bluuebunny/crossref_metadata_2025 huggingface dataset:
FDM: a graph-based statistical method to detect differential transcription using RNA-seq data
Authors: Singh Darshan , Orellana Christian F. , Hu Yin , Jones Corbin D. , Liu Yufeng , Chiang Derek Y. , Liu Jinze , Prins Jan F.
Published: August 2011

Motivation: In eukaryotic cells, alternative splicing expands the diversity of RNA transcripts and plays an important role in tissue-specific differentiation, and can be misregulated in disease. To understand these processes, there is a great need for methods to detect differential transcription between samples. Our focus is on samples observed using short-read RNA sequencing (RNA-seq).

Methods: We characterize differential transcription between two samples as the difference in the relative abundance of the transcript isoforms present in the samples. The magnitude of differential transcription of a gene between two samples can be measured by the square root of the Jensen Shannon Divergence (JSD*) between the gene's transcript abundance vectors in each sample. We define a weighted splice-graph representation of RNA-seq data, summarizing in compact form the alignment of RNA-seq reads to a reference genome. The flow difference metric (FDM) identifies regions of differential RNA transcript expression between pairs of splice graphs, without need for an underlying gene model or catalog of transcripts. We present a novel non-parametric statistical test between splice graphs to assess the significance of differential transcription, and extend it to group-wise comparison incorporating sample replicates.

Results: Using simulated RNA-seq data consisting of four technical replicates of two samples with varying transcription between genes, we show that (i) the FDM is highly correlated with JSD* (r=0.82) when average RNA-seq coverage of the transcripts is sufficiently deep; and (ii) the FDM is able to identify 90% of genes with differential transcription when JSD* >0.28 and coverage >7. This represents higher sensitivity than Cufflinks (without annotations) and rDiff (MMD), which respectively identified 69 and 49% of the genes in this region as differential transcribed. Using annotations identifying the transcripts, Cufflinks was able to identify 86% of the genes in this region as differentially transcribed. Using experimental data consisting of four replicates each for two cancer cell lines (MCF7 and SUM102), FDM identified 1425 genes as significantly different in transcription. Subsequent study of the samples using quantitative real time polymerase chain reaction (qRT-PCR) of several differential transcription sites identified by FDM, confirmed significant differences at these sites.

Availability:  http://csbio-linux001.cs.unc.edu/nextgen/software/FDM

Contact:  darshan@email.unc.edu

Supplementary information:  Supplementary data are available at Bioinformatics online.
Polycomb eviction as a new distant enhancer function
Authors: Vernimmen Douglas , Lynch Magnus D. , De Gobbi Marco , Garrick David , Sharpe Jacqueline A. , Sloane-Stanley Jacqueline A. , Smith Andrew J.H. , Higgs Douglas R.
Published: August 2011

Remote distal enhancers may be located tens or thousands of kilobases away from their promoters. How they control gene expression is still poorly understood. Here, we analyze the influence of a remote enhancer on the balance between repression (Polycomb—PcG) and activation (Trithorax—TrxG) of a developmentally regulated gene associated with a CpG island. We reveal its essential, nonredundant role in clearing the PcG complex and H3K27me3 from the CpG island. In the absence of the enhancer, the H3K27me3 demethylase (JMJD3) is not recruited to the CpG island. We propose a new role of long-range regulatory elements in removing repressive PcG complexes.
A snRNP's ordered path to maturity: Figure 1.
Authors: Valadkhan Saba
Published: August 2011

The U5 snRNP (small ribonucleoprotein) contains several functionally crucial splicing factors that form an extensive interaction network both in the snRNP and within the spliceosome. In this issue of Genes & Development, Weber and colleagues (pp. 1601–1612) shed light on the dynamic assembly of this critical spliceosomal component and elucidate the molecular interactions underlying the ordered addition of Brr2, a pivotal spliceosomal helicase, to the U5 snRNP.
‘Inc-miRs’: functional intron-interrupted miRNA genes
Authors: Zhang Huibin , Maniar Jay M. , Fire Andrew Z.
Published: August 2011

The discovery of microRNAs (miRNAs) lin-4 and let-7 as temporal regulators in Caenorhabditis elegans led to broader searches for novel miRNAs and their biological roles. Unlike protein-coding genes and some long noncoding RNAs, canonical metazoan miRNAs are not known to contain introns within their genomic precursor sequences. Because the short length of miRNAs complicates a statistically definitive assignment of split genes in RNA sequencing data sets, we took an experimental approach toward testing the compatibility of splicing and functional miRNA biogenesis. To definitively evaluate the possibility that miRNAs could derive from interrupted genes, we constructed intron-interrupted variants of C. elegans lin-4 and assayed for their miRNA-encoding capability and biological activity in the developing organism. Our studies indicate that (1) intron-containing miRNAs (inc-miRs) can be efficiently spliced and processed to produce miRNAs with normal termini, and (2) these miRNAs can be functional in full rescue of developmental phenotypes in null mutants lacking endogenous lin-4. This study provides the first evidence to support the ability of intron-interrupted miRNA precursors to produce functional regulators and identifies an additional modality available for metazoan miRNA production.
Copy number variation analysis in the great apes reveals species-specific patterns of structural variation
Authors: Gazave Elodie , Darré Fleur , Morcillo-Suarez Carlos , Petit-Marty Natalia , Carreño Angel , Marigorta Urko M. , Ryder Oliver A. , Blancher Antoine , Rocchi Mariano , Bosch Elena , Baker Carl , Marquès-Bonet Tomàs , Eichler Evan E. , Navarro Arcadi
Published: August 2011

