However, GM-CSF priming shifted the release of lipid mediators by BMDMs, resulting in a significant decrease of PGE2 production in response to the same stimuli. The decrease of PGE2 production from primed BMDMs was accompanied by a decrease in PGE-synthase mRNA expression and an increase in TNF-α and nitric oxide (NO) production. Moreover, some GM-CSF effects were potentiated by the addition of IFN-γ. Using a variety of TLR2 ligands, we established that PGE2 release by GM-CSF-primed BMDMs was dependent on TLR2 co-receptors , CD14, MyD88 and the nuclear translocation of NFκB but was not dependent on peroxisome proliferator-activated receptor-γ (PPAR-γ) activation. Indeed, GM-CSF priming enhanced TLR2, TLR4 and MyD88 mRNA expression and phospho-IκBα formation. These findings demonstrate that GM-CSF drives BMDMs to present a profile relevant to the host during infections.In response to pathogen recognition by Toll-like receptors (TLRs) on their cell surface, macrophages release lipid mediators and cytokines that are widely distributed throughout the body and play essential roles in host responses. Granulocyte macrophage colony-stimulating factor (GM-CSF) is important for the immune response during infections to improve the clearance of microorganisms. In this study, we examined the release of mediators in response to TLR2 ligands by bone marrow-derived macrophages (BMDMs) primed with GM-CSF. We demonstrated that when stimulated with TLR2 ligands, non-primed BMDMs preferentially produced PGE The increased release of LT results from GM-CSF priming through increase of arachidonic acid (AA) release, enhancement of 5-Lipoxygenase (5-LO) activation The innate immune response is initiated by microbe recognition, phagocytosis and activation of numerous cells; macrophages seem to be at the focal point of this scenario. Through the production of inflammatory mediators macrophages exert a broad variety of functions GM-CSF is an immune regulatory cytokine produced by macrophages, endothelial cells, alveolar epithelial cells and T cells in response to pro-inflammatory cytokines, activating a variety of cells, such as dendritic cells, neutrophils and macrophages 3CSK4), mycoplasma diacylated lipoprotein , lipoarabinomannan (AraLAM), zymosan and protozoan GPI anchors Microbes and their components activate innate immune responses through interaction of their PAMPs with TLRs. TLR2 recognizes bacterial components, such as peptidoglycan (PGN), bacterial triacylated lipoprotein , are widely recognized for their pro-inflammatory activities 2 and LTB4 by GM-CSF primed BMDM and the correlation with other pro-inflammatory mediators, such as TNF-α and Nitric Oxide (NO).However, in contrast to the wealth of data exploring the TLR-dependent cytokine expression and activation of oxidant production, the mechanisms governing the TLR-mediated activation of lipid mediators production in primary macrophages is unclear. The production of eicosanoids and the macrophage differentiation/maturation process play an important role in innate immunity. Here, we address the effect of a set of TLR2-ligands on the production of PGE2/O2 excess atmosphere, and all efforts were made to minimize suffering.Six- to 8-week old C57Bl/6 mice deficient for TLR2 6/ml) for 7 days (37°C and 5% CO2) in Dulbecco’s minimal essential medium (DMEM) supplemented with 10 mM L-glutamine (Sigma), 100 U/mL penicillin (Invitrogen) and 100 U/mL streptomycin (Invitrogen), 20% horse serum and 30% L929 cell-conditioned medium as a source of macrophage colony-stimulating factor (M-CSF). After suspending in cold phosphate-buffered saline (PBS), washing, and re-culturing for 3 days in fresh medium, the cell preparation contained a homogenous population of macrophages (97–98% CD11b+F4/80+). The bone marrow derived macrophages (BMDM) were plated in 48-well microculture plates (Nunc) at a density of 5×105 cells/well in DMEM supplemented with 10 mM L-glutamine, 100 U/mL penicillin and 100 U/mL streptomycin and10% fetal bovine serum (FBS) and cultured at 37°C in a humidified atmosphere of 5% CO2 for 18 h. BMDM were primed (or not) with 10% J558L cell-conditioned medium as a source of granulocyte macrophage colony-stimulating factor (GM-CSF)  for 24 h before stimulation or in a combination with interferon-γ (IFN-γ) (500 UI/ml). Subsequently, BMDM were stimulated with increased concentrations of TLR-ligands as AraLAM-MS (lipoarabinomannan from Mycobacterium smegmatis) (InvivoGen) (5–5000 ng/mL), LM-MS (lipomannan from M. smegmatis) (InvivoGen) (5–5000 ng/mL), Malp2 -cysteine-GNNDESNISFKEK)  , synthetic bacterial lipopeptide Pam3-CSK4 ((S)-2, 3-bis-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH)tri-hydrochloride, EMC Microcollections) (5–5000 ng/ml) or LPS  (50–1000 ng/ml) diluted in DMEM for 24 h at 37°C in a CO2 atmosphere. To determine the time of stimulation, we performed a time-response titration experiment and chose 24 h of stimulation based on a maximal production of TNF-α and NO (data not shown). Medium (DMEM) was used as negative control. Also, to neutralize potential endotoxin presence in the stimulus solutions, they were pretreated with Polymyxin-B (Sigma) at 10 µg/mL for 20 min and then added to the cell culture. In other experiments, to investigate the participation of peroxisome proliferator-activated receptor-γ PPAR-γ, BMDM were treated for 30 min before stimulation with an antagonist of PPAR-γ (GW9662) (Sigma) at 1 µM at 37°C and stimulated with the TLR-ligands above or infected with BCG  or heat-killed M. tuberculosis H37Rv (HK H37)  during 24 h. Vehicle (DMSO 0.01% in DMEM) was used as control. The culture supernatants were harvested and analyzed immediately or stored at 20°C until further use. The absence of cytotoxicity of the stimuli was controlled using the incorporation of MTT -2,5-diphenyltetrazolium bromide) (Sigma).Murine bone marrow cells were isolated from femurs and cultivated . The detection limit for both cytokines was 7 pg/mL.p-aminobenzene sulfonamide, 1% N-1-napthyl ethylenediamide) as previously described Nitrite concentrations in the cell supernatants were measured using the Griess reaction .LTB2 Profiler PCR Array kit (Qiagen). Total RNA from BMDM cells was isolated using the RNeasy Mini kit (Qiagen), and the reverse transcription of 3 µg of RNA was performed using the RT2 HT First Strand kit (Qiagen). Each cDNA sample was processed in 96-well plates containing sets of pre-defined genes , including Actb and Gapdh as endogenous internal controls in all sets. Amplification was performed duplicates in an Eppendorf Mastercycler ep realplex 4 (Eppendorf). The 2–ΔΔCt method was used in the analysis of the RT-PCR data.Expression of mRNA in BMDM cells after treatment with GM-CSF for 4, 8 and 18 h was evaluated using a custom RT6 cells were lysed in cell lysis buffer . Cell lysates were analyzed, and proteins were semi-quantified using the PathScan® total-NFκBp65, phospho-NFκBp65 (Ser536) and total-IκBα, phospho-IκBα (Ser32) sandwich ELISA kits, according to the manufacturer’s instructions . Brieﬂy, the lysates were diluted (10 µg/mL), and 100 µL of lysate was added to wells pre-coated with primary antibody. The plate was incubated overnight at 4°C and then washed four times. After washing the plate, the detection antibody was added to the wells, and the plate was incubated for 1 h at 37°C. After a second washing process, horseradish peroxidase (HRP)-linked secondary antibody was added, and the plate was incubated for 30 min at 37°C. Substrate was added, and the plate was incubated for 30 min at room temperature. Finally, the reaction was stopped, and the samples were read at an absorbance of 450 ηm. The results were expressed as the percentage of relative expression (absorbance) of control versus that produced by primed BMDMs stimulated with LM.Following priming and LM (5000 ng/mL) stimulation, 3×102. Cells