is the median radial basis function (MRBF) neural network. An error criterion function derived from the probability estimation theory and expressed as a function of the moving scene model is used as the cost function. Each basis function is activated by a certain image region. Marginal median and median of the absolute deviations from the median (MAD) estimators are employed for estimating the basis function parameters. The image regions associated with the basis functions are merged by the output units in order to identify moving objects.

Object classification in 3-D images using alpha-trimmed mean radial basis function network.

We propose a pattern classification based approach for simultaneous three-dimensional (3-D) object modeling and segmentation in image volumes. The 3-D objects are described as a set of overlapping ellipsoids. The segmentation relies on the geometrical model and graylevel statistics. The characteristic parameters of the ellipsoids and of the graylevel statistics are embedded in a radial basis function (RBF) network and they are found by means of unsupervised training. A new robust training algorithm for RBF networks based on alpha-trimmed mean statistics is employed in this study. The extension of the Hough transform algorithm in the 3-D space by employing a spherical coordinate system is used for ellipsoidal center estimation. We study the performance of the proposed algorithm and we present results when segmenting a stack of microscopy images.

Prediction and tracking of moving objects in image sequences.

We employ a prediction model for moving object velocity and location estimation derived from Bayesian theory. The optical flow of a certain moving object depends on the history of its previous values. A joint optical flow estimation and moving object segmentation algorithm is used for the initialization of the tracking algorithm. The segmentation of the moving objects is determined by appropriately classifying the unlabeled and the occluding regions. Segmentation and optical flow tracking is used for predicting future frames.

Median radial basis function neural network.

Radial basis functions (RBFs) consist of a two-layer neural network, where each hidden unit implements a kernel function. Each kernel is associated with an activation region from the input space and its output is fed to an output unit. In order to find the parameters of a neural network which embeds this structure we take into consideration two different statistical approaches. The first approach uses classical estimation in the learning stage and it is based on the learning vector quantization algorithm and its second-order statistics extension. After the presentation of this approach, we introduce the median radial basis function (MRBF) algorithm based on robust estimation of the hidden unit parameters. The proposed algorithm employs the marginal median for kernel location estimation and the median of the absolute deviations for the scale parameter estimation. A histogram-based fast implementation is provided for the MRBF algorithm. The theoretical performance of the two training algorithms is comparatively evaluated when estimating the network weights. The network is applied in pattern classification problems and in optical flow segmentation.

[A new method of molecular testing in the differential diagnosis of hereditary hemochromatosis]. / Uj, molekuláris genetikai módszer az öröklödö haemochromatosis differenciáldiagnosztikájában.

Hereditary hemochromatosis is an autosomal, recessive disorder of the iron metabolism. The hemochromatosis gene (HFE) was previously located on chromosome 6 and recently identified by positional cloning. A point mutation, C282Y, was found to be present in the HFE gene in homozygous form in 64 to 100% of patients with established hemochromatosis. The relationship of a second polymorphic variant of the HFE gene, H63D to the formation of iron overload is debated. Although hemochromatosis is one of the most common inherited disorders among Caucasians, in the absence of specific signs it is rarely diagnosed. In order to obtain comparable epidemiological data for Hungary, we tested 1271 and 277 randomly selected, unrelated, healthy subjects for C282Y and H63D respectively. In addition C282Y testing was carried out in 58 patients suffering from liver cirrhosis, and in 191 individuals with suspected hemochromatosis. For C282Y and H63D mutation analyses polymerase chain reaction technique followed by Rsa I and Bcl I restriction enzyme digestion was used. We developed an alternative method for the detection of C282Y based on an amplification-generated Kpn I restriction site. The allele frequencies were 3.8% and 12.3% for C282Y and H63D respectively in the normal Hungarian population. There was no significant difference in C282Y allele frequencies between liver disease patients (1.7%) and the normal population. We identified 15 homozygous and 25 heterozygous individuals among 191 individuals with suspected hemochromatosis. The C282Y and the H63D allele frequencies in the normal Hungarian population were found to be similar to the allele frequencies observed in other European populations, indicating that there is a large number of individuals susceptible for iron overload in Hungary (1:700). Mutation analysis is a novel, non-invasive method in the diagnostics of hereditary hemochromatosis, which increasingly becomes part of the routine clinical work.

GnRH Antagonist IVF Protocol in PCOS.