Copy number variants (CNVs) are increasingly acknowledged as an important source of evolutionary novelties in the human lineage. However, our understanding of their significance is still hindered by the lack of primate CNV data. We performed intraspecific comparative genomic hybridizations to identify loci harboring copy number variants in each of the four great apes: bonobos, chimpanzees, gorillas, and orangutans. For the first time, we could analyze differences in CNV location and frequency in these four species, and compare them with human CNVs and primate segmental duplication (SD) maps. In addition, for bonobo and gorilla, patterns of CNV and nucleotide diversity were studied in the same individuals. We show that CNVs have been subject to different selective pressures in different lineages. Evidence for purifying selection is stronger in gorilla CNVs overlapping genes, while positive selection appears to have driven the fixation of structural variants in the orangutan lineage. In contrast, chimpanzees and bonobos present high levels of common structural polymorphism, which is indicative of relaxed purifying selection together with the higher mutation rates induced by the known burst of segmental duplication in the ancestor of the African apes. Indeed, the impact of the duplication burst is noticeable by the fact that bonobo and chimpanzee share more CNVs with gorilla than expected. Finally, we identified a number of interesting genomic regions that present high-frequency CNVs in all great apes, while containing only very rare or even pathogenic structural variants in humans.
Inhibition of Protein Synthesis by TOR Inactivation Revealed a Conserved Regulatory Mechanism of the BiP Chaperone in Chlamydomonas
Authors: Díaz-Troya Sandra , Pérez-Pérez María Esther , Pérez-Martín Marta , Moes Suzette , Jeno Paul , Florencio Francisco J. , Crespo José L.
Published: August 2011

The target of rapamycin (TOR) kinase integrates nutritional and stress signals to coordinately control cell growth in all eukaryotes. TOR associates with highly conserved proteins to constitute two distinct signaling complexes termed TORC1 and TORC2. Inactivation of TORC1 by rapamycin negatively regulates protein synthesis in most eukaryotes. Here, we report that down-regulation of TOR signaling by rapamycin in the model green alga Chlamydomonas reinhardtii resulted in pronounced phosphorylation of the endoplasmic reticulum chaperone BiP. Our results indicated that Chlamydomonas TOR regulates BiP phosphorylation through the control of protein synthesis, since rapamycin and cycloheximide have similar effects on BiP modification and protein synthesis inhibition. Modification of BiP by phosphorylation was suppressed under conditions that require the chaperone activity of BiP, such as heat shock stress or tunicamycin treatment, which inhibits N-linked glycosylation of nascent proteins in the endoplasmic reticulum. A phosphopeptide localized in the substrate-binding domain of BiP was identified in Chlamydomonas cells treated with rapamycin. This peptide contains a highly conserved threonine residue that might regulate BiP function, as demonstrated by yeast functional assays. Thus, our study has revealed a regulatory mechanism of BiP in Chlamydomonas by phosphorylation/dephosphorylation events and assigns a role to the TOR pathway in the control of BiP modification.
The Small GTPase Rab5a Is Essential for Intracellular Transport of Proglutelin from the Golgi Apparatus to the Protein Storage Vacuole and Endosomal Membrane Organization in Developing Rice Endosperm
Authors: Fukuda Masako , Satoh-Cruz Mio , Wen Liuying , Crofts Andrew J. , Sugino Aya , Washida Haruhiko , Okita Thomas W. , Ogawa Masahiro , Kawagoe Yasushi , Maeshima Masayoshi , Kumamaru Toshihiro
Published: August 2011

Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as larger precursors, which are then transported via the Golgi to the protein storage vacuole (PSV), where they are processed into acidic and basic subunits. Three independent glutelin precursor mutant4 (glup4) rice lines, which accumulated elevated levels of proglutelin over the wild type, were identified as loss-of-function mutants of Rab5a, the small GTPase involved in vesicular membrane transport. In addition to the plasma membrane, Rab5a colocalizes with glutelins on the Golgi apparatus, Golgi-derived dense vesicles, and the PSV, suggesting that Rab5a participates in the transport of the proglutelin from the Golgi to the PSV. This spatial distribution pattern was dramatically altered in the glup4 mutants. Numerous smaller protein bodies containing glutelin and α-globulin were evident, and the proteins were secreted extracellularly. Moreover, all three independent glup4 allelic lines displayed the novel appearance of a large dilated, structurally complex paramural body containing proglutelins, α-globulins, membrane biomarkers for the Golgi apparatus, prevacuolar compartment, PSV, and the endoplasmic reticulum luminal chaperones BiP and protein disulfide isomerase as well as β-glucan. These results indicate that the formation of the paramural bodies in glup4 endosperm was due to a significant disruption of endocytosis and membrane vesicular transport by Rab5a loss of function. Overall, Rab5a is required not only for the intracellular transport of proglutelins from the Golgi to the PSV in rice endosperm but also in the maintenance of the general structural organization of the endomembrane system in developing rice seeds.
Seed Storage Oil Mobilization Is Important But Not Essential for Germination or Seedling Establishment in Arabidopsis
Authors: Kelly Amélie A. , Quettier Anne-Laure , Shaw Eve , Eastmond Peter J.
Published: August 2011

