A recently published article describing a case report on a patient affected by Fibrodysplasia Ossificans Progressiva (FOP) treated with Rosiglitazone has prompted interest for careful analysis of the rational basis of such treatment. This article reviews the effects of PPARγ agonists in relationship with various pathogenic steps that occur during the course of FOP by reviewing the particularly rich literature on the effects of Rosiglitazone, to underscore their relevance to FOP and to consider possible adverse effects.Thiazolidinediones (TZDs), among which Rosiglitazone, are known agonists of the peroxisome-proliferator-activated receptor  Fibrodysplasia Ossificans Progressiva (FOP) MIM 135100) is a rare and extremely disabling disorder, characterized by congenital malformation of the great toes and progressive heterotopic ossification 35100 is .Heterotopic ossification generally starts in the infancy and continues in an episodic and progressive way. Typically, the disorder presents with alternate quiescent periods, that sometimes last also years, and acute phases (flareups) through which ectopic bone formation at the level of axial and appendicular skeleton occurs and causes progressive and severe joint ankyloses and formation of an ectopic “second skeleton” .ACVR1/ALK2 gene, that encodes a Type I receptor for bone morphogenetic proteins (BMPs) [ACVR1/ALK2 mutation, accurately characterized by in vitro studies for the most frequently occurring R206H mutation, is anomalous ligand-independent activation and hyper-response to BMP ligands of the SMAD-dependent signaling pathway [ACVR1/ALK2 gene.The molecular defect causing the disorder is a gain of function mutation in the s (BMPs) . The lars (BMPs) . Much les (BMPs) , 6. The  pathway –9. Thus,The quite mild congenital malformation in the great toes can be interpreted in the context of the complex interplay of developmental pathways, including the BMP pathway, controlling development of limb elements. The most critical and still poorly understood point in the pathogenic mechanism is the type of stimuli that trigger flareup episodes and subsequent ossification. Flareups seem to be induced by trauma, medical or surgical or dental interventions, intramuscular injections, and infections. However, in some cases flareups start with no apparent stimulus. The disorder is highly variable in term of onset, frequency, and severity of ectopic ossification episodes . Pathological findings described after analysis of bioptic specimens from misdiagnosed patients have highlighted two important points: the phases of tissue composition at the sites of flareup and the type of bone produced in ectopic lesions –14.Bone formation is preceded by the appearance of bumps that very often cause misdiagnosis by erroneous attribution of the initial lesion to cancer or other disorders like aggressive juvenile fibromatosis or lymphoedema . The hisFOP ectopic bone appears as a normal endochondrally formed lamellar bone with normal bone marrow elements. Radiological and scintigraphic characterization of ectopic bone in 47 FOP patients showed characteristics of resistance, stress response, and remodeling comparable to normal bone in unaffected people .Thus, an inflammatory stimulus that in some way reaches a mutated receptor and stimulates a hyper-responsive signaling pathway are essential elements in the pathogenic process. Many efforts and expectations are directed to find approaches to interfere with the most critical steps of the pathogenic mechanism that could beneficially affect patients' quality of life.Very recently an article that describes successful results of treatment of an FOP patient with Rosiglitazone has been published , which sThe aim of this article was to review a particularly rich literature on the effects of Rosiglitazone and try to underscore their relevance to FOP.Rosiglitazone (ROSI) belongs to the class of Thiazolidinediones (TZDs) and has been approved as drug for treatment of hyperglycemia . Other TRosiglitazone is mainly used as an orally active hypoglycemic agent for the treatment of noninsulin-dependent diabetes mellitus or Type 2 Diabetes (T2D). γ), a ligand dependent transcription factor that belongs to the family of nuclear hormone receptors [γ is expressed in tissues that are key targets for insulin action such as adipose tissue, skeletal muscle, and liver. PPAR-γ activation positively regulates transcription of insulin responsive genes involved in glucose control of synthesis, transport, and utilization, thus contributing to restore sensitivity to insulin at target organs and to glycemia control.One of the characteristics of T2D is impaired response to normal circulating insulin levels at target tissues . Pancreatic beta cells partly compensate this resistance by increasing their own mass and/or efficiency of insulin secretion, however, when this compensatory ability is insufficient, hyperglycemia occurs. ROSI acts by the highly selective activation of the Peroxisome-Proliferator-Activated Receptor gamma  or negative (transrepressing), is modulated both by the interaction with a ligand and by posttranslational modifications. Interaction with the ligand induces heterodimerization with RXR and DNA binding. The simultaneous release of repressor cofactors and recruitment of coactivators also occur. PPAR-γ can interact with different endogenous ligands, such as fatty acid oxidized metabolites and 15-deoxi-D-prostaglandine [PPAR-glandine , 23, or glandine .γ phosphorylation at specific residues, Ser 82/112 and Ser 84 in the AF1 domain, by different kinases , has a negative effect on transcriptional activation both ligand-dependent or independent [PPAR-ependent , 21. γ. Sumoylation causes formation of a complex including PPAR-γ, a nuclear receptor corepressor (NcoR) and histone deacetylase-3 (HDAC3). This complex can be localized at PPREs in promoters of proinflammatory genes related to the NFκB that are therefore repressed [Another mechanism of transcriptional repression is due to ligand-dependent sumoylation at the ligand-interaction domain of PPAR-epressed . γ is normally expressed in antigen presenting cells in the immune system, as monocytes, macrophages, and dendritic cells [γ agonists such as TZDs causes suppression of inflammatory cytokines production [γ exerts a general repressive action on a wide range of target genes inducible by LPS or Interferon gamma (IFNγ) [PPAR-ic cells –28. Treaoduction , 30. Acca (IFNγ) .γ conditional knock out mice in macrophages further underscored its importance in control of inflammation and oxidative metabolism [Generation of PPAR-tabolism .γ and promotes a proinflammatory response to elimininate pathogens [γ is involved in such alternative maturation process and therefore plays an important anti-inflammatory role. Macrophages represent a first line defense against pathogens and, in response to microenvironment stimuli, may become activated and differentiate by different ways. The classic activation is induced by Interferon-athogens . An alteathogens . PPAR-γ γ. Animal models of inflammatory and autoimmune disorders as asthma, rheumatoid arthritis, multiple sclerosis, and type 2 diabetes associated nephropathy [γ agonists in improving several signs associated with these diseases. As an example, Pioglitazone results equally efficient as common treatments with corticosteroids for murine models of asthma [γ agonists in clinical trials for treatment of multiple sclerosis and ulcerative colitis and for already approved trials for treatment of patients with rheumatoid arthritis and asthma [Other evidences underscore the anti-inflammatory role of PPAR-hropathy , 25, 33,f asthma . These sd asthma –37.γ (Pro12Ala) nonsynonymous polymorphism that causes substitution of the conserved Pro12 residue in the ligand-independent DNA binding domain and other less common variants have been evaluated in association studies and also investigated by functional studies [Together with the above evidences, a common PPAR- studies . The var studies ; (b) mal studies ; (c) pat studies .γ was also reported as a target of 5-aminosalicylic acid (5-ASA) anti-inflammatory effects in an animal model of inflammatory bowel disease [Besides mediating the antidiabetic and anti-inflammatory effects of TZD agonists, PPAR- disease .Osteoblasts and adipocytes share common progenitors, mesenchymal stem cells.γ expression is early induced by different key factors in the adipogenesis pathway and is considered the master transcriptional regulator of adipogenesis [γ cDNA and murine myoblasts transdifferentiate to mature adipocytes [PPAR-ogenesis . Fibroblipocytes . Phenotyγ knock out mouse models were critical for defining the effects of changes in PPAR-γ levels in vivo.Generation of PPAR-γ agonists. The protective state in heterozygotes was associated with a nearly 2-fold higher leptin expression under high fat diet. Studies on these mice to examine the effect of PPAR-γ deficiency in bone metabolism [γ restored adipogenesis and decreased osteogenesis indicating a coordinated opposite regulation of the two processes by PPAR-γ. The observation of heterozygous mice confirmed an apparent normal growth under standard diet, including the length of trunk and long bones. However, radiological and densitometric analyses show a 40% increase of trabecular bone mass and concomitant reduction of marrow adipocyte number in long bones and vertebrae. Accordingly, primary bone marrow cells from heterozygotes showed increased osteoblastogenesis in the absence of differentiating agents, with upregulation of key molecules for osteoblast differentiation, Runx2, osterix, and LRP5. Although the switching mechanism between the adipogenic and osteogenic differentiation pathways from common progenitors was not fully clarified, these studies clearly indicated that PPAR-γ acts as a potent suppressor of commitment and differentiation to the osteoblastic lineage. Another contribution by the study of PPAR-γ deficient mice [γ signaling does not seem to be involved in osteopenia caused by estrogen deficiency while seemed to be involved in age-related bone loss possibly due to gradual increased adipogenic differentiation at the expense of osteoblastogenesis.Null homozygous mice showed lethality during embryo development at 10.5–11.5 dpc because of placental dysfunction . Interestabolism  showed tent mice  was the γhyp/hyp, in which a hypomorphic mutation at the PPAR-γ locus causes absence of PPAR-γ expression in white adipose tissue (WAT), has highlighted the contribution of PPAR-γ to bone homeostasis [γ activity in fat enhances bone formation. Increased osteoblast activity was consistent with increased expression of osteoblast-specific transcription factors and markers. In association to lipodystrophy, strong reduction of leptin levels was observed, known to enhance bone formation in mice and humans [Another mouse model, the PPAR-eostasis . The homd humans . These mγ agonists, both in animal models and in clinical studies in diabetic human patients, the question whether these drugs, besides affecting osteoblastogenesis, also affect osteoclastogenesis also arises. Osteoclasts are multinucleated cells derived from hematopoietic precursors, contrary to osteoblasts that derive from mesenchymal stem cells. Considering the observed reduced bone mass after treatment with TZD PPAR-γhyp/hyp   mice showed increased osteoclast activity documented by increased expression of genes typical of the osteoclastic lineage, tartrate-resistant acid phosphatase (Trap) and cathepsin K (CathK) [As mentioned above, PPAR- (CathK) .