were primed with GM-CSF for 24 h and subsequently stimulated with TLR2-ligands as described above for 2 h at 37°C and 5% CO2. Next, the monolayer was washed by gentle agitation with PBS, fixed in 4% paraformaldehyde, permeabilized with Triton X-100 (0.5%) and used for immunolocalization of NFκB as described BMDM were adhered to cover slides and cultured for 18 h at 37°C in a humidified atmosphere of 5% COp<0.05 were considered statistically significant.Results were expressed as mean ± SEM. Statistical significance was determined with Graph Pad Prism software  by unpaired t-test analysis of variance. Values of 2 and LTB4 from macrophage after priming with GM-CSF. Cells were primed, or not primed, with GM-CSF and then stimulated with the TLR-agonists AraLAM, LM, Malp2, Pam3-CSK4 or LPS TLR4 ligand as a positive control. As shown in 3-CSK4 and LPS produced high levels of PGE2 as compared with the negative control; however, the response to AraLAM stimulation was slightly weaker. GM-CSF priming resulted in a significant decrease in PGE2 production by stimulated macrophages. The kinetics of stimulation was similar to all ligands, with the maximum response obtained after 24 h of incubation (data not shown). Macrophage priming with GM-CSF resulted in a small increase in LTB4 release as compared with unprimed cells (4 production (4 release after TLR-agonist stimulation in the same conditions (data not shown). Thus, GM-CSF priming decreased PGE2 release by TLR-activated macrophages.We characterized the capacity of different TLR2 ligands derived from bacteria to induce the release of PGEed cells . Howeveroduction . Similar3-CSK4 . Therefore, in all subsequent experiments, TNF-α and NO was measured at this time point. We also evaluated the concentration-dependent effect of TLR-ligands and observed that Malp2 , LM Fig and Pam33-CSK4  induced 3-CSK4 . GM-CSF 3-CSK4 .3-CSK4  analysis from these BMDMs revealed that GM-CSF priming increased COX-2 mRNA expression (threefold) after 4 h of incubation  components.To determine the effects of GM-CSF on steady-state mRNA encoding for 5-LO, FLAP, COX-2, PGEcubation , and thicubation  and 18 hcubation  of incubrophages . Howeverrophages . As showrophages , which wrophages . Thus, G3-CSK4 and LPS resulted in IL-10 secretion. However, AraLAM and Malp2 had a very small effect on IL-10 induction (data not shown). Subsequently, the impact of TLRs and co-receptors on IL-10 production after GM-CSF priming was examined. As shown in −/−, CD14−/− and MyD88−/− cells. Indeed, TLR2−/−, TLR1−/− and MyD88−/− cells were largely unresponsive to Pam3-CSK4 in terms of IL-10 release, and TLR6−/− macrophages were partially responsive  NFκBp65, total IκBα, and phospho-IκBα in BMDM lysate after priming the cells with GM-CSF, IFN-γ or GM-CSF plus IFN-γ in the presence or absence of LM stimulus (2 h) using PathScan® ELISA. The results showed that all priming had no effects on total and phospho-NFκBp65 expression  or total2 production by TLR-stimulated macrophages primed with GM-CSF using a specific PPAR-γ antagonist (GW9662). Primed macrophages were pre-treated with GW9662 and then stimulated with AraLAM, LM, Malp2, Pam3-CSK4, and LPS, using heat-killed M. tuberculosis H37Rv (HK H37) and M. bovis BCG as positive controls. Pre-treatment with the PPAR-γ antagonist GW9662 had no effect on the production of TNF-α  or the TLR4 ligand LPS. Although this reduction was accompanied by an increase in COX-2 mRNA expression, PGE-synthase mRNA expression was downregulated at the time of the analyses, and this downregulation may have led to the decrease in PGE2 production. Several reports demonstrated that PGE2 contributes to immune suppression 2 secreted from activated macrophages in response to pro-inflammatory stimuli acts on macrophages themselves and exhibits an inhibitory function in a negative feedback loop 4 and cysteinyl leukotrienes (Cys-LT). Here, although the effect of GM-CSF priming on BMDM release of LTB4 was demonstrated and 5-LO mRNA expression was upregulated in BMDMs after 8 h of GM-CSF priming, no further increase in LTB4 was observed after stimulation with TLR2 ligands or LPS. These results suggest that there is additional regulation at other levels in the 5-LO pathway and may reflect the activation/deactivation of the cell signaling response to a stimulatory assay As an activator of cell functions, GM-CSF has been shown to upregulate LTs release by alveolar macrophages (AM) in rats 2 release. NO has been reported to suppress COX activity and PGE2 production 4 and the decreased PGE2 production by GM-CSF-primed BMDMs after TLR-ligand stimulation.Priming with GM-CSF was also critical for the formation of NO by BMDMs. In GM-CSF-primed macrophages, the level of NO production was strongly increased with all the stimuli evaluated. The increase in NO production may contribute to the decrease in PGE2, GM-CSF-primed BMDMs subsequently stimulated with TLR ligands produced higher concentrations of TNF-α, as compared with unprimed cells. These data confirm the results of a study demonstrating that the main priming factor for TNF-α release by human PMNs was GM-CSF 3-CSK4 or LPS was reduced in GM-CSF-primed macrophages (data not shown). Our results demonstrate that GM-CSF priming enhances phospho-IκBα expression after TLR2 agonist stimulation and that IFN-γ potentiates this phenomenon. Increased IκBα phosphorylation and elevated levels of nuclear NFκB components p50 and p65 by GM-CSF-priming was also demonstrated in monocytes Different from that observed with PGE2 and IL-10 production by BMDMs primed with the combination of GM-CSF and IFN-γ, in comparison to BMDMs primed with GM-CSF alone. Indeed, GM-CSF and IFN-γ have immunostimulatory properties; they not only prime resting monocytes but also restore monocytic functions after LPS tolerization We addressed the effect of GM-CSF priming in combination with IFN-γ in macrophage stimulation. IFN-γ is a major activation signal for macrophages. The priming or concomitant exposure of macrophages to IFN-γ dramatically increases their responsiveness to microbial products, such as LPS, in terms of the production of NO, cytokines and co-stimulatory molecules M. smegmatis, and we described the pattern of recognition of GM-CSF-primed BMDMs by TLR2, TLR1, TLR6, TLR4 and CD14 agonists. The release of TNF-α from AraLAM-stimulated cells in culture is mediated through TLR2 and CD14. However, NO production and PGE2 production are mediated by only TLR2. As we demonstrated here, LM stimulation of PGE2 formation occurs by activating a complex cluster of receptors mediated through CD14, TLR2 and TLR1, which may function as heterodimers. The lipopeptides induce signaling in the cells of the immune system through TLR2–TLR1 or TLR2–TLR6 heterodimers 2 release is strongly dependent on TLR2–TLR6 and the adaptor protein MyD88 in GM-CSF-primed macrophages, while Pam3-CSK4 stimulation of PGE2 release depends mostly on the TLR2 and MyD88 pathway.The concept of TLR-ligand interactions suggests that individual TLR proteins recognize a set of microbial products 2 production, thereby affecting the host response to infection 2 release by macrophages. However, PPAR-γ inhibition by GW9662 treatment did not affect TNF-α and PGE2 release in response to TLR agonists, heat-killed M. tuberculosis, or BCG infection.NFκB plays a central role in the regulation of many of the genes responsible for the generation of inflammatory mediators. 2, which was more prominent than that of LTB4, was strongly decreased by GM-CSF priming, indicating a compensatory function for these cells in a high inflammatory environment. Our data suggest that the GM-CSF produced when a host is infected by a pathogen may be responsible for the macrophages exhibiting a pro-inflammatory profile. Additionally, GM-CSF drives host immune defense mechanisms, which favor phagocytosis and killing, as a result of a shift in the production of lipid mediators from PGE2 to LTB4.Together, our results demonstrate that GM-CSF priming of BMDMs enhances the capacity to drive the pro-inflammatory response against bacterial components by increasing TNF-α and NO production and by altering lipid mediator release. In particular, the production of PGE