Triacylglycerol (TAG) is a major storage reserve in many plant seeds. We previously identified a TAG lipase mutant called sugar-dependent1 (sdp1) that is impaired in TAG hydrolysis following Arabidopsis (Arabidopsis thaliana) seed germination (Eastmond, 2006). The aim of this study was to identify additional lipases that account for the residual TAG hydrolysis observed in sdp1. Mutants were isolated in three candidate genes (SDP1-LIKE [SDP1L], ADIPOSE TRIGLYCERIDE LIPASE-LIKE, and COMPARATIVE GENE IDENTIFIER-58-LIKE). Analysis of double, triple, and quadruple mutants showed that SDP1L is responsible for virtually all of the residual TAG hydrolysis present in sdp1 seedlings. Oil body membranes purified from sdp1 sdp1L seedlings were deficient in TAG lipase activity but could still hydrolyze di- and monoacylglycerol. SDP1L is expressed less strongly than SDP1 in seedlings. However, SDP1L could partially rescue TAG breakdown in sdp1 seedlings when expressed under the control of the SDP1 or 35S promoters and in vitro assays showed that both SDP1 and SDP1L can hydrolyze TAG, in preference to diacylglycerol or monoacylglycerol. Seed germination was slowed in sdp1 sdp1L and postgerminative seedling growth was severely retarded. The frequency of seedling establishment was also reduced, but sdp1 sdp1L was not seedling lethal under normal laboratory growth conditions. Our data show that together SDP1 and SDP1L account for at least 95% of the rate of TAG hydrolysis in Arabidopsis seeds, and that this hydrolysis is important but not essential for seed germination or seedling establishment.
Evaluation of antitumor effects following tumor necrosis factor‐α gene delivery using nanobubbles and ultrasound
Authors: Horie Sachiko , Watanabe Yukiko , Ono Masao , Mori Shiro , Kodama Tetsuya
Published: August 2011

The antitumor effects of tumor necrosis factor (TNF‐α) were evaluated following transfection of TNF‐α plasmid DNA into solid mouse tumors using the nanobubbles (NBs) and ultrasound (US) gene delivery system. Murine breast carcinoma (EMT6) cells expressing luciferase (1 × 106 cells) were injected intradermally into the flanks of 6–7‐week‐old male SCID mice on day 0. Ten microliters of TNF‐α (5 μg/μL) or TNF‐α mock plasmid DNA (5 μg/μL) with/without NBs (15 μL) and saline was injected intratumorally in a total volume of 30 μL, and tumors were exposed to US (frequency, 1 MHz; intensity, 3.0 W/cm2; duty cycle, 20%; number of pulses, 200; and exposure time, 60 s) on days 2, 4, 7, and 9. Changes in tumor size were measured with an in vivo bioluminescent imaging system and a mechanical caliper. Changes in tumor vessel area were quantified using contrast‐enhanced US imaging with Sonazoid and a high frequency US imaging system (40 MHz) and immunohistochemistry (CD31). At the mRNA level, expression of TNF‐α, caspase‐3, and p53 were quantified using real‐time quantitative RT‐PCR. At the protein level, expression of caspase‐3 and p53 were confirmed by immunohistochemistry. We show that repeated TNF‐α gene delivery using NBs and US can lead to the local production of TNF‐α. This results in antitumor effects, including activation of p53‐dependent apoptosis, decrease in tumor vessel density, and suppression of tumor size. In this study, we showed the effectiveness of using NBs and US for TNF‐α gene delivery into tumor cells. (Cancer Sci 2011; 102: 2082–2089)
Downregulation of GRIM‐19 promotes growth and migration of human glioma cells
Authors: Zhang Yanmin , Hao Hongbo , Zhao Shidou , Liu Qian , Yuan Qiuhuan , Ni Shilei , Wang Fuwu , Liu Shangming , Wang Liyan , Hao Aijun
Published: August 2011

It has become increasingly clear that there are notable parallels between normal development and tumorigenesis. Glioma is a classic model that links between tumorigenesis and development. We evaluated the expression of GRIM‐19, a novel gene essential for normal development, in various grades of gliomas and several human glioma cell lines. We showed that GRIM‐19 mRNA and protein expression were markedly lower in gliomas than in control brain tissues and negatively correlated with the malignancy of gliomas. Downregulation of GRIM‐19 in glioma cells significantly enhanced cell proliferation and migration, whereas overexpression of GRIM‐19 showed the opposite effects. We also showed that the activation of signal transducer and activator of transcription 3 (STAT3) and the expression of many STAT3‐dependent genes were regulated by the expression of GRIM‐19. In addition, GRIM‐19 exerted its role probably through the non‐STAT3 signaling pathway. Collectively, our data suggest that most gliomas expressed GRIM‐19 at low levels, which may play a major role in tumorigenesis in the brain. (Cancer Sci 2011; 102: 1991–1999)
Involvement of retrotransposition of long interspersed nucleotide element‐1 in skin tumorigenesis induced by 7,12‐dimethylbenz[a]anthracene and 12‐O‐tetradecanoylphorbol‐13‐acetate
Authors: Okudaira Noriyuki , Goto Motohito , Yanobu‐Takanashi Rieko , Tamura Masato , An Akihiro , Abe Yukiko , Kano Shigeyuki , Hagiwara Shotaro , Ishizaka Yukihito , Okamura Tadashi
Published: August 2011