γ in hematopoietic and endothelial cells showed evident splenomegaly with megakaryocyte accumulation, extramedullary hematopoiesis, pale bones, increased bone volume with reduced marrow cavity [TRAP), calcitonin receptor , carbonic anhydrase 2 (Car2), cathepsin K (Ctsk), resulted defective due to lack of RANKL direct transcriptional activation by PPAR-γ and consequent defect of an essential pathway for differentiation of precursor cells to mature osteoclasts [γ defective mice. The defect of the RANKL pathway appeared associated with signaling through c-fos, since the differentiation block could be released when the mutant cells were infected with a viral vector expressing exogenous c-fos cDNA. The extramedullary hematopoiesis in the mutant mice could be corrected by transplantation of bone marrow from normal mice, whereas the reverse transplantation of bone marrow from PPAR-γ mutant mice to normal mice caused appearance of extramedullary hematopoiesis.On the other hand, mice generated with conditional knock out of PPAR-w cavity . After heoclasts . Treatmeγ in osteoclast differentiation and bone remodeling.In summary, although with some contradiction in published data  male nondiabetic C57BL/6 mice [Dlx5 and Runx2 and the osteoblast-specific marker Col1A1. In contrast the mRNA for the adipocyte-specific fatty acids binding protein aP2 was increased.The examination of the L/6 mice  showed dAnother article described the effect of treatment of adult (5 months old) male and female mice with rosiglitazone, providing other informations on the mechanism of bone loss . A decreRunx2, Osterix, Col1A1, and osteocalcin, in favor of markers specific of adipogenesis.Morphologic changes in bone and cell differentiative potential were accompanied by changes in markers specific of bone development, as ROSI is approved and currently utilized as antidiabetic drug in patients affected by type 2 diabetes. Data on efficacy and adverse events are available in large studies in which, besides evaluating glycemic control, the most frequent adverse events are reported: risk of cardiovascular events, weight gain, fluid retention and bone fracture. We focus especially on the risk of bone fractures since other adverse events are more connected with diabetes complications while the effect on bone metabolism has a direct relevance to FOP disease.A large comparative study in which participated 500 centers in North America, Canada, and Europe, known as A Diabetes Outcome Progression Trial (ADOPT), involved an initial group of 4360 patients [The first published results of ADOPT  reportedIn a more recent study, the effect of Pioglitazone and ROSI was evaluated in a group of 1819 male and female over 40 years old patients who underwent bone fracture and TZD drug exposure . The conA confirmation of risk of bone fracture in patients treated with TZD drugs is reported in a recently published article describing results of a cross-sectional study .Another article by the group of authors who participated in the ADOPT study, described results of measures of bone biomarkers. Although not providing conclusive explanation for the clinical findings, it would suggest an effect of TZD drugs primarily on increased bone resorption . The samACVR1/ALK2 gene, undergo a process of endochondral ossification in response to a mandatory inflammatory trigger. The identity of such cells is still not completely understood and it is likely that both resident progenitor cells of endothelial origin and circulating hematopoietic stem-cells contribute to the heterotopic bone formation [FOP is one of the most disabling disorders of heterotopic ossification, considered as a “stem-cell disease” in which progenitor cells harbouring a mutated ormation , 58. γ transcription factor.Keeping in mind these aspects of FOP pathogenesis, the biological rationale of the use of ROSI in the treatment of FOP is mainly based on the potent inducing effect of ROSI and, in general, TZD drugs, on the PPAR-γ can significantly counteract the inflammatory stimuli responsible for acute phases of the disease preliminary to ossification episodes. Moreover, a relevant PPAR-γ effect on bone differentiation and metabolism, based on the ability to trigger and switch differentiation of progenitor cells towards adipogenesis at the expense of osteogenesis, and possibly on mechanisms involving osteoclast-mediated bone resorbtion, is also taking place.The anti-inflammatory properties of PPAR-Whether rosiglitazone, due to both anti-inflammatory effects and effects on ossification pathways, can be a better treatment than commonly used glucocorticoids or other anti-inflammatory drugs might be deduced from results of carefully designed clinical experimentation that should take into account potential benefit and undesired effects. ROSI is a well known antidiabetic drug for which a number of adverse effects have been widely described in the scientific literature. Therefore, these have to be carefully considered first in term of awareness and then in term of ability to limit and control such adverse events when proposing ROSI as a potential treatment of FOP.


Chemoprophylaxis and therapeutic intervention of the HIV epidemic requires understanding the mechanisms by which newly assembled HIV-1 virions are released from infected cells. This knowledge may prove diagnostic in identifying unsuspected sites for intervention and plausible strategies for targeted drug delivery. It is imperative, urgent, and ethical to uncover reliable and selective inhibitors of progeny virus release Vpu, an accessory membrane protein from the AIDS-associated virus HIV-1 We propose that it is feasible to increase the effective cell surface density of Vpu-free tetherin by designing transmembrane peptide decoys that target the Vpu TM α-helix TM peptide decoy design involves identification of sequences that would fold in the proximity of the Vpu TM helix and conform complementary matching surfaces to secure tight binding to Vpu and thereby generate unproductive, membrane-embedded heteromers. It requires exploring the TM sequence landscape to identify optimal Vpu TM matchmakers that would self-assemble into unproductive heterooligomers, thereby overcoming the restrain on tetherin and the consequent restriction of virion release from infected cells. A more refined design would entail identifying sequences that effectively embody intrinsically unfolded conformers Tetherin + channel, markedly impairs the ability of Vpu to enhance virion release The rationale underlying this hypothesis is strengthened by a number of additional facts: (1) The intact TM of Vpu is required to enhance virus release + human T cell line A3.01 or HeLa cells infected with HIV-1 clone NL4-3 This hypothesis naturally leads us to outline an initial itinerary to identify the minimal sequence determinants for the folding and coupling between decoy and Vpu TM modules that would minimally entail the following: (1) Identification of Vpu TM matchmakers by sequence analysis. The selection criteria consider experimentally determined TM α-helices or predicted single TM spans with a high propensity to fold into α-helices, and a high sequence similarity to the Vpu TM α-helix. A truncated alignment of relevant TMs with the target sequence of Vpu TM α-helix, as determined by NMR spectroscopy The guiding principle underpinning this hypothesis outlines a new way of thinking about disrupting protein–protein interactions by focusing on the TM anchors of the partners, a hitherto unexplored path towards the development of more effective blockers of virus release that may generate novel leads that could be developed into specific, realistic, and effective medications.


UPAR) has been shown to have clinical relevance in various cancers. We have recently identified UPAR as an asthma susceptibility gene and there is evidence to suggest that uPAR may be upregulated in lung diseases such as COPD and asthma. uPAR is a key receptor involved in the formation of the serine protease plasmin by interacting with uPA and has been implicated in many physiological processes including proliferation and migration. The current aim was to determine key regulatory regions and splice variants of UPAR and quantify its expression in primary human tissues and cells , airway smooth muscle (HASM) and peripheral cells).Expression of the urokinase plasminogen activator receptor (Using Rapid Amplification of cDNA Ends (RACE) a conserved transcription start site (-42 to -77 relative to ATG) was identified and multiple transcription factor binding sites predicted. Seven major splice variants were identified , including multiple exon deletions and an alternative exon 7b . Variants were differentially expressed, with a high proportion of E7b usage in lung tissue and structural cells (55–87% of transcripts), whereas classical exon 7 (encoding the GPI-linked protein) was preferentially expressed in peripheral cells (~80% of transcripts), often with exon 6 or 5+6 deletions. Real-time PCR confirmed expression of uPAR mRNA in lung, as well as airway and peripheral cell types with ~50–100 fold greater expression in peripheral cells versus airway cells and confirmed RACE data. Protein analysis confirmed expression of multiple different forms of uPAR in the same cells as well as expression of soluble uPAR in cell supernatants. The pattern of expression did not directly reflect that seen at the mRNA level, indicating that post-translational mechanisms of regulation may also play an important role.e.g. alternative E7b results in a soluble isoform due to the loss of the GPI anchor and exon deletions may affect uPA (ligand) and/or integrin binding and therefore influence downstream pathways. Expression of different isoforms within the lung should be taken into consideration in studies of uPAR in respiratory disease.We have identified multiple uPAR isoforms in the lung and immune cells and shown that expression is cell specific. These data provide a novel mechanism for uPAR regulation, as different exon splicing may determine uPAR function  The plasminogen pathway and specifically the urokinase plasminogen activator receptor (uPAR) has been of interest in the cancer field for some time . The The uPAR protein is a modulator of the plasminogen pathway, which cleaves and activates urokinase (uPA) . uPAR-uPuPAR or PLAUR) as an asthma susceptibility gene [UPAR determine the rate of decline in FEV1 and are also determinants of soluble uPAR in the serum of asthma subjects [A key feature of many respiratory diseases, including asthma, is airway remodelling . From thity gene . We havesubjects . In addisubjects , whilst subjects . A gene subjects . TherefoThe uPAR protein has three domains and is bound to the membrane via a glycosylphosphatidylinositol (GPI) anchor; however it may also be found as a soluble form due to cleavage of the GPI anchor and both membrane bound and soluble forms may be cleaved by various proteases, including uPA to give D1 and D2/3 fragments . The humUPAR gene consists of seven exons. Exon 1 encodes the 5'UTR and a signal peptide, whilst exons 2–3, 4–5 and 6–7 encode the homologous protein domains D1, D2 and D3 [The 2 and D3 . Use of 2 and D3 . Variant2 and D3 ,23. The UPAR gene and identify cell specific transcription initiation sites as well as to identify and quantify splice isoforms in cells and tissues from the lung and periphery. These data provide definitive answers regarding the type and extent of uPAR splice variation and generate a greater insight into the molecular mechanisms underlying the expression and activity of uPAR in the normal lung and immune cells, which may in turn have implications for the role of this receptor in airway disease.Due to the increasing interest in uPAR expression in lung diseases, the current study aimed to characterise the UPAR were determined by 5' RACE in a panel of six human tissues/cell types  for s Figure . In lungs Figure .To identify splice variants and determine the exon structure of uPAR, 5' and 3' RACE were completed in six different tissues/cell types Figures  and 3. 