We showed that alr is expressed in liver throughout hepatogenesis. Knockdown of alr through morpholino antisense oligonucleotide (MO) leads to suppression of liver outgrowth while overexpression of alr promotes liver growth. The small-liver phenotype in alr morphants results from a reduction of hepatocyte proliferation without affecting apoptosis. When expressed in cultured cells, zebrafish Alr exists as dimer and is localized in mitochondria as well as cytosol but not in nucleus or secreted outside of the cell. Similar to mammalian ALR, zebrafish Alr is a flavin-linked sulfhydryl oxidase and mutation of the conserved cysteine in the CxxC motif abolishes its enzymatic activity. Interestingly, overexpression of either wild type Alr or enzyme-inactive AlrC131S mutant promoted liver growth and rescued the liver growth defect of alr morphants. Nevertheless, alrC131S is less efficacious in both functions. Meantime, high doses of alr MOs lead to widespread developmental defects and early embryonic death in an alr sequence-dependent manner. These results suggest that alr promotes zebrafish liver outgrowth using mechanisms that are dependent as well as independent of its sulfhydryl oxidase activity. This is the first demonstration of a developmental role of alr in vertebrate. It exemplifies that a low-level sulfhydryl oxidase activity of Alr is essential for embryonic development and cellular survival. The dose-dependent and partial suppression of alr expression through MO-mediated knockdown allows the identification of its late developmental role in vertebrate liver organogenesis.Augmenter of Liver Regeneration (ALR) is a sulfhydryl oxidase carrying out fundamental functions facilitating protein disulfide bond formation. In mammals, it also functions as a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage or partial hepatectomy. Whether ALR also plays a role during vertebrate hepatogenesis is unknown. In this work, we investigated the function of  ALR was first purified and cloned from rat liver as a secreted protein of 125 amino acids In mammals, ALR has an additional function, i.e. stimulating hepatocyte proliferation and liver regeneration as a cytokine. In adult rat liver, ALR is believed to be predominantly and constitutively produced and stored in hepatocytes in an inactive form. Upon partial hepatectomy or other hepatic damage, ALR is activated and secreted out of hepatocytes into circulation ALR R194H mutation has been identified as an autosomal-recessive infantile mitochondrial disorder presenting myopathy with cataract and combined respiratory-chain deficiency Recently, the first human disease due to Despite many studies demonstrating various functions of ALR during liver regeneration, its developmental role has not been studied. Based on the fact that ALR exists in large amount in fetal livers of both rat and human Zebrafish offers great promise as a model organism to study embryonic liver development and liver diseases hnf4, hhex, and prox1 are expressed in the liver bud at this stage. Beyond 32 hpf, liver bud begins to express differentiated hepatocyte markers such as ceruloplasmin (cp), transferrin (tfa), and liver fatty acid binding protein (lfabp). Around 50 hpf liver bud completely delaminates from gut tube. Subsequently, liver enters a rapid growth phase during which it becomes vascularized and expands rapidly. By 5 days post fertilization (dpf), liver has crossed the midline to reach the right side of the body In vertebrate, liver develops from the anterior endoderm. In both zebrafish and mammals, Fgfs, Bmps, and the Wnt/β-catenin pathway are the primary signaling pathways required for zebrafish hepatogenesis Fish maintenance and experimental protocols were approved by Institutional Animal Care and Use Committee (IACUC) of National University of Singapore (Protocol 007/06). Embryos were collected and staged as described alr (NM_001089386.1) was obtained by high fidelity RT-PCR using Advantage High Fidelity 2 (HF2) PCR kit (Clontech) using pools of RNA from mixed stages of zebrafish embryos. The PCR product was cloned into pGEM-T vector (Promega) and validated by sequencing. The alr gene was subcloned into pCS2(+) (Addgene) vector for in vitro transcription. The alr ORF was cloned into pEF6/V5-His-TOPO (Invitrogen) vector to transiently express Alr with a C terminal V5 epitope in cultured cells. After PCR addition of the restriction enzyme sites on zebrafish alr coding sequence, the cDNA was cloned back into pGEM-T vector (Promega), and subcloned into pEGFP-N1 (Clontech) vector to create an ALR-EGFP fusion protein. Cys 131 of zebrafish Alr was mutated into Ser by QuickChange site-directed mutagenesis kit (Stratagene) in pCS2-alr plasmid. Both the wild type and mutant alr genes are cloned into pET28b vector (Novagen), with a N-terminal His-tag for protein expression and purification from E.coli.The full length cDNA of zebrafish cp, prox1, alr, insulin, foxa3 according to the Zebrafish Book WISH was performed using digoxigenin labeled RNA antisense probe for the following genes: Tg  or local wild type embryos to monitor liver formation. The alr morpholinos used are:Morpholino antisense oligonucleotides were purchased from Gene Tools, and were dissolved in sterile water at the concentration of 1 mM. A total amount of 5–10 ng morpholino per embryos was injected into either 5′-CGTGTGCAGCTGCCATGTTGTTATG, 5 bp mismatch control: 5′-CcTGTGgAGCTcCCATcTTcTTATG;Translation blocking morpholino (ATG morpholino): 5′-TCATTCATAATTGTTCACCTGCACC, 5 bp mismatch control: 5′-TgATTgATAATTcTTCAgCTcCACC;Splicing inhibiting morpholino targeting first exon and first intron splicing junction (E1I1 morpholino): 5′-CTCTCCTGTACAACATATCACGTTG, 5 bp mismatch control: 5′-CTCTCgTcTACAAgATATgACcTTG.Splicing inhibiting morpholino targeting first intron and second exon splicing junction (I1E2 morpholino): Total RNA from zebrafish embryos and adult tissues was extracted using TRI reagent (Ambion) following manufacturer's instruction. Adult zebrafish tissues samples were extracted from a pool of 5–10 fishes. The livers were extracted from 100 five-days old fishes, 50 two-week old fishes, 30 three-week old fishes, 20 four-week old fishes, 10 six-week old fishes and 5 adult fishes (three-month and nine-month old fishes) respectively. For fishes younger than 5 dpf, total RNA were extracted from a pool of 30–50 embryos.alr were: foward 5′-GGGTCGTCTCCACATAGC-3′ and reverse 5′-CTCTCCATCGCTCATCCACCCT-3′. The one-step RT-PCR conditions were: 50°C 30 min; 95°C 15 min; 95°C 30 sec, 55°C 30 sec, 72°C 40 sec, for 30 cycles; 72°C 5 min. The zebrafish ribosomal protein S18 (rps18) or β-actin were used as internal control. The cycle numbers used for each gene are selected within the exponential amplification phase of that gene. The relative signal intensity of alr bands were determined using the ImageJ software by normalizing to the respective rps18 band.RT-PCR was performed using one-step RT-PCR kit (Qiagen) and 0.5 µg total RNA per reaction. The primers used to amplify alr and alrC131S were synthesized using mMessenger mMachine kit (Ambion) and injected into fertilized embryos at 1–2 cell stages. For overexpression, 1.6 ng alr mRNA was injected into each 1–2 cell stage embryos. To rescue morphants, 5 ng E1I1 morpholino and 1.6 ng alr mRNA were co-injected into 1–2 cell stage zebrafish embryos.5′-capped mRNA of zebrafish prox1 WISH to label the liver were used for liver size quantification. Photos were taken for these embryos from dorsal view by