Konsensuální doporučení ČNHP (Českého národního hemofilického programu) pro diagnostiku a léčbu pacientů s vrozenou hemofilií a s inhibitorem FVIII/FIX

Cílem standardu je stanovit základní diagnostické a především terapeutické postupy v péči o pacienty s vrozenou hemofilií a s inhibitorem koagulačního faktoru VIII, resp. faktoru IX.The aim of the guidelines is to set-up and standardize diagnostic as well as therapeutic approaches to care about patients with congenital haemophilia and inhibitors to coagulation factor VIII or factor I

Insights into the evolution of mammalian telomerase: Platypus TERT shares similarities with genes of birds and other reptiles and localizes on sex chromosomes

Background The TERT gene encodes the catalytic subunit of the telomerase complex and is responsible for maintaining telomere length. Vertebrate telomerase has been studied in eutherian mammals, fish, and the chicken, but less attention has been paid to other vertebrates. The platypus occupies an important evolutionary position, providing unique insight into the evolution of mammalian genes. We report the cloning of a platypus TERT (OanTERT) ortholog, and provide a comparison with genes of other vertebrates. Results The OanTERT encodes a protein with a high sequence similarity to marsupial TERT and avian TERT. Like the TERT of sauropsids and marsupials, as well as that of sharks and echinoderms, OanTERT contains extended variable linkers in the N-terminal region suggesting that they were present already in basal vertebrates and lost independently in rayfinned fish and eutherian mammals. Several alternatively spliced OanTERT variants structurally similar to avian TERT variants were identified. Telomerase activity is expressed in all platypus tissues like that of cold-blooded animals and murine rodents. OanTERT was localized on pseudoautosomal regions of sex chromosomes X3/Y2, expanding the homology between human chromosome 5 and platypus sex chromosomes. Synteny analysis suggests that TERT co-localized with sex-linked genes in the last common mammalian ancestor. Interestingly, female platypuses express higher levels of telomerase in heart and liver tissues than do males. Conclusions OanTERT shares many features with TERT of the reptilian outgroup, suggesting that OanTERT represents the ancestral mammalian TERT. Features specific to TERT of eutherian mammals have, therefore, evolved more recently after the divergence of monotremes.Radmila Hrdličková, Jiří Nehyba, Shu Ly Lim, Frank Grützner, Henry R Bose J

Regulation of Telomerase Activity by Interferon Regulatory Factors 4 and 8 in Immune Cells▿

Telomerase activity is downregulated in somatic cells but is upregulated during the activation of cells of the immune system. The mechanism of this reactivation is not well understood. In this study, we demonstrated that interferon regulatory factor 4 (IRF-4) and, to a lesser extent, IRF-8 induce telomerase activity. The suppression of IRF-4 results in decreased levels of TERT (telomerase reverse transcriptase) mRNA and telomerase activity and reduces cell proliferation. The overexpression of TERT compensates for this proliferation defect, suggesting that telomerase contributes to the regulation of cell proliferation by IRF-4. The induction of telomerase by IRF-4 and IRF-8 correlates with the activation of the TERT promoter. IRF-4 binds the interferon response-stimulated element and the gamma interferon-activated sequence composite binding site in the TERT core promoter region in vivo. Additionally, the binding of Sp1, Sp3, USF-1, USF-2, and c-Myc to the TERT promoter is elevated in cells expressing IRF-4. IRF-4, but not IRF-8, synergistically cooperates with Sp1 and Sp3 in the activation of the TERT promoter. Collectively, these results indicate that IRF-4 and IRF-8, two lymphoid cell-specific transcription factors, increase telomerase activity by activating TERT transcription in immune cells

A Novel Interferon Regulatory Factor (IRF), IRF-10, Has a Unique Role in Immune Defense and Is Induced by the v-Rel Oncoprotein

The cloning and functional characterization of a novel interferon regulatory factor (IRF), IRF-10, are described. IRF-10 is most closely related to IRF-4 but differs in both its constitutive and inducible expression. The expression of IRF-10 is inducible by interferons (IFNs) and by concanavalin A. In contrast to that of other IRFs, the inducible expression of IRF-10 is characterized by delayed kinetics and requires protein synthesis, suggesting a unique role in the later stages of an antiviral defense. Accordingly, IRF-10 is involved in the upregulation of two primary IFN-γ target genes (major histocompatibility complex [MHC] class I and guanylate-binding protein) and interferes with the induction of the type I IFN target gene for 2′,5′-oligo(A) synthetase. IRF-10 binds the interferon-stimulated response element site of the MHC class I promoter. In contrast to that of IRF-1, which has some of the same functional characteristics, the expression of IRF-10 is not cytotoxic for fibroblasts or B cells. The expression of IRF-10 is induced by the oncogene v-rel, the proto-oncogene c-rel, and IRF-4 in a tissue-specific manner. Moreover, v-Rel and IRF-4 synergistically cooperate in the induction of IRF-10 in fibroblasts. The level of IRF-10 induction in lymphoid cell lines by Rel proteins correlates with Rel transformation potential. These results suggest that IRF-10 plays a role in the late stages of an immune defense by regulating the expression some of the IFN-γ target genes in the absence of a cytotoxic effect. Furthermore, IRF-10 expression is regulated, at least in part, by members of the Rel/NF-κB and IRF families