5In order to confirm and quantify the expression of different uPAR variants at the mRNA level, a series of real-time PCR assays were designed to amplify uPAR exons 1–2 , 1–7 and 1–7b, whilst a series of deletion-specific probes allowed detection of variants Table . Two difReal-time PCR was performed using cDNA from eight tissues/cell types in total, from either one (lung and brain) or two  donors, or prepared from RNA extracted on two different occasions for cell lines (THP1 (monocyte), BEAS2B ). Total uPAR expression was confirmed in the lung tissue and varied in a cell-type specific pattern; PMN ≥ PBMC>>lung>HBEC≈HASM>BEAS2B≈THP-1 (ANOVA p = 0.013), the HASM and HBEC expression levels being ~100 fold lower than primary peripheral cells (PMN and PBMC) Figure . ExpressOverall, expression levels of classical uPAR (exon 7) correlate with total uPAR levels, PMN ≥ PBMC>>lung>HBEC≈HASM>BEAS2B≈THP1 , suggesting that this may be the dominant expressed variant Figure  and 7B. e.g. PBMC/PMN vs. lung/HASM/HBEC as shown in the absolute expression data  form to total uPAR(E7b) expression, suggests lower expression in lung, PBMC and THP1 than HASM, HBEC and BEAS2B  were assayed . Representative results are shown in Figure B Figure .Predicted sizes for all variants detected by real-time PCR suggested that multiple variants between 60–100% of the full length classical uPAR should be detected in all cell types Table . It shouFull-length uPAR protein has a predicted molecular weight of 31 kDa, however differential glycosylation patterns result in a size range of 40–60 kDa. Recombinant uPAR expressed in a mouse melanoma cell line was included and was detected between 45–60 kDa with both antibodies. The D2 specific antibody also detected a higher molecular weight protein (70–75 kDa), possibly representing a dimerisation or aggregation product. A lower molecular weight protein was also observed (30–35 kDa), which probably corresponds to a D2/3 proteolytic fragment.e.g. exon 6) deletions of classical uPAR . A protein at 37 kDa is detected in all cell types except HBEC, being particularly strongly expressed in PMN and PBMC. This could represent dual exon deletions , although its pattern of expression does not completely correspond to that seen at the mRNA level. The smallest protein seen (30 kDa) may represent an alternative exon 7b, exon 4+5 deleted form , although this might be expected to be expressed in a greater number of cell types.The D1 antibody detected fewer proteins than the D2 antibody (including for the recombinant protein) Figure . D1 protThe D2 antibody detects three proteins in the recombinant sample, probably reflecting a dimerisation, full-length uPAR and a D2/3 proteolytic fragment Figure . The larThe expression of soluble PAR was detected in the supernatants of all cells tested Figure . OverallWestern blotting using two monoclonal antibodies for uPAR detected multiple bands. To confirm that these bands represent uPAR variants, siRNA was performed in undifferentiated HBEC cells. Cells were treated with siRNA  or a uPAR-specific panel (mix)) for 24 hours before RNA and protein were extracted and analysed by real-time PCR and Western blotting Figure . Total uUPAR, identify all common splice variation and quantify expression in the normal lung and in specific lung and peripheral cells at the mRNA and protein levels. We have identified a localised TSS with some cell specificity and confirmed expression of uPAR in lung and peripheral cells. At the mRNA level, multiple uPAR splice variants were identified including alternative E7 (E7b) and deletions of E3, E5+6 and E6, and their patterns of expression in different tissues/cell types characterised. Primary peripheral cells (PMN and PBMC) expressed multiple exon deleted forms of membrane bound uPAR, whilst lung cells including epithelium and airway smooth muscle expressed a greater proportion of an alternative soluble uPAR with and without an exon 4+5 deletion. Protein analyses confirmed expression of multiple differentially expressed forms of uPAR in all cell extracts, and soluble uPAR was detected in the supernatants of cultured HASM, HBEC, THP1 and BEAS2B cells. Specificity of the Western blotting analyses was confirmed by siRNA. Our data provide a novel insight into the molecular mechanisms that potentially regulate uPAR expression and activity in the airways and the periphery which has implications regarding the potential role of uPAR in airway disease biology.The expression and functional activity of uPAR is of interest in both cancer and respiratory disease. The current study aimed to characterise the key regulatory regions of UPAR is 52 bp upstream of the ATG, with a minor start site around -77 bp in U937 (monocytes) and HeLa  cells [i.e. -46 to -52 is the dominant TSS in most cells and lung tissue but not in HBEC where the -75 to -78 locus is dominant. The functional significance of the cell specific TSS usage remains to be resolved although it is interesting to note that functional studies using promoter-reporter approaches have suggested that most transcriptional activity is found in the first 220 bp upstream of the ATG in HeLa, HCT116 and RKO (colon cancer) cells [Previous analyses in cell lines suggested the TSS of l) cells . We haver) cells -31. Funcr) cells ,30. We ur) cells . TGFβ1 iThe second objective of the current study was to determine uPAR splice variation in airway and peripheral cells using real-time PCR. Two donors were used for each cell type (but not tissues), to allow for inter-individual differences. We observed expression of the previously identified uPAR E7b soluble splice variant , which iIn addition to the exon7 variants we also identified a series of internal exon deletion variants expressed within the context of both the membrane bound and soluble receptor. These included exon 3, 4+5, 5+6 and 6 deletions. Exon deleted forms of classical uPAR were found most frequently in the primary peripheral cells, PMN and PBMC. Exon 6 deletion was the most widely expressed. Loss of exon 6 would disrupt the structure of D3, particularly as it includes one of the key cysteines involved in disulphide bonding. This would be expected to lead to reduced uPA binding affinity  as two ai.e. exon 5 deletions) may be of significance in normal and disease mechanisms.Repeated epithelial wounding/repair leading to airway remodelling has been implicated in asthma pathogenesis . Epithelet al. [At the protein level, multiple forms of uPAR were detected in cell lysates. Overall, our data suggest that multiple forms of uPAR are expressed in the panel of cells studied and there is some evidence of differential expression. This corresponds to the findings of Brooks et al. , who sho52) [e.g. the presence of a 50 kDa protein potentially representing the alternative exon7b observed in all cell types except PMN and PBMC, as well as a 45 kDa form seen only in PBMC and PMN which may represent single exon deletions of classical uPAR. Concordance between the two assays may be affected by differing efficiencies of the real-time PCR assays or antibody specificity. However, it will also reflect genuine biological differences, as post-translational control mechanisms including glycosylation and proteolytic cleavage play a key role in uPAR protein maturation [Although the majority of high abundance predicted uPAR variants might be expected to be detected using either the D1 or D2 antibody, the patterns of expression observed were markedly different. This could reflect differing affinities of the two antibodies for different variants. It should be noted that the epitope for the D1 antibody (amino acids 52–60) corresponds to one of the potential glycosylation sites for the protein (Asn52) . The preturation ,25. Our e.g. BEAS2B and THP1 had elevated alternative uPAR(E7b) mRNA and soluble uPAR protein. Therefore, the elevated levels of uPAR in the sputum of asthma and COPD subjects may reflect expression of soluble uPAR from both epithelial and smooth muscle cells.All cultured cells tested showed expression of soluble uPAR. This assay does not distinguish between classical uPAR released from its GPI anchor, proteolytic fragments and alternatively spliced (exon 7b) soluble uPAR. However, all of these cell lines expressed relatively high proportions of alternative uPAR(E7b) mRNA and the level of mRNA correlates with soluble uPAR protein expression UPAR in the airway and highlighted potential regulatory transcription factor binding sites. We have identified multiple splice variants with potentially different functional activity including soluble expression and/or the loss of key binding domains, such as those involved in ligand binding or integrin interactions. The expression patterns of these splice variants in airway and peripheral cells provide an insight into the molecular mechanisms regulating uPAR expression and activity. These data highlight the importance of uPAR splice variants, which should be taken into consideration in future work to determine the role of uPAR in respiratory disease.We have identified the key promoter region of Primary human airway smooth muscle (HASM) cells were isolated and prepared as described previously  and cultFor downstream analysis, cells were lysed and RNA extracted using silica columns . For protein analysis, cell lysates were prepared using cytobuster buffer  with benzonase (Merck) and complete protease inhibitor , as directed by the manufacturer. Ethical approval for the use of primary cells was obtained from the Nottingham University Hospitals local ethical committee.uPAR was targeted for knockdown in undifferentiated HBEC cells using siRNA specific to exon 1 (S032) or a panel of four siRNAs specific to exons 1–2, 7 and 7b (mix). Silencer select negative control #1 was used as a control. siRNA sequences available on request. Silencer select siRNAs were obtained from Applied Biosystems . Cells were plated in 6-well plates at 100,000 cells per well and transfected after 48 hours with 10 nM total siRNA using 5 μl Liopfectamine 2000 as directed by the manufacturer .Whole blood was collected from healthy volunteers (45 ml). Red blood cells were removed using dextran sedimentation, before separating the neutrophils (PMN) and mononuclear cells (PBMC) by centrifugation in a discontinuous percoll gradient. Slides were prepared by cytospin for characterisation of cell populations. PMN were >95% pure (some eosinophils and basophils), whilst PBMC consisted mostly of lymphocytes (44%) and monocytes (39%) with the remainder eosinophils and neutrophils as defined by morphology. Ethical approval was obtained from the Nottingham University Hospitals local ethical committee. RNA was prepared as described previously. RNA and lysates from PMN and PBMC were also obtained commercially .. Alignments were made against reference sequences NM_002659.2  and NM_001005376 .RACE-ready cDNA was synthesised from total RNA (1 μg) ) using the GeneRacer kit as directed (Invitrogen). 