microscope under the same magnification and then analyzed in Photoshop CS3 software. The liver size in 2-D dimension was represented by the number of pixels in liver region. Data was presented as mean ± standard deviation (SD). Student's t-test was used to analyze the data and p<0.05 is considered significant.Embryos at 48 hpf that have gone through Embryos were fixed in 4% paraformaldehyde at 4°C for overnight. Frozen sections of 10 µm were collected. After blocking with 3% BSA for 1 h at room temperature, the sections were incubated with rabbit anti-proliferating cell nuclear antigen (PCNA)  or rabbit anti-phospho histone H3 (p-H3) antibody  at 4°C overnight. Secondary antibody of Alexa Fluor 568 conjugated anti-rabbit IgG (Invitrogen) was then incubated for 1 h at room temperature. The stainings were imaged with fluorescent microscope.To calculate the percentage of PCNA/p-H3 positive hepatocytes per embryo, number of stained hepatocytes and total hepatocytes were counted on each section. For PCNA staining of 4 dpf embryos, 3 livers, 7 sections per liver were counted. For p-H3 staining of 4 dpf embryos, 4 livers, 7 sections per liver were counted. For p-H3 staining of 48 hpf embryos, 5 livers, 7 sections per liver were counted. Data was presented as bar graph of mean ± standard deviation (SD) and p<0.05 was considered significant as analyzed by student's t-test.TUNEL assay was performed using Roche in situ cell death detection kit following the manufacture's instruction and quantified similarly.The expression plasmids for Alr-V5 and Alr-EGFP were transfected into human hepatocellular carcinoma cells (HepG2) and zebrafish liver cells (ZFL) For western blot analysis, cells were lysed using RIPA lysis buffer at 48 hour post transfection. The culture medium was collected and cold acetone was used to precipitate proteins from the medium. Mitochondria isolation from the cultured cells was carried out using Mitochondria Isolation Kit for Cultured Cells (Pierce).Western blot was performed using standard method and probed with mouse anti-V5 antibody (Invitrogen), mouse anti-GFP antibody (Millipore), rabbit anti-VDAC/porin antibody (Santa Cruz), mouse anti-α tubulin (Sigma) and mouse anti-β actin (Santa Cruz) respectively.C131S were expressed in E. coli BL21-DE3 strain. Bacterial pellet were collected, lysed in lysis buffer, and soluble proteins were subjected to Ni-NTA resin purification under native condition (Promega). Imidazole in the purified protein solution was removed by dialysis.The wild type pET28b-alr and mutant pET28b-alrLysozyme (Sigma) was reduced and used as substrate as described before alr cDNA was cloned by 5'RACE from local wild type embryos, and it codes for a protein of 191 amino acids. Sequence alignment showed that zebrafish Alr protein was 62% identical to human and mouse short form ALR, 48% identical with their long forms  and the liver expression persists and become intensified during the liver growth phase from 3–5 dpf (days post fertilization)  mediated gene knockdowns were performed. As illustrated in alr gene has three exons separated by two introns. Three morpholinos were designed, targeting the translation starting site (ATG), exon1-intron1 boundary (E1I1) and intron1-exon2 boundary (I1E2) respectively. Morpholinos were microinjected into 1–2 cell stage embryos, using 5 bp mismatch morpholinos as controls.To investigate the developmental functions of alr mRNA expression in a dose-dependent manner. At 26–28 hpf, a stage in which liver has just budded from the anterior endoderm and alr is expressed in the budding liver, significant reductions of endogenous alr mRNA were demonstrated in morphants injected with 5 ng morpholino per embryo  leads to death of embryos within 24 hpf. When injected at 5 ng morpholino per embryo, embryos are morphologically normal, except for a mild developmental delay. Thus all functional studies presented in this paper were carried out with this morpholino dose.When injected at ≥10 ng morpholino per embryo, embryos showed severe morphological defects including a curved body, small head with high level of apoptosis , no circulation, and cardiac edema (data not shown). In comparison, embryos injected with the same amount of 5 bp mismatch control morpholino did not produce such morphological defects. This was more obvious with the translation blocking morpholino (data not shown). It therefore seems that the maternally supplied alr knockdown on liver formation was monitored using the transgenic line Tg. In this transgenic line, liver-specific expression of DsRed (red fluorescence) is easily visible after 60 hpf while the exocrine pancreas is labeled green with EGFP from 4 dpf onwards alr lead to an obvious reduction in liver size in morphants from 3–5 dpf compared to control morpholino injected embryos at the same stage . In contrast, the endoderm rod marked by foxa3 and the endocrine pancreas marked by insulin were not affected  expression in the liver primordial region was detected at 26 hpf in alr morphants, suggesting that specification of liver progenitor cell hepatoblasts was not affected  and 3 dpf (51% of embryos). In comparison, the development of lens is not affected in alr morphants  despite the high prox1 expression in lens. Since prox1 is expressed in hepatoblast and hepatocyte, the small liver phenotype in alr morphants suggests the possibilities of alr function in differentiation of hepatoblast to hepatocyte or proliferation of hepatocyte.During zebrafish liver organogenesis, competent endoderm cells become specified into bipotential hepatoblasts upon induction and later differentiate into hepatocytes or cholangiocytes affected . Consist budding . Neverthcp which is expressed in the liver bud from 32–34 hpf onwards. It is also expressed in the yolk syncytial layer cp expression was observed in alr morphants (data not shown). From 48 hpf onwards, an obvious reduction of liver size was observed in alr morphants using cp as a marker while its expression in the yolk syncytial layer is not affected  were present in the liver budding region at 26–30 hpf in alr morphants, but marker genes for hepatocytes  showed that the liver is much smaller compared to control between 48 hpf to 5 dpf. Altogether, the above results indicate that alr plays a major role in liver outgrowth, but has negligible influence on hepatoblast determination or differentiation to hepatocyte.As a summary of the knockdown experiment, hepatoblast markers  and phosphorylated histone 3 (p-H3). As shown in alr morphants compared to control embryos (injected with same amount of 5 bp mismatch control morpholino) using both proliferation markers.As hepatocytes are the parenchymal cells in liver which constitute more than 80% of the liver, the small liver phenotype in alr morphants was observed as determined by TUNEL assays. A similar low level hepatocyte apoptosis was observed in 4 dpf alr morphants and control embryos, with only a couple of cells stained positive on each section  and ALR205 (long form) can stimulate hepatoma cell proliferation as an extracellular growth factor Subcellular localization is important for protein function. In mammals, two protein isoforms of ALR exist: the long form and the short form. While the short from have been shown to be localized in the nucleus, the long form is localized in the intermembrane space of mitochondria and the cytosol in vitro transcribed Alr-EGFP mRNA into 1–2 cell stage zebrafish embryos and detection of Alr-EGFP fusion protein in the 6 hpf embryos by anti-EGFP antibody staining also indicated a predominant cytosol localization of the fusion protein , HEK293T (human embryonic kidney cell) as well as ZFL cells (zebrafish liver cell). Live cell imaging demonstrated that Alr-V5 protein is mainly localized in the cytosol but not in the nucleus . Co-loca protein . Cell fr protein . No Alr- protein . In compArabidopsis, and yeast ALR is known as a “cytozyme”, bearing both cytokine and enzymatic activity. Several members of the ERV1/ALR family are sulfhydryl oxidases including those from human, rat, C131S proteins from E. coli under native condition. The purified recombinant Alr proteins were relatively pure as shown in coomassie blue stained SDS-PAGE gel . Notably, overexpression of Alr (at 1.6 ng mRNA/embryo) significantly enhanced liver growth, with embryos in the overexpression group showing a 40% increase in average liver size comparing to WT embryos at 48 hpf as determined by WISH  (Embryos injected with  by WISH . Intereser size) . Neverthalr or C131Salr mRNA  was effectively rescued by co-injection of either 31S mRNA . In alr  embryos . These rC131S promoted liver growth less efficiently comparing to the WT Alr , the average liver size of AlrC131S rescued embryos is obviously smaller than that of the WT Alr rescued embryos  cascade through its cell surface receptor independent of its sulfhydryl oxidase activity in vivo functional assay Although we did not detect any secreted zebrafish Alr-V5 or Alr-EGFP fusion protein in the media of cultured cells, it is probable that Alr can be released by hepatocyte under specific environmental conditions such as after liver injury. Incidentally, although low level ALR was detected in medium of primary rat hepatocytes by ELISA C131S may form functional heterodimers with the residual wild-type Alr protein in alr morphants, thus partially rescued the morphant phenotype and promoted liver growth. However, in alr morphants with liver growth defects, the amount of alr mRNA is almost undetectable up to 5 dpf . Future study of AlrC131S in an alr complete knockout zebrafish (if possible) would help to exclude this second possibility.Alternatively, it can be speculated that exogenously introduced Alralr morpholino, the morphants exhibited severe defects in multiple organs and embryonic death (data not shown). Under low dose morpholino, it is likely that the residual amount of Alr is sufficient for early embryonic development, but not sufficient to support normal liver growth. Accordingly, alr morphants only showed a small-liver phenotype, but not completely lack of liver growth. The clear correlation of knockdown levels with different phenotypes and their severeness supports the current model that Alr performs different functions at different cellular locations and developmental stages.There are two isoforms of ALR protein in mammals, with long form contain about an additional N-terminal non-conserved region. Both isoforms contain no signal peptide at the N-terminal. The composition of the secreted ALR that stimulate hepatocyte proliferation is still not very clear up to now alr is also expressed at high levels ubiquitously in early embryos (before segmentation) and later in the developing brain and pharyngeal arches  (long), NP_075527 (Mus musculus) (long), EDM03859 (Rattus norvegicus) (long), NP_001082855 (Danio rerio), NP_011543 (Saccharomyces cerevisiae). All the cysteines are highlighted in red. In human, mouse and rat, methionines labeled by blue are the starting amino acids of the short form ALR proteins; in zebrafish, the conserved methionine at same position is also highlighted by blue. Grey brackets mark the Erv1/ALR domain. Green brackets indicate the known intra-molecular disulfide bonds while green arrows indicate the cysteines residues that form the inter-molecular disulfide bonds. The conserved Arginines, which correspond to the position of the R194 mutation in human ALR, are highlighted in purple.(TIF)Click here for additional data file.Figure S2Zebrafish Alr is the ortholog of mammalian ALR and yeast Erv1p. A. Phylogenetic tree was constructed using MEGA version 4 . The branches were validated by bootstrap analysis from 1000 replications, which were represented by percentage in branch nodes. The scale bar under the tree indicates the p-distance. ALR protein sequences used in this analysis are: NP_005253 (Homo sapiens) (long), P55789 (Homo sapiens) (short), NP_075527 (Mus musculus) (long), P56213 (Mus musculus) (short), EDM03859 (Rattus norvegicus) (long), NP_037354 (Rattus norvegicus) (short), XP_414848 , AAH97922 (Xenopus laevis), CAF89716 (Tetraodon nigroviridis), NP_001082855 (Danio rerio), NP_608353 (Drosophila melanogaster), NP_490690 (Caenorhabditis elegans), NP_011543 (Saccharomyces cerevisiae) (Erv1p), NP_015362 (Saccharomyces cerevisiae) (Erv2p). B. Synteny analysis of alr (gfer) with neighbor genes in zebrafish, chicken, mouse and human genomes. Only one copy of alr gene was found in the genomes of the four species. Homologous genes are labeled by the same color. Arrow head shows the direction of that gene. Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24:1596–1599.(TIF)Click here for additional data file.Figure S3Expression of alr in zebrafish exocrine pancreas. Cross-sections of 4 dpf embryos after WISH with alr probe were presented. A, cross section of embryo at the position of liver, dash line circles the liver. B, cross section of embryo at the position of anterior pancreas, the pancreas is circled by dash line. Expression of alr is found in exocrine pancreas. C, cross section of embryo at the position of posterior pancreas, dash line circles the exocrine pancreas. L: liver; P: pancreas.(TIF)Click here for additional data file.Figure S4Liver growth is significantly inhibited in alr morphants. Cryostat section was obtained from 5 dpf Tg embryos. Red color is from the DsRed expressed under lfabp promoter, indicating the liver. Blue color is the nucleus staining by DAPI. Images in the same column are sections from similar anterior-posterior position of liver.(TIF)Click here for additional data file.Figure S5Hepatocyte apoptosis is not elevated in alr morphants. A–F, TUNEL assay performed on 4 dpf embryo liver sections. White dashed lines outline the liver. White arrowheads indicate some of the positively stained cells, which are undergoing apoptosis. Very low levels of apoptosis are found in the developing livers of wild type embryos and alr morphants. G, DNase treated sample from 30 hpf embryos, as a positive control. H, brain section from 30 hpf embryos, treated by heat shock  to induce apoptosis, as a positive control. L: liver; I: intestine.  Yabu, T., Todoriki, S., Yamashita, M., 2001. Stress-induced apoptosis by heat shock, UV and γ-ray irradiation in zebrafish embryos detected by increased caspase activity and whole-mount TUNEL staining. Fisheries Science 67, 333–340.(TIF)Click here for additional data file.Figure S6Cellular localization of Alr-EGFP in zebrafish embryo and cultured cells. A. Alr-EGFP is mainly localized in the cytoplasm in zebrafish embryo. The plasmid expressing Alr-EGFP fusion protein under the CMV promoter, was injected into zebrafish 1-cell stage embryos and these embryos were fixed at shield stages (6 hpf) and processed for sectioning. The cryo-sections were stained with mouse anti-GFP primary antibody and Alexa Fluor 568 conjugated anti-mouse IgG secondary antibody. DAPI was used to stain nucleus. Red color shows the predominant presence of Alr-EGFP fusion protein in cytoplasm, but not nucleus. B. Alr-EGFP is localized in both the cytosol and mitochondria. HEK293T cells were transfected with Alr-EGFP expressing plasmid. Cell fractionation followed by Western blot using anti-EGFP antibody revealed that Alr-EGFP was localized in both the cytosol and mitochondria in transfected HEK293T cells. The mitochondrial porin voltage-dependent anion channel (VDAC) was used as the mitochondria marker while α-tubulin was used as the cytosolic marker. C. Alr was not secreted outside of cell. Alr-EGFP is detected by anti-GFP antibody Western blot. The β-actin was used as loading control. L, cell lysate; M, conditioned medium.(TIF)Click here for additional data file.