Tumor development induced by 7,12‐dimethylbenz[a]anthracene (DMBA) plus 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) is a well‐characterized model of multistep carcinogenesis. DMBA mutates the Ha‐ras gene, whereas TPA promotes the growth of transformed cells by activating cellular signaling molecules. It remains to be clarified how repeated TPA treatment endows transformed cells with autonomous cell growth. Long interspersed nucleotide element‐1 (L1) is an endogenous retroelement, and 80–100 copies of L1 function as autonomous mobile elements. Although the L1 retrotransposition (RTP) has been found in various human tumors, implying the possible mobility of L1 during carcinogenesis, little is known about how L1‐RTP arises in tumor cells, owing to a lack of experimental models. To dissect the mechanism of L1‐RTP during carcinogenesis, we established a line of transgenic mice carrying human L1 and enhanced green fluorescent protein (hL1‐EGFP mice) and subjected them to DMBA/TPA‐induced skin tumorigenesis. Of 15 skin tumors examined, 13 were positive for L1‐RTP; L1‐RTP was not detected in normal skin tissues adjacent to the tumors. Moreover, nine L1‐RTP‐positive tumors were positive for activated Ha‐ras, and immunohistochemical analysis revealed cells positive for both L1‐RTP and phosphorylated Stat3, a marker of tumor cells. Additional in vivo experiments suggested that L1‐RTP occurred during tumor promotion by TPA. This is the first report on the involvement of L1‐RTP in chemical carcinogenesis. We propose hL1‐EGFP mice as a versatile system for investigating the mode of L1‐RTP in tumor development and discuss the possible role of L1‐RTP in tumorigenesis. (Cancer Sci 2011; 102: 2000–2006)
Urinary mRNA markers of epithelial–mesenchymal transition correlate with progression of diabetic nephropathy
Authors: Zheng Min , Lv Lin‐Li , Cao Yu‐Han , Zhang Jian‐Dong , Wu Min , Ma Kun‐Ling , Phillips Aled O. , Liu Bi‐Cheng
Published: August 2011

Objective  Epithelial–mesenchymal transition (EMT) plays an important role in the pathogenesis and progression of diabetic nephropathy (DN). Quantification of messenger RNA (mRNA) expression in urinary sediment is emerging as a noninvasive method of screening DN‐associated biomarkers. The aim of our study was to examine whether urinary mRNA profile of EMT‐associated genes may provide valuable clinical insight into the different stages of DN.

Design and methods  Diabetic nephropathy patients (n = 44) and healthy controls (n = 12) were enrolled in this study. DN patients were divided into three groups according to the levels of estimated glomerular filtration rate (eGFR): Group A (eGFR > 60 ml/min per 1·73 m2, n = 27), Group B (45 < eGFR < 60 ml/min per 1·73 m2, n = 9), and Group C (eGFR < 45 ml/min per 1·73 m2, n = 8). Relative mRNA abundance of α‐smooth muscle actin (α‐SMA), fibronectin, FSP1 and matrix metalloproteinase‐9 (MMP‐9) were quantified, and correlations between target mRNAs and clinical parameters were examined.

Results  The urinary mRNA levels of α‐SMA, fibronectin and MMP‐9 were significantly higher in the DN group compared with controls (P < 0·05), and mRNA levels increased with DN progression. Urinary mRNA levels of all target genes positively correlated with both urinary albumin excretion (UAE) and blood urea nitrogen (BUN). Moreover, the expression of α‐SMA, fibronectin and MMP‐9 mRNA correlated with serum creatinine levels (r = 0·514, r = 0·53 and r = 0·469, all P < 0·001) and eGFR levels (r = −0·374, r = −0·392 and r = −0·487, all P < 0·01).

Conclusions  Quantification of EMT‐associated genes in urinary sediment may be a novel approach for searching new biomarkers of DN.
Plasticity of the Parental Brain: A Case for Neurogenesis
Authors: Lévy F. , Gheusi G. , Keller M.
Published: August 2011

Profound behavioural changes occur in the mother at parturition, together with extensive remodelling of neural circuits. These changes include neurochemical, morphological and functional plasticity. The continuous generation of new neurones in the hippocampus and the olfactory system is an additional form of neuroplasticity that contributes to motherhood. This review describes the reciprocal relationships between hippocampal and olfactory neurogenesis and parental behaviour. Studies in rodents demonstrate that parturition and interactions with the young affect both cell proliferation and survival in a different manner across neurogenic zones. Species in which an individual recognition of the offspring is formed, such as sheep, show a down‐regulation of neurogenesis during the perinatal period. This would function to decrease cell competition, favouring the selection of newborn neurones involved in olfactory recognition of the young. Also, in biparental species, increases in olfactory neurogenesis occur in the father in response to pup exposure during the early postpartum period. Oestradiol, corticosterone and prolactin changes associated with parturition are the main physiological factors involved in the regulation of neurogenesis that have been determined so far. In the father, prolactin mediates an enhancement of olfactory neurogenesis. Contradictory evidence indicates a functional link between neurogenesis and parenting behaviour. Mice receiving focal irradiation of the olfactory neurogenic subventricular zone show few disturbances in the expression of maternal behaviour, whereas a reduction of both hippocampal and olfactory neurogenesis as a result of the infusion of an anti‐mitotic agent induces behavioural deficits. Disrupting prolactin signalling abolished increased paternal neurogenesis and offspring recognition by the father, and rescuing this neurogenesis restored recognition behaviour. More studies that selectively suppress the changes of neurogenesis are needed to confirm the role of new neurones in regulating parenting behaviour.
Differential Changes in the Hypothalamic‐Pituitary‐Adrenal Axis and Prolactin Responses to Stress in Early Pregnant Mice
Authors: Parker V. J. , Menzies J. R. W. , Douglas A. J.
Published: August 2011