5' and 3' RACE used GeneRacer primers and uPAR exon 4 specific nested primers for amplification Table . Full-le-ΔCt method and normalised to a variant specific positive control plasmid. Alternative splice variants were also normalised to total uPAR to identify proportional expression and allow comparison to RACE data.mRNA levels of total uPAR and splice variants were quantified using a series of real-time PCR assays. Amplicons spanning exons 1–2, 1–7 and 1–7b were used in combination with a series of splice-variant specific FAM/TAMRA labelled TaqMan probes to define up to 16 different variants  0.25 μg/ml (1:400) and uPAR D2 monoclonal: 3932  2.5 μg/ml (1:200) with an appropriate HRP-conjugated secondary antibody  (1:1000). Binding was visualised by enhanced chemiluminescence . Membranes were stripped using Restore Western blot stripping buffer  and re-probed for β-actin expression  as a loading control. Antibody dilutions were based on manufacturers' recommendations. Densitometry of protein bands was completed using ImageJ 1.41 The uPAR Duoset ELISA kit (R&D systems) was used to quantify soluble uPAR in cell culture supernatants, following the manufacturer's recommendations. uPAR was quantified using tetramethylbenzidine (R&D systems) and a MultiscanEX plate reader (Thermo Fisher Scientific) at 450 nm with 570 nm background subtraction. The detection limit of the assay was 30 pg/mL sample.Transcription factors relevant to respiratory disease and asthma were identified by reference to the literature ,42. A 40post hoc test. Data were analysed using Prism v.5.01 . P < 0.05 was considered significant.Differences between RACE results were evaluated using contingency tables (Chi-square test). Differences in expression in real-time PCR experiments were evaluated using ANOVA and Tukey's COPD: chronic obstructive pulmonary disease; D1–3: domain 1–3 (of uPAR protein); ECM: extracellular matrix; ELISA: enzyme-linked immunosorbent assay; GPI: glycosylphosphatidylinositol; HASM: human airway smooth muscle cell; HBEC: human bronchial epithelial cell; HPRT1: hypoxanthine guanine phosphoribosyltransferase 1; PBMC: peripheral blood mononuclear cell; PCR: polymerase chain reaction; PMN: polymorphonuclear cell (neutrophil); RACE: rapid amplification of cDNA ends; SEM: standard error of the mean; TSS: transcriptional start site; uPA: urokinase plasminogen activator (PLAU); uPAR: urokinase plasminogen activator receptor (PLAUR); UTR: untranslated region.CS participated in the design of the study, performed the benchwork and statistical analyses and drafted the manuscript. IS conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Thus far, proposed mechanisms for carcinogeneis associated with this syndrome include aberrant apoptosis, oxidative stress, and pseudohypoxic drive. At this time, the majority of accumulating data support a role for pseudohypoxic drive in tumour development. The link between FH mutation and pseudohypoxic drive may reside in the biochemical alterations resulting from diminished/absent FH activity. These biochemical derangements may interfere with oxygen homeostasis and result in a cellular environment conducive to tumour formation.Hereditary leiomyomatosis and renal cell cancer is a recently described hereditary cancer syndrome in which affected individuals are predisposed to the development of leiomyomas of the skin and uterus. In addition, this clinical entity also can result in the development of biologically aggressive kidney cancer. Affected individuals harbour a germline mutation of the  VHL gene  is a disease with varying genetic basis. Biologic characterisation of this disease has been greatly aided by the investigation of families with multiple members affected by RCC. Probably the earliest and best-characterised familial form of kidney cancer is von-Hippel Lindau (VHL). von-Hippel Lindau families are susceptible to the development of clear cell histology RCC owing to inherited alterations of the FH allele in leiomyomas and kidney tumours have been identified   have been implicated in the development of a cancer syndrome referred to as hereditary paraganglioma and pheochromocytoma (HPGL). Succinate dehydrogenase catalyses the conversion of succinate to fumarate. In contrast to FH, which functions as a homotetramer  cycle would be at a disadvantage. However, FH is not the only TCA enzyme whose inactivation is linked to neoplasia. Mutations of tetramer . MutatioPostulated mechanisms for these inherited cancer syndromes include aberrant apoptosis, oxidative stress, and pseudohypoxic drive . Of thesVHL is a tumour suppressor gene involved in the development of clear cell RCC. The VHL protein product, hereafter referred to as pVHL, is part of a complex signalling cascade that modulates a cell's oxygen-dependent gene expression. In normoxic conditions, pVHL is known to associate with several proteins including Cul-2, Elongin B, and Elongin C to form the VHL complex (α (HIF-1α) and HIF-2α are two of the proteins targeted for degradation by the VHL complex under normoxic conditions , glucose transporter 1 (GLUT1), platelet-derived growth factor (PDGF), transforming growth factor-α (TGF-α), and erythropoietin (EPO). VHL complex recognition of HIF requires the hydroxylation of conserved proline residues of the HIF protein  in patients with HLRCC. Their findings in kidney tumours were compelling for activation of hypoxic pathways. In all renal tumours examined, they noted strong HIF-1α staining in the nuclei of tumour cells. This finding was also confirmed by immunoblotting, as HIF-1α was easily detected in tumour cell lysate with no detectable HIF-1α in normal kidney lysate  as well as sporadic uterine leiomyomas (VEGF (1.4–3.5-fold) as compared to patient-matched normal myometrium. In addition, other hypoxia-responsive gene changes were also found in HLRCC leiomyomas including downregulation of TSP1, a known antiangiogenesis factor  from HPGL patients. Paragangliomas from patients with known germline SDHB mutations have been found to have elevated succinate levels  via fumarate accumulation. By a similar mechanism, elevated levels of succinate may be responsible for pseudohypoxic drive in the context of paragangliomas/pheochromocytomas in patients with germline SDH mutations. These studies provide a unique molecular basis for the Warburg effect. The Warburg effect refers to the preferential derivation of energy by rapidly dividing cancer cells from glucose metabolism via glycolysis rather than oxidative phosphorylation despite adequate intracellular oxygen levels. We have shown that experimental depletion of FH results in acute upregulation of GLUT1 protein, glucose uptake, and lactate production in normoxic cells (Hereditary leiomyomatosis and renal cell cancer is a hereditary cancer syndrome predisposing individuals to the development of aggressive kidney cancers. These individuals are known to harbour a germline mutation of


In all TB patients a mixed Th1/Th2 profile was noted. Necrotic granulomas were more evident in HIV positive patients with a clear association between TNF-α and necrosis. This study demonstrates immune dysregulation which may include TNF-α-mediated immunopathology at the site of disease in HIV infected pleural TB patients.Although granulomas may be an essential host response against persistent antigens, they are also associated with immunopathology. We investigated whether HIV co-infection affects histopathological appearance and cytokine profiles of pleural granulomas in patients with active pleural tuberculosis (TB). Granulomas were investigated in pleural biopsies from HIV positive and negative TB pleuritis patients. Granulomas were characterised as necrotic or non-necrotic, graded histologically and investigated for the mRNA expression of IL-12, IFN-γ, TNF-α and IL-4 by  Mycobacterium tuberculosis (Mtb) than other forms of the disease due to the fact that a high proportion of individuals can recover without antibiotic therapy Granulomas represent a distinctive pattern of chronic inflammation that involves adaptive immunity, and are the hallmark of many human diseases of significance, such as tuberculosis (TB) MtbGranuloma formation is considered to be an essential component of protective immune responses against Mtb. It has been hypothesized that quantitative and qualitative T-helper responses may impact on AIDS pathogenesis. Although the supposed role of modulation of T helper responses in HIV pathogenesis remains controversial, a switch in the predominant response from Th1 to Th2 and the production of associated cytokines may facilitate disease progression Mtb due to insufficient macrophage, T cell and cytokine responses Mtb can also modulate HIV replication by generating a cytokine microenvironment favouring viral infection and spread in local mononuclear cells HIV infected people develop TB at a rate of 10% per year, rather than the norm of 10% per lifetime There is a delicate and complicated interplay between different cytokines and there are numerous reports of beneficial and detrimental effects of individual cytokines. High levels of pro-inflammatory cytokines, including IFN-γ and TNF-α in situ. In addition we compared the cytokine profiles of necrotic and non-necrotic granulomas from HIV-positive and -negative patients in order to describe the association between cytokine production, necrosis and HIV positivity.Understanding the human host response is important, as this would assist in the development of new vaccines and immunomodulatory adjuvants for the prevention or treatment of TB. We chose to investigate the Th1/Th2 profile in pleural TB patients by measuring the mRNA expression levels of the cytokines IL-12, IFN-γ, TNF-α and IL-4 A total of 12 patients with pleural TB were recruited from Tygerberg Academic Hospital, including 6 HIV sero-negative (group 1) and 6 HIV sero-positive (group 2) people. HIV serology was done on all patients by ELISA. HIV positive patients were in the early stage of the disease and had no opportunistic infections besides TB. All patients presented with symptoms of pleuritis and had radiological confirmation of the effusion. None of the patients had had TB previously nor were any on treatment at the time of the study. Pleural biopsy tissue was obtained for diagnostic purposes. The diagnosis of TB was confirmed by the presence of granulomatous inflammation and Ziehl-Neelsen (ZN) positive bacilli in biopsy material This was performed essentially as described previously This was performed essentially as described previously Briefly, consecutive 5 µm sections were applied to RNase-free slides previously coated with aminopropyltriethoxysilane , deparaffinized in xylene, rehydrated through graded ethanols and diethyl pyrocarbonate-treated water and incubated in phosphate-buffered saline (PBS). The sections were treated with proteinase K in Tris-HCl-EDTA, washed with PBS, refixed in paraformaldehyde and acetylated in triethanolamine-acetic anhydride. The slides were then rinsed, dehydrated and air-dried before hybridization. Sections were incubated in a mixture of dextran sulphate, Tris-HCl, Denhardt's solution, EDTA, dithiothreitol, herring sperm DNA, tRNA and deionized formamide. Sections were hybridized for 18 hours in a humidified chamber at 50°C, washed in saline sodium citrate, incubated in Tris-HCl-NaCl containing sheep serum and then washed. Thereafter, sections were incubated with antidigoxigenin antibody conjugated to alkyline phosphatase (Boehringer Mannheim) and the signal detected with BCIP-NBT-INT (Dako). After the appearance of a brown colour slides were counterstained with Mayer's hematoxylin (Sigma Aldrich), rinsed in distilled water, mounted with Dako Faramount and viewed under a light microscope.For dual labelling, slides were rinsed and non-specific protein blocked with milk powder and Triton-X 100 in PBS. Sections were incubated with antibody diluted in goat serum, washed and labelled with secondary biotinylated goat anti-mouse antibody diluted in goat serum and Triton-X 100. Sections were washed again, incubated with streptavidin conjugated to alkyline phosphatase, washed in PBS and incubated with a solution of fast red (Vector Laboratories). Slides were counterstained with hematoxylin and mounted with Dako Faramount.Images were captured using a Zeiss light microscope with light parameters optimized for the actin stained slide. These settings were kept constant in order to maintain comparability between slides. Control slides were added parallel to each staining procedure to show that the positive colour was specific and that the mRNA was not degraded . The num>75% of cells in a granuloma positive: grade 3 positivity25% to 75% of cells in a granuloma positive: grade 2 positivity<25% of cells in a granuloma positive: grade 1 positivityno cells positive: grade 0 positivityAll granulomas were regarded as independent parameters.Statistical analysis on samples from patients with pleural TB was performed by applying the Pearson Chi square and Spearman rank R tests to the following parameters:The association between HIV serology and presence of necrosis.The association between HIV and individual cytokinesComparisons in association patterns between various cytokines between HIV negative and HIV positive granulomas.The association between necrosis and individual cytokines.Comparisons in association patterns between various cytokines between necrotic and non-necrotic granulomas.In addition, to test for association between