V12 can reduce colony formation in a semi-solid matrix and focus formation on a cell monolayer. The ability of Paip2 to bind to Pabp is required to suppress the transformed phenotype mediated by hRasV12. These observations indicate that Paip2 is able to function as a tumor suppressor.Controlling translation is crucial for the homeostasis of a cell. Its deregulation can facilitate the development and progression of many diseases including cancer. Poly (A) binding protein interacting protein 2 (Paip2) inhibits efficient initiation of translation by impairing formation of the necessary closed loop of mRNA. The over production of Paip2 in the presence of a constitutively active form of hRas Uncontrolled cell growth, the loss of contact inhibition and the ability of cells to grow in semi-solid matrix are all characteristics of transformed cells and the beginning of oncogenesis. These processes are dependent upon cell metabolism. Regulation of protein synthesis, a critical component of cell metabolism, is a critical component of cellular transformation and oncogenesis.The cell regulates protein synthesis at many different steps, such as initiation of translation by a variety of mechanisms. One mean governs the recruitment of the ribosome, and the proteins required for initiation of translation to the RNA. Formation of the closed loop of mRNA is necessary for efficient translation initiation is also another point of regulation. The circularization of the mRNA brings together of the 5′- and 3′-ends of the mRNA. Several initiation proteins, and the poly (A) tail, a stretch of adenosine residues that varies in length found at the 3′-end of the most eukaryotic mRNAs, mediate formation of the closed loop. The eukaryotic initiation factor (eIF) complex 4F binds the 5′-end of the mRNA. Three proteins, eIF4E, the cap binding protein, eIF4G, a large scaffolding protein and the RNA bidirectional DEAD-box helicase eIF4A make up this complex. Within the amino-terminus (N-) of eIF4G is the binding site for eIF4E; thus, the complex is tethered to the 5′-end of the mRNA. The binding site for poly (A) binding protein (Pabp) lies within this region of eIF4G as well Two proteins, poly (A) binding protein interaction protein (Paip) 1 and 2, regulate the interaction between Pabp and the poly (A) tail, and Pabp and eIF4G The de-regulation and over production of many proteins involved in mRNA translation such as eIF4E has been implicated in cellular transformation and oncogenesis Understanding the biological function of proteins can be investigated either by deleting the encoding gene or by the over production of the protein. To decipher how Paip2 alters the biology of the cell, the protein was over produced in NIH3T3 cells . IncreasV12 and assayed for the ability to form foci on a monolayer and growth in soft agar. Increased levels of Paip2 impeded the ability of hRasV12 producing cells from growing in soft agar and forming foci on a monolayer  tail and eIF4G, and by preventing free Pabp from interacting with eIF4G, thereby disrupting formation of the closed loop of mRNA necessary to efficiently initiate translation. Alteration of the glutamic acids in the Pam2 domain of Paip2 reduced its ability to bind Pabp. The over production of wild type Paip2 suppresses cellular transformation mediated by the oncogene hRasFormation of the closed loop of mRNA necessary for efficient translation initiation can be regulated at both the 5′-end and the 3′-end of the RNA. The translation inhibitors, eIF4E binding proteins (4E-BPs) prevent formation of the eIF4F complex at the 5′-end of the mRNA by preventing the eIF4G-eIF4E interaction Previous work has demonstrated that the concentration of Paip2 within the cell is mediated by its interaction with Pabp. When not bound to Pabp, Paip2 can be targeted to the proteosome for degradation by ubiquitination by the E3 ligase EDD1. Activity of EDD1 is cell cycle regulated; it is high during G1/S and low during mitosis, suggesting that the level of Paip2 would be higher during mitosis than G1/S. Higher levels of Paip2 during mitosis may lead to in the dramatic reduction of 5′-end dependent initiation known to occur during this phase of the cell cycle. Aberrant degradation of Paip2 may lead to a higher concentration of free Pabp within the cytoplasm. Increased levels of Pabp correlate with the development of several cancers V12 was maintained by the addition of puromycin at every passage. NIH3T3 cells stably expressing murine HA-Paip2 or HA-Paip2 (9A) were grown in the presence of hygromycin. NIH3T3 cells stably producing HRasV12 and HA-Paip2 or HA-Paip2 (9A) were maintained by the addition of puromycin and hygromycin at every passage. Phoenix-293-T cells were grown in Dulbecco's modified Eagle medium, 10% fetal calf serum and (Invitrogen) and 1% penicillin-streptomycin (Invitrogen). Plasmids p-BABE hRasV12 and pWzl were generous gifts of Scott Lowe, Cold Spring Harbor Laboratory, NY.NIH3T3 cells were grown in Dulbecco's modified Eagle medium (Sigma) 10% bovine calf serum (Invitrogen) and 1% penicillin-streptomycin (Invitrogen). Selection of NIH3T3 cells stably expressing hRasV12, HA-Paip2 and HA-Paip2 (9A) were generated by introducing the appropriate encoding plasmid into phoenix-293-T cells by calcium phosphate transfection. Viruses were harvested 48 hours post-transfection and filtered (0.45 mm-pore-size filter). NIH3T3 cells were infected with the appropriate virus or viruses and 48 hours postinfection were selected by the addition of puromycin, hygromycin or puromycin and hygromycin. Production of hRasV12, HA-Paip2 and HA-Paip2 (9A) was confirmed by Western blot analysis.Cell lines over producing of hRasCells were collected following stimulation and lysed in cold buffer containing 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, and 10% glycerol (v/v), and a mixture of protease inhibitors (Roche Diagnostics). Protein content was determined using the Bio-Rad Protein Assay . Cellular extracts (1 mg of protein) were incubated end-over-end with 50% slurry of protein G-sepharose for 1 hour at 4°C, and with mouse monoclonal anti-HAII antibody overnight at 4°C. The mixture was centrifuged at 3000 rpm for 30 seconds and the resin was washed three times in 0.5 ml of the above buffer. Proteins were eluted with 2× Laemmli sample buffer and resolved by 15% SDS-PAGE. The separated proteins transferred onto a nitrocellulose membrane . Membranes were incubated at room temperature in 5% nonfat milk dissolved in blot buffer  for 1 hour and incubated at 4°C overnight with one of the following specific antibodies: anti-HAII, anti-Pabp and anti-β-actin was from Sigma-Aldrich. Proteins were then detected with anti-mouse (sheep) or anti-rabbit (donkey) IgG horseradish peroxidase-linked antibodies  and subsequent visualization by Western Lightning-chemiluminescent substrate system (Perkin Elmer).NIH3T3 cells and NIH3T3 cells producing HA-Paip2 and HA-Paip2 (9A) were grown to 40% confluency in presence 10% bovine calf serum. Cells were washed twice with 1× PBS and grown for 72 hours in presence of 0.1% bovine calf serum in order to synchronchrous their position within the cell cycle. Cells were harvested at various time after the addition of medium containing 10% bovine calf serum, washed twice in cold 1× PBS, fixed in ethanol, stained with prodium iodine, and analyzed by flow cytometry.4 cells were resuspened in 2.5 ml of 0.35% (wt/vol) agar solution, containing DMEM plus 20% BCS and onto a 0.5% (wt/vol) agar solution containing DMEM plus 20% BCS in a 60-mm plate. 2 days after incubation, 3 ml of DMEM supplemented with 20%BCS was added. Colonies were counted 21 days after plating.Approximately 5×10V12 and HA-Paip2 or HA-Paip2 (9A) were on a monolayer of NIH3T3 in 1000∶1 ratio for 28 days. Foci were fixed in methanol and stained by methylene blue.NIH3T3 cells stably producing HRasAll data are the mean ± the standard of error of the mean from three independent experiments.