The aim of the present study was to compare the GnRH agonist long protocol with the flexible GnRH antagonist protocol in infertile PCOS women undergoing COS in terms of clinical pregnancy rate (CPR), with special reference to the incidence of OHSS. Materials and Methods. The study was conducted at the Hospital Obstetrics and Gynecology Cuza Voda Iasi and Fertility Reproductive Medical Center Omini Clinic Iasi from June 1, 2010, to September 31, 2012. PCOS as defined by the Rotterdam 2003 consensus, i.e. presence of two of the following three features: presence of oligo- and/ or anovulation, clinical and/or biochemical signs of hyperandrogenism, polycystic ovaries and exclusion of other endocrinopathies. Results. No differences were observed in clinical pregnancy rate (CPR) in the agonist and antagonist protocols, respectively. Incidence of OHSS was lower in the antagonist compared with agonist group (4% versus 28%). Duration of stimulation (13,80 + 1,4 vs 11,85 + 2,4 p < 0,001) and total gonadotrophin required (2435,5 + 884,5 versus 2005, 5 + 545,5 IU p < 0.003) were also lower in the antagonist compared with agonist protocol. Conclusions. The current study suggests that the flexible GnRH antagonist protocol is associated with a similar ongoing pregnancy rate, lower incidence of OHSS grade II, lower gonadotrophin requirement and shorter duration of stimulation, compared with GnRH agonist. The GnRH antagonist might be the treatment choice for patients with PCOS undergoing IVF.

The effect of new adamantane derivatives on experimental influenza infection in mice.

Two newly synthesized derivatives of 1-aminoadamantane hydrochloride--CF.01.01 and CF.01.06--were assayed for their anti-influenza efficacy. Aqueous solutions of the drugs (200 gamma/0.1 ml) were administered intranasally to mice at 24 hours and 5 hours or at 24 hours, 5 hours and 1 hour prior to intranasal inoculation of influenza virus A/PR/8/34 (H1N1). Compound CF.01.06 caused a significant decrease of the HA titer in the lungs of virus-infected mice. There was no change in mortality rate and the increase in mean survival time was at the limit of statistical significance.

Genotype screening for hereditary hemochromatosis among voluntary blood donors in Hungary.

Hereditary hemochromatosis (HH) is a common genetic disorder. Although it is inherited in an autosomal recessive manner, heterozygous individuals are believed to be protected against iron deficiency. Screening to estimate the prevalence of HH was frequently performed among blood donors, not considering that carriers of the HH gene mutations may be present in higher proportion in this population. To examine the allele frequencies of the HH gene (HFE) point mutations, C282Y and H63D genotyping was carried out in 996 consecutive, first-time, and regular Hungarian blood donors by PCR-RFLP techniques. Iron parameters of the first-time donors and the identified C282Y heterozygotes and age, gender, and number of previous blood donation-matched wild-type donors were also determined. We were not able to demonstrate a significant increase in the frequency of C282Y and H63D alleles among regular blood donors, compared to first-time blood donors. However, there was a trend of higher C282Y allele frequency among women with higher number of previous blood donations (2.2 +/- 1.5% in female blood donors with 0-8 previous blood donations compared to 4.8 +/- 2.3% in women with more than 8 previous blood donations, P = 0.06). No detectable phenotypic differences were observed in serum iron, ferritin, and transferrin saturation values between C282Y wild-type and heterozygous groups. However, the single identified C282Y homozygous male (age 21) showed definite signs of iron overload. Our observations suggest that the protective effect of C282Y heterozygosity against iron deficiency may be less significant than other environmental (e.g., iron-rich diet) or genetic factors.

A haemophilia A and B molecular genetic diagnostic programme in Hungary: a highly informative and cost-effective strategy.

Our aim was to set up a protocol in order to provide carrier and prenatal diagnosis to Hungarian haemophilia A (HA) and B (HB) patients and their relatives. For HA, a combination of direct mutation detection and some indirect marker analyses were used: the detection of the inversion mutation and analysis of three polymorphisms, BclI, IVS13 (CA)n and P39(CA)n. In severe cases, direct mutation detection was performed first. In inversion-negative severe cases and in moderate and mild cases, indirect methods were used. For carrier and prenatal diagnosis in HB, four polymorphisms, DdeI, TaqI, XmnI, and HhaI were examined. Our DNA bank contains samples from 50 HA families (34 severe, 15 moderate and one mild) and seven HB families from different parts of the country. In 100% of the HA cases either the gene inversion and/or at least one of the polymorphisms was found to be informative for carrier or prenatal diagnosis. In the HB cases, an informative marker was found in 95% of the cases (19 of 20). We conclude that these strategies are sufficient to make genetic diagnosis available to almost all HA and HB families in the region. This approach is highly informative and cost-effective, so it can be very useful in countries where direct sequencing of genes for factor VIII and IX is not available for routine diagnosis.