pairs of cytokines and necrosis a two-tailed Fisher's exact test was used.Mtb.There were 2 male and 4 female TB patients in the HIV positive group with a mean age of 34.5 years compared to 5 males and 1 female with a mean age of 38 years in the HIV negative group. All patients presented with symptoms of pleuritis. All pleural biopsies were subsequently found to be culture positive for drug-sensitive p<0.01).Of the 166 granulomas present in pleural biopsies from the 12 patients with pleural TB, 125 were from HIV negative patients and 41 from HIV positive patients. Necrosis was more prevalent in granulomas of HIV positive patients . In the in situ hybridization (ISH). Representative images illustrate the presence of both Th1 and Th2 cytokine mRNA expression, all of which were more prevalent in necrotic granulomas (Pleural biopsy tissue was examined for IL-12, IFN-γ, TNF-α and IL-4 mRNA expression using anulomas . The extanulomas .While the presence of necrosis did not significantly influence the grade of IL-12 mRNA positivity, IFN-γ, TNF-α and IL-4 mRNA expression were markedly upregulated in necrotic granulomas . SimilarThere was a strong positive correlation between all cytokines expressed in non-necrotic granulomas and granulomas from HIV negative TB patients . By contet al found the opposite This work characterised cytokine expression at the site of disease during the granulomatous response in pleural TB, in HIV infected and uninfected individuals and in necrotic and non-necrotic granulomas. Patients with pleural TB presented with a mixed Th1/Th2 profile and this contradicts a characterisation of HIV negative pleural TB as Th1 dominant The investigation of immune responses at the site of disease is extremely valuable because cytokine responses do not necessarily mimic those found in the peripheral blood Mtb, and is necessary for granuloma formation, it does cause immunopathology such as caseous necrosis Mtb infection, secondary necrosis may be common Mtb-mediated lysis of macrophages which triggers the macrophage cell death pathway. This not only allows dissemination of the bacilli but also contributes to the formation of necrotic lesions Although the presence of TNF-α is beneficial in the immune response against Mtb of its intracellular sanctuary and inhibit its growth and survival Mtb after Fas ligand or TNF-α mediated apoptosis of immune cells is controversial with both reduced and unchanged bacterial numbers reported Although apoptosis is an important host defence mechanism to deprive Previous studies that investigated correlations between granuloma histology and cytokine mRNA expression Mtb. We found increased IFN-γ expression in necrotic granulomas and, although, like TNF-α, it is crucial in defence against Mtb, high effector cytokine levels during chronic phases of disease may contribute to immunopathology, especially in the presence of high TNF-α expression The cytokine pattern observed in patients with pleural TB reflects a model by Wigginton and Kirscher et alBy contrast, non-necrotic granulomas with a high number of IL-12 positive cells also expressed high levels of IL-4 and this was not associated with necrosis presumably because Th2 cytokines mediate local tissue inflammation and necrosis through their effect on TNF-α induced cytotoxicity Interestingly very few necrotic granulomas expressed a high level of IFN-γ-positive cells in the presence of high IL-12 expression. This may suggest a breakdown in the regulatory function of IL-12 which usually regulates the expression of IFN-γ et alIn summary, intracellular bacteria that can survive within macrophages lead to granuloma formation, which benefits the host by controlling local and systemic dissemination of pathogens but facilitates destructive pulmonary pathology. In TB, this manifests as caseous necrosis with tissue destruction and subsequent fibrosis. This facilitates disease transmission and therefore blocking any mechanism responsible for necrosis would also limit person-to-person disease transfer Mtb.In conclusion, this study reports on the expression of Th1 and Th2 immune phenotypes at the site of disease in TB pleuritis. It is shown that HIV positivity results in increased necrosis associated with elevated TNF-α mRNA expression. Future studies should also investigate the expression of other cytokines, chemokines and their receptors to allow a more comprehensive understanding of factors that determine the fine balance between protective and immunopathological responses after infection with


While the phylogenetic relationships of selenoprotein families and the evolution of selenocysteine usage are well documented, the evolutionary history of SECIS binding proteins has not been explored.The co-translational incorporation of selenocysteine into nascent polypeptides by recoding the UGA stop codon occurs in all domains of life. In eukaryotes, this event requires at least three specific factors: SECIS binding protein 2 (SBP2), a specific translation elongation factor (eEFSec), selenocysteinyl tRNA, and a in vitro. An in-depth phylogenetic analysis of the conserved L7Ae RNA binding domain suggests an ancestral relationship with ribosomal protein L30. In addition, we describe the emergence of a motif upstream of the SBP2 RNA binding domain that shares significant similarity with a motif within the pseudouridine synthase Cbf5.In this report we present a phylogeny of the eukaryotic SECIS binding protein family which includes SBP2 and a related protein we herein term SBP2L. Here we show that SBP2L is an SBP2 paralogue in vertebrates and is the only form of SECIS binding protein in invertebrate deuterostomes, suggesting a key role in Sec incorporation in these organisms, but an SBP2/SBP2L fusion protein is unable to support Sec incorporation Our analysis suggests that SECIS binding proteins arose once in evolution but diverged significantly in multiple lineages. In addition, likely due to a gene duplication event in the early vertebrate lineage, SBP2 and SBP2L are paralogous in vertebrates. Eukaryotic SECIS elements are stable stem-loop structures that are comprised of two helices separated by a kink-turn containing a conserved GA quartet (SECIS core) and an apical AAR motif that is present either as a terminal loop (Form 1) or a terminal bulge (Form 2)  UTP-labeled SECIS elements and the complexes resolved by non-denaturing gel electrophoresis. This amount of CT-SBP2 is within the linear range of this binding assay (data not shown). As expected, CT-SBP2 shifted wild-type but not mutant SECIS elements in which the AUGA motif in the SECIS core was deleted . Using Figure Having established the phylogenetic relationship of metazoan SBPs, we sought to determine whether we could elucidate the origins of SBP2. As mentioned above, SBPs contain an L7Ae RNA binding motif. This motif was identified by Koonin and colleagues  as a 32 de novo. Regardless of its origin, the conservation of the SID core motif in the eukaryotic taxa presented here (K517-R543) suggests that it arose once in eukaryotic evolution and that organisms such as D. discoideum and Plasmodium vivax with poorly conserved SID sequences likely diverged to adapt to as-yet unidentified unique requirements . Notably, the only region of significant similarity is that between RFQDR654-658 and the universally conserved Gly residue in the L7Ae RNA binding motif (G676). Figure Caban and colleagues recently performed structure/function studies on the SBP2 RNA binding domain including its L7Ae motif . In thisynthase  upstream of the  at NCBI  for thesD Figure . AdditioThis report represents the first phylogenetic analysis of SBPs across eukaryotic taxa. Since vertebrates possess two SBPs, we annotated determinants that serve as identifying motifs in SBP2L and SBP2. We showed that SBP2L is the likely progenitor of mammalian SBP2 and as the only form of SECIS binding protein in invertebrate deuterostomes, SBP2L is likely required for Sec incorporation in these organisms. However, the role of SBP2L in vertebrates will require further experiments to elucidate its function, and we expect forthcoming experimental evidence to support this hypothesis. Additionally the retention and loss of LSAD and PVFQ motifs in several taxa suggest that SBPs are subject to diverse evolutionary pressures. Conservation of the SID and RBD in unicellular eukaryotes and metazoans suggests that SBP2 arose once in eukaryotic evolution.A. aegypti, showed that its SID and RBD are coded by separate genes. Future use of A. aegypti cell culture could provide a unique system in which to study Sec incorporation. Based on the taxa sampled here, we propose that SECIS binding proteins arose once in eukaryotic evolution and diverged substantially as in the case of P. vivax, T. gondii, and D. discoideum. Additionally, the presence of SBP2L in some protostomes (C. sp. I and L. gigantia) but not in others (i.e. insects) suggests that SBP2L sequences occurred in the last common ancestor of protostomes and deuterostomes followed by divergence in different lineages. Lastly, a gene duplication event, possibly as the result of whole genome duplication, generated the paralogous pair of SBP2 and SBP2L in vertebrates.The origins of the SID and RBD, the essential domains for SBP2 function, may provide insight into the mechanism of Sec incorporation. The apparent phylogenetic relationship between the SBP L7Ae motif and RPL30 could provide insight into SBP2-ribosome interactions or reiterate a possible role for RPL30 in Sec incorporation as suggested by Chavatte . The oriab initio gene prediction using GenomeScan or the FGenesH webserver -Met as directed by the manufacturer. Protein quantitation was performed by PhosphorImager analysis as previously described -labeled wild-type and mutant GPX4 SECIS elements were transcribed as described by Caban [2007]. SECIS binding of in vitro translated proteins was assayed by incubating 8 fmol with 20 fmol of wild-type or mutant (ΔAUGA) SECIS probes in 1 × PBS supplemented with 250 μg/mL yeast tRNA (Sigma), 10 mM DTT, and 5 μg soybean trypsin inhibitor (Sigma). Final reaction volumes were 20 μL. EMSA reactions were incubated for 30 min at 37°, resolved on 4% non-denaturing polyacrylamide gels, and visualized by PhosphorImaging. Percent shifted probe was determined with ImageQuant software by subtracting background from the RRL only lane and normalizing to CT-SBP2.[aa: Amino acid; Sec: Selenocysteine; SECIS: Sec Insertion Sequence; SBP: SECIS binding protein; SBP2L: SBP2-like protein; SID: Sec incorporation domain; RBD: RNA binding domain; RP: Ribosomal protein; EMSA: Electrophoretic mobility shift assay; RRL: Rabbit reticulocyte lysate; UTR: Untranslated region; EST: Expressed Sequence Tag;JD performed all wet-lab experiments, sequence alignments, gene predictions and phylogenetic analysis, contributed to the writing of the manuscript and preparation of figures and PRC initiated the project, performed sequence alignments and phylogenetic analyses and contributed to the writing of the manuscript and preparation of figures. Both authors read and approved the final version.Multiple sequence alignment of all SBP2 and SBP2L sequences used for Figures Click here for fileA generalized workflow for identification of SBP2 or SBP2L in organisms for which these proteins were not annotated.Click here for fileList of SBP2/2L sequences used in this paper. Accession numbers are provided for sequences available at NCBI and predicted sequences are provided in full. Underlined residues indicate regions supported by ESTs. NCBI annotated sequences marked with an asterisk were refined based on EST and/or genomic DNA data . Note that some predicted sequences are likely to be partial due to lack of EST coverage or inability of gene prediction programs to correctly identify a start codon.Click here for fileAlignment of the core L7Ae motifs used to generate the phylogenetic tree in Figure Click here for file