Sleeping Beauty (SB) transposon system has been used for germline transgenesis of the diploid frog, Xenopus tropicalis. Injecting one-cell embryos with plasmid DNA harboring an SB transposon substrate together with mRNA encoding the SB transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that SB transposons stably integrated into the frog genome are effective substrates for remobilization.The SB10 transposase were bred with SB transposon-harboring animals to yield double-transgenic 'hopper' frogs. Remobilization events were observed in the progeny of the hopper frogs and were verified by Southern blot analysis and cloning of the novel integrations sites. Unlike the co-injection method used to generate founder lines, transgenic remobilization resulted in canonical transposition of the SB transposons. The remobilized SB transposons frequently integrated near the site of the donor locus; approximately 80% re-integrated with 3 Mb of the donor locus, a phenomenon known as 'local hopping'.Transgenic frogs that express the SB transposons integrated into the X. tropicalis genome are effective substrates for excision and re-integration, and that the remobilized transposons are transmitted through the germline. This is an important step in the development of large-scale transposon-mediated gene- and enhancer-trap strategies in this highly tractable developmental model system.In this study, we demonstrate that  Xenopus genus are particularly well suited for embryological study as these animals adapt well to captivity and the females can be induced to lay large numbers of eggs throughout the year. The most commonly used amphibian model is the South African clawed frog, X. laevis. Genetic manipulation of this species is not practical due to the long generation time (> 1 year) and the pseudo-tetraploid nature of the genome. Another species of the Xenopus genus, X. tropicalis, shares the embryological advantages of its South African cousin and is better suited for genetic studies as it is a true diploid and has a relatively short generation time (approximately 6 months). The potential of applying modern genetics to this classical embryological model system has resulted in the rapid development of genomic tools for X. tropicalis in recent years , and thiewed in .Our studies have focused on using the class II DNA 'cut-and-paste' transposable elements to modify the frog genome for gene- and enhancer-trapping and for insertional mutagenesis -9. TransTol2 transposon system in fish and frogs . T. TX. troin (RFP)  reportern Figure . The pres Figure . We usedng frogs . Injecter Figure . Reversestage 40  express e Figure . As expes Figure . SB10 isSB10;γcRFP 2M line was outcrossed with SB transposon transgenic animals that express GFP under the control of the CAGGS promoter (pT2βGFP [2 'hopper' frogs (ubiquitous GFP and lens-specific RFP) were outcrossed with wild-type frogs and the progeny (F3) were either analyzed for remobilization events or raised and outcrossed  will be described in detail below.The CAGGS-pT2βGFP ). DoubleβGFP . DopT2βGFP 8F founder harbors two independently-segregating alleles: a concatemer of three SB transposons integrated at a single locus on scaffold 57, at base number 2456981 (57:2456981) of the JGI X. tropicalis genomic sequence v4.1 assembly, and another allele with a single-copy transposon integration [2 hopper frogs inherited either one, or both, of the 8F integration events. Southern blot analysis of progeny from 8Fhopper♂35 indicated that this double-transgenic hopper had inherited the trimeric concatemer of pT2βGFP on scaffold 57 alone. Double-transgenic (RFP+/GFP+) progeny (F3) from the outcross of 8Fhopper♂35 were raised and outcrossed, and the resulting progeny (F4) were analyzed for modification of the parental pT2βGFP locus . An orgn Figure  likely rpT2βGFP8F:CAGGS-SB10;γcRFP) 8F hopper frogs was analyzed by Southern blot. Digestion of genomic DNA from pT2βGFP 8F tadpoles with BglII resulted in three bands when the blot was hybridized with a GFP probe  from the remobilized tadpoles . From the 677 GFP-positive progeny analyzed by Southern blot, we identified four excision-only events and two remobilizations. Samples of genomic DNA where changes were evident by Southern blot analysis were used in EPTS LM-PCR to clone the integration site of the remobilization events. In this experiment, the remobilization frequency was 0.3% (two remobilization events out of 677 GFP-positive tadpoles). These data indicated that the actual remobilization frequency may be somewhat higher than that estimated by simple visual inspection of the GFP-positive progeny. The observed rate of excision-only events in this outcross population was 4 out of 677, that is, 0.6%.Pre-sorting tadpoles based on GFP intensity may underestimate the total remobilization activity if the re-integration event resulted in GFP expression that was not markedly different from the parental expression. To test this, we outcrossed a double transgenic hopper frog (8Fhopper♂51) and analyzed all of the GFP-positive progeny by Southern blot. The 8Fhopper♂51 frog inherited both of the Scoring the outcross progeny of hopper frogs for changes in GFP intensity may also overestimate the remobilization frequency, as this method may not distinguish between remobilization events and excision-only events. We analyzed 25 GFP-bright tadpoles from the outcross of 8F and 7M (see below) hopper frogs, by Southern blot analysis and by cloning the novel insertion sites by EPTS LM-PCR. Only one GFP-bright tadpole had an excision-only modification of the parental transposon donor locus (4%); 24 GFP-bright tadpoles (96%) had re-integration events that were evident by novel bands on the Southern blot and by cloning the sequences flanking the canonical re-transposition events. Thus, while it is possible to identify excision-only events by changes in GFP expression, the vast majority of GFP-bright progeny represent re-integration events.SB transposons from a high-copy number (approximately 30 copies) concatemer resulted in frequent alteration of the genomic sequences flanking the transposon donor locus; nine out of nine remobilized pedigrees examined displayed genomic alterations spanning 105 bp to 107 bp flanking the donor site [SB remobilization in the frog resulted in similar genomic alterations near the donor locus, we examined the sequences flanking the 8F donor locus by PCR. Genomic DNA samples from eight remobilization events and eight excision-only events were used to amplify the sequences flanking the 5' and 3' ends of the 8F concatemer on chromosome 6. In each case, genomic PCR using primers that amplify the 5' and 3' junctions of the 8F locus generated the appropriate sized products (data not shown), indicating that the sequences directly flanking the donor locus are intact following excision of pT2βGFP transposons from the donor site.Remobilization of transposons resident in the genome may result in chromosomal rearrangements near the donor locus -41. In mSB integration in mammalian genomes [Tc1/mariner family, including SB, integrate at TA dinucleotides. The consensus sequence for SB integration in frogs, as in mammals, is a palindromic ATATATAT sequence, where the canonical TA target is in bold, although none of the re-integration events observed in the frog have this exact palindrome.Sequence analysis of the re-integration target sites indicated a similar base distribution flanking the canonical TA dinucleotide to that observed with  genomes ,43 . The novel re-integration event was on the same chromosome as the donor locus , approximately 1 cM from the parental 8F concatemer, and represents a local hop (data not shown). The 62E3 integration event was in the 3' UTR of a muscle-related coiled coil protein (GenBank accession number XM_002935280.1) gene.GFP-bright progeny from the outcross of 8F hopper frogs were raised to the adult stage, and outcrossed to demonstrate that the remobilized transposon alleles are stably transmitted through the germline. Genomic DNA was harvested from GFP-positive and GFP-negative siblings and used for Southern blot analysis and for cloning the novel integration site by EPTS LM-PCR. For example, remobilized female frog 62E3 produced GFP-bright progeny and integration site analysis showed a single copy of the pT2βGFP 7M founder had a concatemer of 8 to 10 pT2βGFP