Transfusion-related Acute Lung Injury: Report of Two Cases

Transfusion-related acute lung injury (TRALI) is a severe life-threatening complication of blood transfusion, characterized by acute lung injury developing within 2–6 h of transfusion. However, TRALI is difficult to diagnose, and the initial report or suspicion of TRALI depends on close collaboration between clinical departments and transfusion centres. A total of 17 adverse post-transfusion reactions were reported to the Blood Centre of the University Hospital Ostrava as suspected TRALI between 2005 and 2010. We report two cases of serious TRALI with different pathogenetic mechanisms

miRNAs targeting TERT regulate genes in the Wnt pathway.

<p>Western blot analysis of endogenously expressed TCF7 (38–80 kDa) and MSI1 (39 kDa and 43 kDa) proteins in DLD-1 cells transfected with scrambled control (SC), and MIX1, MIX2, and MIX3 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086990#pone-0086990-g003" target="_blank">Figure 3</a> or with transfection reagent alone (TR). The mixtures of miRNAs as well as SC were at a concentration of 60 nM. Single transfected miRNAs gave more modest differences than mixtures. Molecular weight markers are shown in the left margin. β-actin served as a loading control. The quantification of Western blot analysis (Table) is shown on the right side. The signals of TCF7 or MSI1 were normalized to β-actin and expressed as a percentage of SC control. The ratio of 43 to 38 kDa MSI1 forms is shown on the picture under MSI1 bands.</p

Selection of miRNA target sites in the 3′UTR of human TERT.

<p>(a) miRNA target sites predicted by TargetScanHuman 5.2 (<a href="http://www.targetscan.org/" target="_blank">http://www.targetscan.org/</a>). The horizontal bar on the right upper corner represents 100 kb scale. (b) miRNAs selected for experimental evaluation. The table identifies the miRNA gene, hairpin strand encoding the mature targeting strand (Str), the evolutionary conservation (EC) of the specific miRNA family: B - conserved in Bilateria, V - across vertebrates, P - in placental mammals. Number of targeting sites in the hTETR 3′UTR is indicated (#) together with the position in the 3′UTR (Pos). Evolutionary conservation of the sites is shown in the chimpanzee (Ptr), rhesus macaque (Mml), and common marmoset (Cja) indicated by black boxes (absolute conservation) or gray boxes (conserved when wobble G-U pairing is considered). Presence of the sites in the 3′UTR of the three genes of the Wnt pathway is indicated by a number in the gray field.</p

Downregulation of luciferase activity of the TCF7, MSI1, and PAX5 3′UTR reporters.

<p>The results are expressed as percent of reporter activity in cells transfected with a scrambled control (SC). Differences between the luciferase activity in SC and transfection agent treated cells were not statistically significant, demonstrating that transfection of miRNA did not induce a nonspecific effect (data not shown). Means and standard errors were calculated from 4 independent experiments. Statistically significant differences relative to the SC control are indicated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086990#pone-0086990-g002" target="_blank">Figure 2</a>. Number of miRNA sites in respective 3′UTR is displayed. let-7g* has no predicted binding sites in the 3′UTRs of these three genes.</p

miRNAs targeting hTERT decrease cell proliferation in the DLD-1 and MCF-7 cell lines.

<p>The cells were transfected by miRNA mimics and cell numbers were determined 48 hours post-transfection. An increase in cell death was not detected (data not shown). Means and standard errors were calculated from 5 (single miRNAs) and 3 independent experiments (mixtures of miRNAs). Statistically significant differences relative to the negative control is expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086990#pone-0086990-g002" target="_blank">Figure 2</a>. Differences between the SC and transfection agent treated cells were not statistically significant (data not shown). <b>(</b>a) Proliferation of DLD-1 cell transfected with miRNA mimic molecules are expressed as the percent of number of cells transfected with a scrambled control (SC). (b) Proliferation of DLD-1 and MCF-7 cells transfected with mixtures of miRNA mimics molecules expressed as a percent of the number of cells transfected with a scrambled control (SC).</p