Peripheral blood is an accessible and informative source of transcriptomal information for many human disease and pharmacogenomic studies. While there can be significant advantages to analyzing RNA isolated from whole blood, particularly in clinical studies, the preparation of samples for microarray analysis is complicated by the need to minimize artifacts associated with highly abundant globin RNA transcripts. The impact of globin RNA transcripts on expression profiling data can potentially be reduced by using RNA preparation and labeling methods that remove or block globin RNA during the microarray assay. We compared four different methods for preparing microarray hybridization targets from human whole blood collected in PAXGene tubes. Three of the methods utilized the Affymetrix one-cycle cDNA synthesis/in vitro transcription protocol but varied treatment of input RNA as follows: i. no treatment; ii. treatment with GLOBINclear; or iii. treatment with globin PNA oligos. In the fourth method cDNA targets were prepared with the Ovation amplification and labeling system.We find that microarray targets generated with labeling methods that reduce globin mRNA levels or minimize the impact of globin transcripts during hybridization detect more transcripts in the microarray assay compared with the standard Affymetrix method. Comparison of microarray results with quantitative PCR analysis of a panel of genes from the NF-kappa B pathway shows good correlation of transcript measurements produced with all four target preparation methods, although method-specific differences in overall correlation were observed. The impact of freezing blood collected in PAXGene tubes on data reproducibility was also examined. Expression profiles show little or no difference when RNA is extracted from either fresh or frozen blood samples.RNA preparation and labeling methods designed to reduce the impact of globin mRNA transcripts can significantly improve the sensitivity of the DNA microarray expression profiling assay for whole blood samples. While blockage of globin transcripts during first strand cDNA synthesis with globin PNAs resulted in the best overall performance in this study, we conclude that selection of a protocol for expression profiling studies in blood should depend on several factors, including implementation requirements of the method and study design. RNA isolated from either freshly collected or frozen blood samples stored in PAXGene tubes can be used without altering gene expression profiles. Gene expression profiling of RNA extracted from peripheral blood is an informative method used to identify biomarkers, examine disease states, and investigate immune responses. However, the relatively high proportion of globin messenger RNA present in total RNA extracted from whole blood can reduce the efficacy of the microarray assay by interfering with the detection of less abundant gene transcripts -3. FurthCommon laboratory practice often includes fractionation of whole blood components prior to RNA extraction. The process allows for the removal of the red blood cells from whole blood isolations and facilitates the study of more homogeneous cell populations. Depending on the fractionation method selected, partial or complete removal of reticulocytes, the primary source of globin RNA, may be achieved. Despite the obstacle posed by globin RNA contamination, however, there are compelling reasons to study gene expression from whole blood rather than from subpopulations such as neutrophils. First, to capture expression profiles that accurately reflect the transcriptome at time of blood collection and to minimize sample handling artifacts, it is preferable to avoid additional processing steps as some degree of cell activation is inevitable during cell fractionation. Second, even when fractionation steps are performed, α and β globin mRNA transcripts are often the most abundant transcripts present in total RNA extracted from leukocyte-enriched populations  and the Several commercially available methods for reducing the impact of globin RNA transcripts in the microarray expression assay have been developed. One approach involves the removal of α and β globin RNAs from total RNA by selective hybridization and magnetic bead separation  prior toEach method has been demonstrated to produce array data in which an increased number of non-globin gene transcripts are detected ,10, and RNA isolated directly from whole blood and processed using standard target preparation methods produces gene expression profiles with increased noise and reduced sensitivity compared with total RNA isolated after various leukocyte isolation protocols. However, the advantages of profiling gene expression from whole blood are compelling: 1) cell fractionation steps that may artifactually alter gene expression patterns are avoided; 2) easy and rapid isolation of RNA from whole blood facilitates clinical studies; 3) even rare subpopulations of cells such as dendritic cells or eosinophils remain included in the transcriptome; and 4) costs are vastly reduced. To increase assay sensitivity and reproducibility when profiling RNA extracted from whole blood, it is necessary to employ a method to reduce the impact of highly abundant globin mRNAs on target hybridization with the microarray. In this study, four methods for RNA preparation and labeling were examined to determine which protocol produced the most sensitive and reproducible results when blood RNA isolated from PAXGene tubes was used as input. In addition, since the option to freeze a whole blood sample allows samples to be collected over time and at different study sites prior to processing, the effects of freezing blood samples were examined by comparing data generated from both fresh and frozen specimens. Freezing blood samples after collection has the advantage of allowing batching of samples for RNA extraction and simplifying the blood collection protocol for multicenter studies.Blood samples were collected from healthy, human donors in PAXGene tubes; RNA was isolated either on day of collection or after freezing and storage. Four different methods of microarray target preparation for whole blood RNA samples were examined Figure . Three oThe quality of total RNA prepared from fresh and frozen PAXGene tubes and from GLOBINclear-treated RNA was assessed using standard UV spectrophotometry and by examining electropherogram images generated with the Agilent Bioanalyzer. Input RNA was of high quality for both fresh and frozen samples  with a slight reduction in average quality metrics for GLOBINclear-treated samples (average RIN score 8.6 and 8.5). Reductions in RIN scores after GLOBINclear treatment of whole blood RNA were also observed by Liu et al. . Each amUsing recommended RNA inputs for each method, average cRNA target yield ranged from 20 μg to 102 μg. The average yield with Method 1 (no depletion_Affymetrix) using RNA from either fresh or frozen PAXGene tubes is typical of yields obtained in our laboratory with this method. We speculate that the high yields for Method 2 (Globin PNAs_Affymetrix) relative to the other Affymetrix one-cycle target labeling methods are, at least partially, the result of the higher input RNA amount (5 μg) required by the Method 2 protocol. The GLOBINclear-treated RNA produced the lowest cRNA yields with the Affymetrix one-cycle labeling protocol, as has been observed previously with this method [Targets produced with each labeling method were hybridized with the GeneChip Human Focus array. Target amounts and array processing steps were performed according to the manufacturers' recommendations for the labeling method and the array format. Overall performance in the array assay was examined in three ways: 1) individual array performance metrics (generated with MAS 5.0); 2) data visualization for assessment of data distribution and variation; and 3) correlation of detected transcripts among targets generated with each method.Array performance metrics Table  indicateExamination of probe cell intensity distributions prior to normalization shows that overall signal distribution patterns Figure  varied wSample-to-sample variation was also examined by measuring similarity among expression profiles from different donor samples processed with the same target preparation method. A high within-method correlation (average r = 0.98) was measured for Methods 1 , 2 (Globin PNAs_Affymetrix) and 4 (no depletion_NuGEN). Method 3 (GLOBINclear_Affymetrix) again showed somewhat poorer performance (average r = 0.96).We assessed the extent of globin transcript depletion in the microarray assay with each labeling method by examining α- and β-globin mRNA signal intensities normalized to actin mRNA signal. All of the globin mRNA depletion methods resulted in decreased α- and β-globin mRNA transcript measurements relative to Method 1 (no depletion_Affymetrix) targets. Method 2 (Globin PNAs_Affymetrix) generated targets that showed the greatest reduction in globin mRNAs Figure . InteresExpression levels of a panel of 84 genes from the NF-κB pathway plus 5 housekeeping genes were measured by quantitative RT-PCR using total RNA from two of the individuals profiled in the microarray component of this study. The NF-κB pathway was chosen because of biological interest to study authors and high overlap of panel genes with genes represented on the Human Focus array. RNA prepared from both frozen and unfrozen PaxGene tubes was analyzed for each donor. All of the genes measured in the RT-PCR assay were scored as present in the microarray profiles produced with targets from one or more of the methods examined in this study.Comparison of the RT-PCR data with array profiles for 75 matched genes demonstrated that Method 2 (Globin PNAs_Affymetrix) produced expression data that most closely correlates with the RT-PCR results, with an average Pearson correlation of 0.77 , Method 3 (GLOBINclear_Affymetrix) and Method 4 (no depletion_NuGEN) is illustrated in the Venn diagram in Figure Overall, the vast majority of gene transcripts detected with each method were also detected by the other three methods. The highest expression concordance was observed between Methods 1 (no depletion_Affymetrix) and 2 (Globin PNAs_Affymetrix). The Globin PNAs_Affymetrix method and the no depletion_NuGEN method each detect a significant number of gene transcripts  not detected by any of the other methods tested in this study. Four of the transcripts detected with only one labeling method (3 in no depletion_NuGEN and 1 one in Globin PNAs_Affymetrix) correspond to genes whose transcription was confirmed in the quantitative RT-PCR analysis. This observation suggests that both the no depletion_NuGEN and the Globin PNAs_Affymetrix method produce microarray data that accurately measures gene expression not detected by the other methods.Microarry performance of RNA extracted from blood collected in PAXGene tubes immediately following a 2 hr room temperature incubation or after a 2 hr incubation and freezing at -80°C was compared. Correlation between array data from fresh and frozen PAXGene preparations from each donor was high across all samples prepared with Methods 1 (no depletion_Affymetrix), 2 (Globin PNAs_Affymetrix) or 4 (no depletion_NuGEN), with the exception of donor A using Method 1, where one of the microarray assays underperformed Table . Method Our results indicate that freezing blood collected in PAXGene tubes prior to RNA extraction has little impact on the RNA profiles measured in the microarray assay. This observation is consistent with the report of Ovstebo et al in whichGiven the overall similarity in fresh and frozen sample pairs as measured in both the microarray and RT-PCR analyses, we elected to use the fresh/frozen pairs for each donor to examine array assay reproducibility among the different assay methods. Method 2 (Globin PNAs_Affymetrix) showed the highest reproducibility in paired donor samples (average r = 0.99). Reproducibility was also high with Methods 1 (no depletion_Affymetrix) and 4 (no depletion_NuGEN) (average r = 0.98), but slightly reduced