Stress can cause pregnancy failure but it is unclear how the mother’s neuroendocrine system responds to stress to impair mechanisms establishing implantation. We analysed stress‐evoked hypothalamic‐pituitary‐adrenal (HPA) axis responses in early pregnant mice. HPA axis secretory responses to immune stress in early‐mid pregnancy were strong and similar to that in virgins, although activation of hypothalamic vasopressin neurones, rather than corticotrophin‐releasing hormone neurones, may be more important in the stress response in pregnancy. The site and mode of detrimental glucocorticoid action in pregnancy is not established. Because circulating prolactin is important for progesterone secretion and pregnancy establishment, we also hypothesised that stress negatively impacts on prolactin and its neuroendocrine control systems in early pregnant mice. Basal prolactin secretion was profoundly inhibited by either immune or fasting stress in early pregnancy. Prolactin release is inhibited by tonic dopamine release from tuberoinfundibular (TIDA) neurones. However, immune stress did not increase TIDA neurone activity in the median eminence in pregnant mice [measured by 3,4‐dihydroxyphenylacetic acid (DOPAC) content and the DOPAC:dopamine ratio]. By contrast, both immune stress and fasting caused weak induction of Fos in TIDA neurones. However, Fos induction does not always reflect dopamine secretion. Taken together, the data suggest that the stress‐evoked profound reduction in prolactin secretion does not involve substantially increased dopamine activity as anticipated. In pregnancy, there was also attenuated recruitment of parvocellular paraventricular nucleus neurones and increased activation of brainstem noradrenergic nuclei after immune stress, indicating that other mechanisms may be involved in the suppression of prolactin secretion. In summary, low prolactin and increased circulating glucocorticoids together may partly explain how a mother’s endocrine system mediates stress‐induced pregnancy failure.
The effectiveness of cognitive‐behavioural therapy for temporomandibular disorders: a systematic review
Authors: LIU H. X. , LIANG Q. J. , XIAO P. , JIAO H. X. , GAO Y. , AHMETJIANG A.
Published: August 2011

Summary Cognitive‐behavioural therapy (CBT) and its effects on temporomandibular disorders (TMD) have been examined in several studies. We are trying to combine results of these studies and to explore the effectiveness. MEDLINE, EMBASE, Cochrane Central Register of Controlled Trial, Pubmed and the Chinese Biomedical Literature Data were searched to collect randomised and semi‐randomised controlled trials (RCTs), comparing CBT with any control group receiving other dental treatments. Two authors independently retrieved, extracted and assessed the quality of included studies. The search strategy resulted in 323 studies, of which five met the inclusion criteria, including three RCTs and two semi‐RCTs. The quality of the included studies was diverse. Meta‐analysis was not performed owing to five studies involving different comparison groups and follow‐up periods. The effect of CBT on patients with TMD is inconsistent among the studies, so no firm conclusion could be drawn in this systematic review. There is insufficient evidence to make firm recommendations for the use of CBT over other intervention for the treatment of TMD. Further high‐quality RCTs are clearly needed for this theme.
The state of hepatitis B and C in Europe: report from the hepatitis B and C summit conference*
Authors: Hatzakis A. , Wait S. , Bruix J. , Buti M. , Carballo M. , Cavaleri M. , Colombo M. , Delarocque‐Astagneau E. , Dusheiko G. , Esmat G. , Esteban R. , Goldberg D. , Gore C. , Lok A. S. F. , Manns M. , Marcellin P. , Papatheodoridis G. , Peterle A. , Prati D. , Piorkowsky N. , Rizzetto M. , Roudot‐Thoraval F. , Soriano V. , Thomas H. C. , Thursz M. , Valla D. , van Damme P. , Veldhuijzen I. K. , Wedemeyer H. , Wiessing L. , Zanetti A. R. , Janssen H. L. A.
Published: August 2011

Summary.  Worldwide, the hepatitis B virus (HBV) and the hepatitis C virus (HCV) cause, respectively, 600 000 and 350 000 deaths each year. Viral hepatitis is the leading cause of cirrhosis and liver cancer, which in turn ranks as the third cause of cancer death worldwide. Within the WHO European region, approximately 14 million people are chronically infected with HBV, and nine million people are chronically infected with HCV. Lack of reliable epidemiological data on HBV and HCV is one of the biggest hurdles to advancing policy. Risk groups such as migrants and injecting drug users (IDU) tend to be under‐represented in existing prevalence studies; thus, targeted surveillance is urgently needed to correctly estimate the burden of HBV and HCV. The most effective means of prevention against HBV is vaccination, and most European Union (EU) countries have universal vaccination programmes. For both HBV and HCV, screening of individuals who present a high risk of contracting the virus is critical given the asymptomatic, and thereby silent, nature of disease. Screening of migrants and IDUs has been shown to be effective and potentially cost‐effective. There have been significant advances in the treatment of HCV and HBV in recent years, but health care professionals remain poorly aware of treatment options. Greater professional training is needed on the management of hepatitis including the treatment of liver cancer to encourage adherence to guidelines and offer patients the best possible outcomes. Viral hepatitis knows no borders. EU Member States, guided by the EU, need to work in a concerted manner to implement lasting, effective policies and programmes and make tackling viral hepatitis a public health priority.
Thrombopoietins and Thrombopoiesis: A Clinical Perspective
Authors: Kuter D.J.
Published: August 2011