with Method 3 (GLOBINclear_Affymetrix) (average r = 0.97).Removing globin transcripts from cRNA hybridization targets prepared from whole blood samples results in increased sensitivity in microarray expression profiles. Sensitivity is also increased when cDNA hybridization targets prepared from undepleted RNA are used instead of cRNA targets. All of the methods tested in this study generate highly reproducible data, except when total RNA is pre-treated to remove globin transcripts (GLOBINclear_Affymetrix method). Comparison of microarray gene level signals with real-time PCR data suggests that the Globin PNAs_Affymetrix method produces the most accurate microarray results, although, all methods produce data that correlate well with the RT-PCR measurements. Performance results for each method are summarized in Table Before selecting a protocol for use in an expression profiling study of whole blood, however, several factors must be considered.This method required relatively low RNA amounts and did not require any additional processing steps. Overall, correlation to real-time PCR data was second only to the Globin PNAs_Affymetrix method (Method 2).It has been well reported that performing microarray profiling of RNA extracted from whole blood using this method without modification results in less sensitive data with high levels of sample-to-sample variability ,4,5,11. Sensitivity was highest with this method. Additionally, this method also produced the lowest relative α- and β-globin intensities when the signal was normalized to a housekeeping control gene, further confirming the effectiveness of the reduction. Among all protocols tested in this study, the globin PNAs method demonstrated the highest reproducibility when correlation values were measured between donor replicates. Finally, microarray RNA expression values produced with the PNA method showed the highest correlation to RT-PCR measurements of a selected gene panel in the same RNA samples compared to all other methods.While targets prepared following the PNA method produced the best overall results in the microarray assay, before selecting this protocol to profile expression patterns of whole blood, it is important to note that, among the protocols examined in this paper, the PNA method is one of the more time-consuming and requires the highest amount of total RNA. Although recent studies in our laboratory show that the PNA method can be successfully applied with RNA inputs as low as 2 μg (unpublished data), the relatively high RNA amounts required by this method could be a confounding factor if the amount of RNA available for expression profiling is low. Furthermore, the PNA sequences supported by Affymetrix for use in this protocol are designed to bind to human α- and β-globin transcripts. Consequently if blood RNA extracted from non-human species is to be analyzed, it would be necessary to design and optimize peptide nucleic acid sequences for the species of interest. Also note that PNA sequences recommended by Affymetrix are no longer sold by Applied Biosystems. The product is now available through Panagene .The third method examined in this study uses a magnetic-bead based hybridization system to extract globin RNA from whole blood total RNA prior to amplification. Beads can be purchased to bind to species-specific globin transcripts (human and rodent), allowing for increased flexibility. Sensitivity is almost as high as that achieved with the PNA method. Globin probe set intensities, compared to the housekeeping controls, were reduced but not to the level observed when using the PNA method.Reproducibility of GLOBINclear targets is lowest of the methods examined. Furthermore, we noted an increase in the ratio of 3' versus 5' housekeeping control probe set intensity, suggesting a slight decrease in the quality of the target prepared using this method. This trend can likely be explained by the increased sample handling required to process the RNA during the bead hybridization and magnetic purification steps. Input requirements are also relatively high for this method, again presenting challenges when sample amounts are limiting. In addition, comparison of microarray expression values generated with this method showed the lowest correlation with the RT-PCR results on the NF-κB pathway genes.Among the four protocols tested, this labeling protocol requires the lowest amount of total RNA (5–100 ng) and does not have a separate globin RNA reduction step. Instead, the hybridization kinetics of the cDNA target appear to be less affected than cRNA targets by the abundant globin RNA present in whole blood extractions. The α- and β-globin RNA intensity measurements in these targets, compared to a housekeeping control, ranked second to the PNA method indicating a reduction in globin transcript signal compared to the standard labeling method with no globin RNA depletion. In addition, because a separate species-specific globin reduction step is not required, the Ovation method is the most flexible cross-species protocol of the alternative methods tested.Microarray expression values generated with this method do not correlate as well with RT-PCR measurements as the Globin PNAs or standard Affymetrix method. In addition, detection sensitivity of targets prepared following the NuGEN Ovation Biotin RNA Amplification and Labeling System v1 protocol is the lowest of the alternative methods tested. However, since we completed this study, NuGEN has released a modified protocol (NuGEN Ovation Whole Blood Labeling System with FLv2) specifically aimed at minimizing the impact of globin transcripts when expression profiling RNA extracted from whole blood. Results from a blood profiling study that used targets generated with the Whole Blood Ovation System show significantly increased transcript detection rates compared to version 1 of this kit . It is likely that the Ovation Whole Blood FLv2 system retains the high reproducibility and implementation advantages of the version 1 system while producing microarray data with detection sensitivity similar to the globin PNAs method. In a study of small sample protocols for microarray analysis using non-blood tissues, another aspect of sensitivity, detection of differential expression between samples, was shown to be highest with the Ovation method compared to other small sample protocols and the standard Affymetrix one-cycle labeling method .Our results and observations can be summarized in five main points: (1) protocols designed to minimize the impact of globin transcripts can significantly improve the performance of whole blood-extracted RNA in an expression profiling microarray study; (2) methods to reduce globin RNA prior to target synthesis may require amounts of RNA that are not feasible in some studies; (3) for nonhuman studies, the requirement for species-specific oligonucleotides in globin RNA depletion protocols may limit use; (4) target preparation protocols can be a major source of variability; (5) if precautions are taken to properly stabilize extracted blood, the impact of freezing specimens prior to RNA extraction is minimal.Unfractionated whole blood collection and RNA isolation were performed using the PAXgene Blood RNA Isolation System . PaxGene tubes contain a proprietary reagent to reduce intracellular RNA degradation and minimize induction of gene expression . With thAmplifications and labeling were performed for each donor using one of the following four methods with RNA extracted from fresh and frozen blood samples: Method 1 – Affymetrix one-cycle cDNA synthesis/Affymetrix in vitro-transcription (IVT) on whole blood RNA (no depletion_Affymetrix); Method 2 – Affymetrix one-cycle cDNA/Affymetrix IVT with globin peptide nucleic acids (PNAs) inclusion during cDNA synthesis (Globin PNAs_Affymetrix); and Method 3-RNA pre-treated with Ambion GLOBINclear beads to reduce globin transcripts prior to labeling with the Affymetrix one-cycle cDNA synthesis/Affymetrix IVT (GLOBINclear_ Affymetrix). In the fourth method tested, cDNA target was synthesized from whole blood RNA using the NuGEN Ovation Biotin RNA Amplification and Labeling System v1 (no depletion_NuGEN). Total RNA input amounts were based on manufacturer's recommendations for each method. with minor modification. Using 1 μg of total RNA as input, messenger RNA was amplified and labeled in two steps. In the first step, mRNA was converted to double-stranded cDNA and purified by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. In the second step, amplified and biotinylated cRNA (the target) was produced by in vitro transcription (IVT). Unincorporated nucleotides were removed using the RNeasy Mini kit (Qiagen) followed by an ethanol precipitation of the labeled target.Method 1 employed the standard laboratory method for amplification and labeling of total RNA. Target synthesis was performed following the Affymetrix GeneChip Expression Analysis Technical Manual, rev. 5 . Globin reduction PNA master mix was prepared by adding equal amounts of the globin PNA stocks. Five μg of total RNA were incubated with oligo-dT primer and 1 μl PNA master mix for 10 minutes at 70°C prior to first strand cDNA synthesis; all subsequent steps for target amplification and labeling were as described for Method 1.The standard Affymetrix amplification and labeling was performed with the following modification: four peptide nucleic acid (PNA) oligonucleotides  designed to anneal to human α- and β-globin mRNA were added to the total RNA preparations immediately prior to initiation of the target synthesis step in order to bind globin mRNA and block cDNA synthesis. The PNA oligonucleotide stocks were prepared on day of use at concentrations recommended by Affymetrix Globin mRNA depletion was performed in three steps using the GLOBINclear Kit . First, species-specific biotinylated oligonucleotides complementary to human α- and β-globin mRNA were hybridized with total RNA (5 μg). Second, magnetic streptavidin-coated beads were added to bind the biotinylated-oligonucleotide:globin RNA complexes. Finally, a magnet was used to remove beads with the selectively bound α- and β-globin transcripts. After globin mRNA depletion, RNA quantity was determined by UV absorbance and RNA integrity was assessed using RNA Nano LabChips as described above. RNA recovery following GLOBINclear treatment ranged from 2–5 μg. Target labeling and amplification steps were then performed as described for Method 1.Ovation v1 labeling and amplification reagents were obtained from NuGEN Technologies, Inc  and biotinylated cDNA targets were prepared according to manufacturer's instructions. Double-stranded cDNA was synthesized from 30 ng total RNA, followed by a linear isothermal amplification (SPIA Amplification™) to produce single-stranded cDNA (target). A proprietary fragmentation and direct labeling process attached biotin to the amplified target. Target purifications were performed using DNA Clean and Concentrator – 25  and the DyeEx 2.0 Spin Kit (Qiagen).Targets labeled with Affymetrix one-cycle cDNA synthesis/Affymetrix IVT  were chemically fragmented as per Affymetrix's recommendations and combined with Affymetrix biotinylated hybridization controls  in hybridization buffer. Hybridizations were performed for 16 hours at 45°C after addition of 6.5 μg of target to the GeneChip Human Genome Focus array (Affymetrix), containing 8700 probe sets. Post-hybridization array processing was performed according to manufacturer's recommendations. The distribution of fluorescent material on the processed array was determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade. Image inspection was performed manually immediately following each scan.For targets labeled with NuGEN Ovation system (Method 4), the hybridization and processing outlined above was performed with the following modification: following NuGEN's recommendations, 1.3 μg of cDNA target were mixed with Affymetrix hybridization controls in hybridization buffer and hybridized with the Human Genome Focus array for 18 hours at 45°C.The array image scan was processed with Affymetrix Microarray Suite, version 5.0, (MAS 5.0) software. GeneChip expression Arrays contain control probe sets for both spiked and endogenous RNA transcripts . Following absolute analysis of the array pattern with MAS, six values were examined to assess overall assay performance: background, noise, average Signal, % Present, ratio of Signal values for probe sets representing the 5' and 3' ends of actin and GAPDH transcripts, and total Signal for probe sets for BioB, BioC, BioD and CreX. Assays demonstrating poor or marginal performance were flagged.2 PCR Array First Strand Kit  following the manufacturer's instructions. The product was combined with SuperArray PCR SYBR Green master mix and pipetted into all wells of a 96-well RT2Profiler™ PCR Array for the human NF-κB pathway (SuperArray APHS-025A) per the manufacturer's instructions. After 10 min at 95°C to activate the HotStart DNA polymerase, the samples were subjected to 40 cycles of 15 seconds 95°C, 35 seconds at 55°C during which time the SYBR Green fluorescence was recorded, and 30 seconds at 72°C with a Chromo4 thermocycler . Cycle threshold (Ct) values were obtained for each sample and were normalized for each plate by subtracting the mean Ct value for five housekeeping genes  to yield ΔCt values.Quantitative RT-PCR was performed on RNA from donors C and D. cDNA was prepared from selected RNA samples (1 μg each) with a RTFor comparison of RT-PCR results with the microarray data, 75 genes from the SuperArray NF-κB pathway panel were matched to probe sets on the Affymetrix Human Genome Focus array.Low-level analysis  of microarray data was performed with both Microarray Suite 5.0 (MAS 5.0) ,18 and RExploratory data analysis was performed on probe cell intensities and summarized probe set signal values. Box plots of signal distributions were generated with Expression Console (Affymetrix). Scatter plots and correlation measurements were performed using Statistica 6.0 . Venn diagrams were generated using VENNY, an interactive tool for comparing lists with Venn diagrams cDNA: complementary deoxyribonucleic acid; Ct: cycle threshold; CTP: cytidine triphosphate; DNA: deoxyribonucleic acid; GAPDH: glyceraldehydes-3-phosphate dehydrogenase; IVT: in vitro-transcription; MAS 5.0: Microarray Suite version 5.0; mRNA: messenger RNA; NF-κB: nuclear factor-kappa B; NK: natural killer; OHSU: Oregon Health and Science University; PCR: polymerase chain reaction; PNA: peptide nucleic acid; r: correlation coefficient; RIN: RNA integrity number; RMA: robust multi-array average; RNA: ribonucleic acid; RT-PCR: reverse transcriptase-polymerase chain reaction; UV: ultraviolet.KV performed the microarray assays and assisted with experimental design, data analysis and preparation of the manuscript; RS assisted with protocol development and microarray assays; AC assisted with protocol development and performed RNA isolations and globin RNA depletion; TJ performed data analysis; SP participated in the design of the study, data analysis and manuscript preparation; DC performed data analysis and participated in critical review of the manuscript; JS and JR participated in the design of the study and critical review of results and the manuscript; CAH conceived of the study, guided its design and coordination, performed data analysis and drafted the manuscript. All authors read and approved the final manuscript.


Objective. To estimate the costs and outcomes of rescreening for group B streptococci (GBS) compared to universal treatment of term women with history of GBS colonization in a previous pregnancy. Study Design. A decision analysis model was used to compare costs and outcomes. Total cost included the costs of screening, intrapartum antibiotic prophylaxis (IAP), treatment for maternal anaphylaxis and death, evaluation of well infants whose mothers received IAP, and total costs for treatment of term neonatal early onset GBS sepsis. Results. When compared to screening and treating, universal treatment results in more women treated per GBS case prevented (155 versus 67) and prevents more cases of early onset GBS (1732 versus 1700) and neonatal deaths (52 versus 51) at a lower cost per case prevented ($8 805 versus $12 710). Conclusion. Universal treatment of term pregnancies with a history of previous GBS colonization is more cost-effective than the strategy of screening and treating based on positive culture results. Intrapartum infection with group B streptococci (GBS) may lead to untoward neonatal sequelae such as pneumonia, septicemia, and meningitis . SeveralRecent longitudinal studies have demonstrated that women colonized with GBS during pregnancy are at an increased risk of colonization in a subsequent pregnancy , 4. RepoWe constructed a decision-analysis model to evaluate the cost-effectiveness of screening-directed versus universal treatment for the prevention of early onset neonatal GBS sepsis in women colonized with GBS in a previous pregnancy. The pathways of events are shown in We compared two strategies described in the literature , 5. WithThree percent to eight percent of individuals in the population report an allergy to penicillin , 10. TheSince publication of the 2002 CDC guidelines, the largest study looking at intrapartum GBS prophylaxis in patients reporting a penicillin allergy, noted of the women who received appropriate antibiotic prophylaxis, 38% were considered low risk for anaphylaxis and given a cephalosporin . Of the streptococcus,” “streptococcus agalactiae,” “pregnancy,” “early onset neonatal group B streptococcal infection,” “maternal colonization with streptococcus B,” and combinations thereof, and the databases queried (January 1966 to January 2009) include MEDLINE, PubMed, Scopus, Knowledge Finder, Cochrane database, and the Centers for Disease Control and Prevention guidelines. The probabilities included in the decision tree are shown in The primary measures of effectiveness of each strategy were (1) the number of early onset neonatal GBS sepsis cases prevented, (2) the total cost of intervention, and (3) overall cost-effectiveness. The baseline efficacy of intrapartum antibiotic prophylaxis, probabilities for each strategy, and outcomes were derived from a systematic review of the English literature, supplemented by bibliographic review. We examined reference lists from studies identified as potentially relevant. The following search terms were used: “group B streptococci,” “group B streptococcus in pregnancy [A societal perspective for this cost analysis was performed. In 2007, there were 4 315 000 live births in the United States of which 2 597 630 (60.2%) were a second child or greater , 27. Of regnancy , 19. We It has been estimated that a colonized gravida who did not receive intrapartum antibiotics would have a probability of 0.016 delivering a neonate with early onset neonatal GBS . A colonEstimates for direct medical costs  were obtPast cost analyses have included expenses associated with cases of neonatal sepsis, but they have not incorporated costs for the pediatric care received by newborns as a result of GBS prevention strategies , 18. It In 2002, the CDC proposed revised guidelines for the management of a newborn whose mother received intrapartum antibiotics for prevention of early onset GBS disease . These gIn the baseline analysis, pathway probabilities were used to calculate costs, number of cases of early onset GBS sepsis prevented, and number of neonatal deaths and morbidity prevented by each strategy. A cost-utility analysis was also performed and reported as cost-effectiveness ratios. The incremental cost-effectiveness ratio represents supplementary costs of changing from a paradigm of using screening with treatment of positive results versus treatment of all previously GBS positive women. The number of life-years was set at 80.4 years or 31.1 years with 3% discounting . Term GBTo calculate the cost per case prevented, we divided the intervention costs by the number of cases of neonatal GBS disease prevented. The benefit-cost ratio was calculated by dividing the monetary savings from preventing disease by the intervention costs. The total costs expected for each strategy were calculated by subtracting the cost for treatment of neonatal GBS disease from the intervention costs of neonatal GBS disease. To determine the expected net benefits, we compared total costs with the costs of treating cases of neonatal GBS disease in the cohort of women if no prevention strategy is used. Data were managed using Excel spreadsheet software . savings of $209 988 (range $113 920 to $329 258) per quality-adjusted life-year gained when compared to treatment directed by screening. In the absence of screening or risk-based strategies, 2003 of the 300 000 newborns were estimated to have early onset GBS disease, of which 60 (3%) would have died , 42, 47,savings per quality-adjusted life-year gained of $154 063 (range $84 804 to $226 553) when compared to treatment directed by screening. The cost for each additional case of early onset GBS sepsis prevented using intrapartum antibiotic prophylaxis directly was $10 226 (range $6371 to $15 708), compared with $13354 (range $8108 to $20 322) for women screened then treated with an annual cost savings for this cohort of $4.96 million (range $2.73 to $7.29 million).The cost for each case of early onset GBS sepsis prevented using intrapartum antibiotic prophylaxis directly was $8805 (range $5 639 to $12 998), compared with $12 710 (range $7773 to $19 105) for women screened then treated. The annual cost savings for the cohort of women in 2007 with direct intrapartum treatment in the U.S. would be $6.35 million (range $3.45 to $9.97 million). When sensitivity analyses were performed for the incidence of recurrent GBS colonization in a subsequent pregnancy, universal treatment remained cost-effective to an incidence as low as 10%. A sensitivity analysis was performed with identical baseline assumptions but presumed that all infants receiving <4 hours of intrapartum antibiotics would have a complete blood count and blood culture, and all penicillin-allergic patients at high risk for anaphylaxis would have GBS screening with GBS isolates being tested for resistance to clindamycin and erythromycin . Universal treatment with intrapartum antibiotics resulted in an incremental cost We considered the results of altering the cost of each variable in This analysis illustrates that universal treatment in subsequent pregnancies of women with a history of GBS colonization is a more cost-effective strategy for reducing neonatal early onset GBS disease. Administration of intrapartum antibiotics to women with history of colonization in past pregnancies could prevent substantial disease and save millions of dollars annually compared to the current screening-directed treatment approach. Cost-effectiveness analysis has been advocated as a basic tool in the evaluation of health care practices . When unEscherichia coli has been raised [Specific concerns about universal antibiotic prophylaxis hinge on the possibility of adverse antibiotic reactions and the development of antimicrobial resistance . Neverthn raised . With mon raised , emerginn raised . Despiten raised . Exposurn raised . Howevern raised . This cuOur study has several limitations. The current analysis focuses on the prevention of early onset GBS neonatal sepsis. No costs or outcomes were included to treat maternal pregnancy-associated disease or for infants who developed late-onset GBS disease. However, the incidence of late-onset GBS neonatal sepsis has not been shown to be impacted by intrapartum antibiotic prophylaxis .The presOur analysis suggests that in this well defined population, universal intrapartum antibiotic prophylaxis in future pregnancies of women with GBS colonization in a prior pregnancy could prevent disease and represent a cost-savings compared to current screening strategies for prevention of early onset GBS infection.