Since the discovery of thrombopoietin four years ago there has been much interest in the clinical use of this growth factor and its impact on platelet transfusions. Two recombinant thrombopoietin molecules are currently under intense clinical investigation. One is a full‐length, glycosylated thrombopoietin (rHuTPO) and the other is a non‐glycosylated, truncated thrombopoietin coupled to polyethylene glycol (PEG‐rHuMGDF). Both bind to the thrombopoietin receptor, c‐mpl, and stimulate megakaryocyte growth and platelet production in vitro and in vivo. In early clinical studies these “Mpl ligands” have been effective in reducing thrombocytopenia after non‐myeloablative but not after myeloablative chemotherapy. In transfusion medicine, they may serve to increase the yield of stem cell harvests, expand progenitor cells ex vivo and stimulate platelet apheresis donors. Their impact on platelet usage is still unclear but may be less than initially estimated.
Transmission of Parasites by Blood Transfusion
Authors: Dodd R.Y.
Published: August 2011

A number of parasitic diseases are known, or suspected to be transmitted by blood transfusion. Of greatest concern are malaria and Chagas' disease, but babesia, leishmania and toxoplasmosis also offer risk in particular locations or circumstances. Some of these parasites may be imported into non‐endemic areas as a result of population movements and in some cases, the natural range of the parasite is increasing as a result of environmental change. Recent research, particularly on Chagas disease and babesiosis will be discussed, along with measures to minimize transmission of these and other parasites via transfusion.
Preparation and Preservation of Red Cells
Authors: Högman C.F.
Published: August 2011

Background: The production of blood components has undergone several changes during the last decades. Methods: Red blood cells will have slightly different properties depending on the way of preparation: whether a hard or soft spin has been used, whether platelets and/or leukocytes have been removed or not, and whether the red cells have been suspended in part of the original plasma or in an additive solution. Automated techniques are now often used for the separation of buffy coats, red cells and plasma. Recently, apheresis techniques have been applied for the preparation of red cells, mostly in combination with plasma or platelets. Continuous addition of the anticoagulant during collection reduces the delay between collection and separation, but the cost is higher and donor time longer. Results: Most of the methods for the preparation and storage of red cells allow 35–42 days of storage with a mean in vivo recovery of >75%. However, the content of erythrocyte 2,3‐DPG is commonly lost within 1–2 weeks, caused by the accumulation of acid metabolites, but can be maintained longer with new systems of storage. Leukodepletion of red cells by filtration is used increasingly, but its importance in the majority of transfusions is still unclear. Conclusion: New options for the preparation and storage of red blood cells are available and undergo continuous evaluation
Changes in Donor Leukocytes during Blood Storage. Implications on Post‐ Transfusion Immunomodulation and Transfusion‐Associated GVHD
Authors: Mincheff M.
Published: August 2011

Freshly drawn peripheral blood granulocytes, monocytes and NK cells express Fas‐L following activation. Fully functional soluble Fas‐L (sFas‐L) is released and found in fresh plasma samples from healthy volunteers coupled to a Fas‐L binding factor (PFBF). In the absence of plasma leukocytes undergo rapid apoptosis with 15% of the granulocytes and 25% of the monocytes showing signs of apoptosis immediately following separation. Apoptosis progresses during refrigerated storage even in the presence of plasma and all granulocytes disintegrate by day 15 of storage. The absence of plasma increases the rate of apoptosis. The resulting increased expression of PS on apoptosing leukocytes enhances blood procoagulant activity. Whereas transfusions of fresh blood lead to alloimmunization, the functional impairment and death of antigen‐presenting cells during storage eliminates their ability to stimulate cytotoxic immune responses. Additionally, loss of function and death of donor T cells during storage preclude the possibility for development of post‐transfusion GVHD. Continued storage leads to the secondary necrosis of apoptosed leukocytes and the release of soluble antigens. Depending on dose, transfusions of such products may lead to immunosuppression due to a T2 bias of the immune response.
Quality Assurance and Standards for Red Cells and Platelets
Authors: Sweeney J.
Published: August 2011

Blood donations vary in composition because of considerable variation in donor cell counts and volume collected. In order to minimize the variation in end products produced, and reduce the likelihood of low quality products, a program called Quality Assurance (QA) is required. Agreement on minimal standards is useful, and a program of quality control (QC) is an important component of QA. However, while QC to ensure compliance with standards is desirable, the objective of manufacturing products of consistently high quality can only be achieved when attention is focused on total process control, self‐ auditing and the energetic management of errors.
Storage and Quality Assessment of Platelets
Authors: Holme S.
Published: August 2011

Platelet concentrates stored for 5 days under currently optimal conditions at 22 C demonstrate approximately a 20–30% loss of viability as determined by CCI measurements in thrombocytopenic patients and radiolabeling studies with healthy volunteers. The functionality of platelets is substantially lost during storage. This loss appears, for most part, to be reversible upon infusion. In view of this, stored platelets should be acceptable for prophylactic treatment of thrombocytopenic patients in whom immediate hemostatic function is not critical. However, whether stored platelets are functionally adequate for immediate arrest of perioperative bleeding in surgical patients remain uncertain. Currently, a fully adequate and appropriate test that evaluates the hemostatic function of a stored platelet product is not available. In view of this, a desired goal in research and development of new platelet products may then be to attain a platelet product that is similar to fresh platelets in various haemostatic functions. Whether this is an obtainable or relevant goal remains to be seen.
Modified Hemoglobin‐based Blood Substitutes: Crosslinked, Recombinant and Encapsulated Hemoglobin
Authors: Chang T.M.S.
Published: August 2011

Native hemoglobin in the form of stroma‐free hemoglobin cannot be used as blood substitute. Hemoglobin has to be modified either molecularly or encapsulated. First generation molecularly modified ultrapure hemoglobins are now in clinical trial ‐ some in Phase III. There are a number of these. Polyhemoglo‐ bin is formed by crosslinking hemoglobin molecules intermolecularly and intra‐ molecularly. A crosslinked single hemoglobin molecule is formed by crosslinking hemoglobin intramolecularly. Recombinant hemoglobin from E.coli is formed by fusion of the subunits of each hemoglobin molecule. Conjugated hemoglobin is formed by crosslinking each hemoglobin molecule to soluble polymers. A second generation system formed by crosslinking hemoglobin‐ superoxide dismutase‐catalase is being developed. A third generation hemoglobin‐based blood substitute is based on microencapsulated hemoglobin, artificial red blood cells, that more closely resemble a complete red blood cell.
Ex Vivo Expansion of Human Hematopoietic Stem Cells: Implications for The Modern Blood Bank
Authors: Hoffman R. , Rozler E. , Chute J. , Nelson M. , Chen L. , Turian J. , Bartholomew A. , Brandt J.E.
Published: August 2011

Pluripotent hematopoietic stem cells (PHSC) are rare cells within the marrow that are capable of self‐renewal and differentiation into multiple hematopoietic lineages. Following myeloablative chemotherapy and radiation therapy and marrow transplantation, hematological reconstitution occurs after a period of 2‐ 3 weeks. Recently, a number of laboratories have shown that both early and delayed phases of engraftment are mediated by PHSC within a graft and that engraftment can be accelerated by transplanting greater numbers of PHSC. Increasing efforts have been directed, therefore, towards developing methods to expand PHSC ex vivo. In this report, we describe an endothelial cell‐based culture system to which exogenous cytokines are added which appears to permit the ex vivo expansion of PHSC. Refinement of these technologies will potentially have a major impact on the ability of blood banks to improve the quality of hematopoietic stem cell grafts.
Blood Cell Precursors in Context. Composition of the Bone Marrow Microenvironment that Supports B Lymphopoiesis
Authors: Kincade P.W. , Yamashita Y. , Borghesi L. , Medina K. , Oritani K.
Published: August 2011

New molecular approaches are rapidly yielding information about the composition of bone marrow and culture studies suggest possible functions for many molecules in that organ. However, the marrow is extraordinarily complex with respect to interactions between cells and their behaviour may not be adequately modeled in culture. It is becoming apparent that post‐translational modifications and function of proteins can be highly influenced by the cellular and extracellular environment. Therefore, in vivo studies will continue to be important for understanding this remarkable organ.
Flowcytometry ‐ Principles and Feasibility in Transfusion Medicine. Enumeration of Epithelial Derived Tumor Cells in Peripheral Blood
Authors: Terstappen L.W.M.M. , Rao C. , Gross S. , Kotelnikov V. , Racilla E. , Uhr J. , Weiss A.
Published: August 2011

Immunomagnetic selection of epithelial cells from peripheral blood was combined with flowcytometric or microscopic analysis to characterize and enumerate epithelium derived tumor cells in the circulation of patients with carcinoma of the breast. Highly significant differences in the number of circulating epithelial cells were found between normal controls and patients with breast cancer. In addition, significant differences in the number of circulating epithelial cells were found in patients in whom the tumor is confined to the primary tumor and those with metastatic disease to local lymphnodes or distant sites [1]. The malignant nature of the cells was demonstrated by their cytology and immunophenotype. In following up the number of epithelial cells in the blood of patients with metastatic carcinoma of the breast, it was shown that it was well correlated with the activity of the disease.
Molecular Genetic Methods: Principles and Feasibility in Transfusion Medicine
Authors: Avent N.D.
Published: August 2011

The scale of the application of molecular biological techniques to modern medicine and research in the biological sciences is vast, and in many instances has captured widespread public appeal. The intention of this review is to summarise the impact of molecular techniques on Transfusion Medicine ranging from diagnostic testing (platelet, granulocyte and red cell genotyping; microbiological testing), stable gene integration into haematopoeitic stem cells (gene therapy), production of blood products in transgenic animals and cell lines, and the inhibition of gene expression using synthetic antisense oligodeoxynucleo‐ tides. All of these techniques involve the manipulation of genes, be it from the relatively simple examination of different alleles to the technically demanding ability to express mammalian genes in culture and other animals.
Immune Thrombocytopenic Purpura ITP
Authors: Imbach P. , Kühne T.
Published: August 2011

Immune thrombocytopenic purpura ITP is characterized by early platelet destruction due to an unbalanced immune response. In acute ITP, a transient increase of HLA‐DR molecules has been detected while in individuals with chronic ITP, in addition, increased serum concentrations of IL‐2 and other cytokines reflecting in vivo T‐cell activation have been observed. Clinically, the hemorrhagic manifestation of ITP rather than the platelet count should define the indication for active intervention. In a staging system a patient with stage III has bleeding signs and platelet counts below 10 or 20 times 109/L and needs treatment, a patient with stage II should be treated on an individual level (prevention of bleeding) and a patient with stage I (no bleeding, platelet count above 50 times 109/L) should be observed only.
CD8+ T Cells of Tc2 Phenotype Mediate a GVL Effect and Prevent Marrow Rejection
Authors: Fowler D.H. , Gress R.E.
Published: August 2011

Donor T cells mediate both beneficial and detrimental immune reactions in the setting of allogeneic BMT (alloBMT). T cells mediate the GVL effect and prevent marrow rejection, but also induce GVHD. In an attempt to favorably influence the balance of these allogeneic responses, we have evaluated the effect of donor CD4+, Thl/Th2 and CD8+, Tcl/Tc2 functional T cell subsets in murine marrow transplantation models. Our studies have identified the CD8+Tc2 population (which is a cytolytic effector secreting the type II cytokines IL‐4, IL‐5, and IL‐10) as a subset capable of mediating the GVL effect and preventing marrow rejection with reduced GVHD. We have also shown that the Tc2 subset can be generated in humans. These studies indicate that administration of donor CD8+ T cells of Tc2 phenotype represents a strategy for improving alloBMT outcome.
Clinical Aspects and Typing of Platelet Alloantigens
Authors: Kroll H. , Kiefel V. , Santoso S.
Published: August 2011

Platelet alloantigens can induce the formation of corresponding alloantibodies when exposed to phenotypically negative individuals. These antibodies are responsible for fetal and neonatal alloimmune thrombocytopenia, posttransfusion purpura, passive alloimmune thrombocytopenia and transplantation‐ associated thrombocytopenia and may contribute to platelet transfusion refractoriness together with HLA antibodies. Besides antibody detection laboratory diagnosis of the clinical syndromes requires alloantigen typing. Furthermore, typed platelet donors are a prerequisite for effective platelet transfusion therapy. Different techniques for phenotyping are well established and easy to perform but they rely on the availability of antisera. Since the molecular genetic background of the clinically most relevant alloantigens has been elucidated during the last years various genotyping methods have been applied to the platelet membrane polymorphisms and thus facilitated widespread platelet alloantigen typing. Generation of antibodies from phage display libraries and of lymphoblastoid cell lines from donors with all genetic variants will allow further developments.
Screening Programmes for Foetomaternal Alloimmune Thrombocytopenia
Authors: Williamson L.M.
Published: August 2011

Screening to detect pregnancies at risk of severe thrombocytopenia due to Foetomaternal alloimmunisation to Human Platelet Alloantigens (FAIT) is not yet a standard of care. Screening may be performed antenatally with the possibility of early intervention/therapy. Post natal measurement of cord platelet count may not always prevent fetal damage but may detect severe thrombocytopenia from other causes. Assessment of FAIT screening against World Health Organisation standards for screening programmes helps define areas where knowledge is still lacking and where research effort should be directed. Limitations to screening have been lack of reliable assays for typing and antibody detection, a limited understanding of the relationship between the presence of alloantibodies and clinical disease, inability to predict non‐invasively which cases will have a severe outcome, and controversy as to which antenatal therapy is optimal for cases at high risk of a poor outcome.
The Transfusion Medicine Consultant and Hospital Transfusion Committee
Authors: Popovsky M.A.
Published: August 2011

Aim: The evolution of blood banking into the broad, multi‐disciplinary specialty of transfusion medicine has created the need for a new type of physician ‐ the transfusion medicine consultant. Both laboratorian and clinician, the transfusion medicine consultant offers a wide range of skills and support to both medical and surgical disciplines. This individual can be based in either the hospital or regional blood center. He is a crucial member of the hospital transfusion committee. The latter can play an activist role in patient care.
Replacement of Massive Blood Loss
Authors: Hiippala S.
Published: August 2011

Treatment of massive blood loss has experienced major changes during the recent decade. The transition towards pure component therapy has been the most significant issue, which has compelled the clinician to revise some of their basic strategics in treatment of massively bleeding patients. The importance of adequate volume resuscitation with crystalloids and colloids is still unrefutable, but the therapy of hemorrhagic derangements has changed. Plasma‐poor red cells (RC) are now commonly used instead of whole blood (WB) or packed red blood cells (PRBC) to correct oxygen carrying capacity during massive blood loss. As the plasma content of RC is minimal, deficit of plasma and coagulation factors develops earlier than during transfusion of WB and PRBC. Hypofibri‐ nogenemia develops first followed by other coagulation factor deficits and later by thrombocytopenia. Therefore the use of fresh frozen plasma (FFP) is the primary intervention to treat abnormal bleeding encountered in the replacement of massive blood loss with RC. As the development of thrombocytopenia is a highly individual phenomenon, the transfusion of platelets should be guided by repeatedly determined platelet counts.
Experiences with “Self Service” Electronic Blood Banking
Authors: Cheng G.
Published: August 2011

Electronic verification of ABO compatibility (computer crossmatching) has been accepted as the crossmatching procedure for patients without clinically significant alloantibodies. Computer crossmatching offers several advantages over the immediate spin crossmatch and enables the setting up of a self service blood banking system. Self service blood banking saves manpower, improves crossmatch/transfusion(C/T) ratio and makes the maximum surgical blood ordering schedule (MSBOS) redundant. Blood banking service can also be provided at satellite hospitals without stationing blood banking staff there. We have also developed a novel self service system that does not require expensive computer hardware and networking.
Hemovigilance in the United States of America
Authors: Menitove J.E.
Published: August 2011

Although hemovigilance is not a word used commonly in the United States, the concept of monitoring morbidity associated with blood transfusion is accepted widely. Currently, quality management focuses on error avoidance rather than error detection in both the “manufacturing” and “clinical” aspects of blood banking. “Manufacturing” is regulated predominantly by current Good Manufacturing Practices while clinical transfusion medicine follows clinical practice guidelines. There are multiple overlapping mechanisms for quality assurance and error reporting among governmental, professional, and non‐profit regulatory agencies. The strategies of each stress the on‐going need for a systems approaches to preventing errors.