transposons at a single locus within a repeat on scaffold 38 , and mapped, by fluorescence in situ hybridization (FISH) analysis, near a telomere on chromosome 10  tadpoles have been identified from 11,646 GFP-positive progeny . The average apparent rate of remobilization was approximately 1%, and is five-times higher than that observed for the 8F hopper animals. The higher rate of remobilization observed in the 7M hoppers compared to the 8F hoppers may be due to the increased number of potential substrate transposons in the donor concatemer (three for 8F compared with 8 to 10 for 7M). A range of remobilization activities, from 0% to 5%, was noted between the different 7M hopper frogs. The 7M hoppers were produced by breeding frogs that were heterozygous for the SB10 enzyme transgene with frogs that were heterozygous for the pT2βGFP 7M allele. Double-heterozygous males  produced offspring with an average remobilization frequency of approximately 0.44%. The frequency of GFP-positive progeny in the outcrosses from these males was approximately 50%, as expected for the Mendelian inheritance of a heterozygous dominant allele. Two 7M hopper male frogs (7Mhopper♂9 and 7Mhopper♂11) produced a much higher rate of remobilized (GFP-bright) tadpoles than their siblings . Intriguingly, these animals appear to be homozygous for both the enzyme  and substrate (pT2βGFP) transgenes; 100% of the outcross progeny were RFP-positive and nearly all (> 98%) were also GFP-positive. Southern blot analysis of the outcross progeny indicated that all of the GFP-positive animals  had inherited the 7M concatemer, and the banding pattern was identical to the parental locus. The GFP-bright tadpoles in the outcross populations of 7Mhopper♂9 and 7Mhopper♂11 showed changes in parental 7M locus indicative of excision and re-integration of a transposon from the substrate donor locus. The rare (< 2%) GFP-negative tadpoles observed in the outcross populations did not inherit the pT2βGFP transgene as determined by Southern blot and genomic PCR for GFP sequences (data not shown). The unexpected non-Mendelian inheritance of hoppers 7M♂9 and 7M♂11 is unexplained; however, this data suggests that increasing the copy number of the transposon substrates in the hopper lines may increase the remobilization frequency observed in the outcross population.The y Figure . GenomicSB10 transgene, is more efficient in the female germline  than in the male germline . With the 8F hoppers, we observed a modest increase in the mean remobilization efficiency with the female hoppers  compared to the male hoppers ; however, due to the small sample size, this may not be statistically significant .To date, four 7M female hopper frogs have been outcrossed, and the mean remobilization rate from these animals is 2.54%. This may reflect individual differences in excision and reintegration activities between different hopper animals, or it may indicate that remobilization, driven by the CAGGS-pT2βGFP concatemer is located on scaffold 38 that maps to chromosome 10  as the transposon donor were cloned from the GFP-bright tadpoles.The 7M 0 Figure  and 7c. SB transposons integrated into the frog genome are effective substrates for remobilization following re-expression of the SB transposase. Unlike the integration events observed in the co-injection strategy that were mediated by a complex non-canonical mechanism, the remobilized SB transposons re-integrated via canonical SB-mediated transposition. The observed frequency of excision and subsequent re-integration of the parental pT2βGFP transposon was low . Our data in X. tropicalis is similar to that observed in other in vitro [in vivo [SB transposon was 'knocked in' to the Hprt gene on the mouse X chromosome and subsequent transient expression of SB transposase (SB10) resulted in a transposition rate of circa 3.5 × 10-5 events per cell per generation [SB transposon donor sites result in a low frequency of remobilization events that are passed through the germline [SB transposon donors result in novel re-integration sites in approximately one embryo in every one hundred (around 1%) in an outcross of double transgenic 'seed' mice [SB transposons to serve as substrates for remobilization. Horie and colleagues observed that low-copy number SB transposon concatemers served very poorly as substrates for remobilization in mice, and that the presence of more copies of the SB transposon in the concatemer acted synergistically to increase the frequency of excision and re-integration. With a donor site that contained around 20 copies of the transposon substrate, a remobilization rate of 1.25 transpositions per genome per animal (125%) was observed [Here, we demonstrate that in vitro  and in v[in vivo ,46,47 sy[in vivo ,46. For neration . In micegermline ,47. For observed . Keng anSB-mediated transpositions. There are several strategies available to increase the frequency of remobilization events in the frog genome. Increasing the copy number of SB transposon substrates will likely significantly increase the frequency of novel re-integration events in the outcross progeny from hopper frogs. Transgenic frogs with multiple copies of the pT2βGFP transposon have been generated (7M and ♀622E) that each harbor more than seven copies of the pT2βGFP transposon [pT2βGFP founders that contain multiple copies of the SB transposon. In addition to donor site transposon copy number, the transgenic transposase enzyme may also influence the remobilization activity in the frog. The transgenic SB enzyme frog described here was generated using the first generation SB transposase . In recactivity  and SB10 to SB10 . In addificiency .SB-mediated methods, that is, injection-mediated and breeding-mediated transposition. First, the concentration of the substrate is vastly higher in the injection method where approximately 75 pg (around 10.8 × 106 copies) of the plasmid harboring the SB transposon substrate was co-injected with SB mRNA. By comparison, the pT2βGFP 8F founder used in the transgenic remobilization strategy contained three copies of the SB transposon. The SB transposase catalyzes transposition as a dimer of dimers (tetramer) bound to the indirect IR/DR elements that flank the transposon [SB activity due to DNA methylation and heterochromatinization [SB transposon-harboring plasmids result in highly efficient transposition by the co-injection method in mammals when compared to non-methylated linear plasmid DNA donors [SB transposition in the developing gametes and the early-cleavage stage Xenopus embryos.There are several possible reasons for the different integration mechanisms used in the two ansposon . The masnization ,51. ReceA donors . FinallySB transposons stably integrated into the frog genome are effective substrates for remobilization is an important step in the development of large-scale insertional mutagenesis and enhancer- or gene-trap screens in the frog. The breeding-based remobilization strategy described here provides a simple and robust method for generating novel transgenic lines without the need for labor- and skill-intensive micro-injection methodologies. The frog provides several important advantages for transposon-based genetic screens. First, each outcross can generate several thousand progeny. The high fecundity of X. tropicalis indicates that, even if the remobilization frequency is low, multiple novel re-integration events can be identified in a single outcross.The demonstration that Xenopus have a long lifespan in captivity that may reach two decades or more, and the animals remain fertile for more than ten years. This has important implications for remobilization strategies, as double transgenic hopper frogs can be maintained and outcrossed at regular intervals for many years; male frogs can be outcrossed every two weeks and females every two months. Laboratories with limited animal holding space can, with a small cadre of hopper frogs, perform large-scale enhancer- or gene-trap screens, keeping only those tadpoles with interesting GFP expression profiles. The long lifespan of the hopper frogs may also have important implications if epigenetic silencing of the transgenic transposase locus is identified over a series of generations. Stably integrated transgenes are frequently subjected to epigenetic silencing over successive generations [A second advantage is that erations ,53,54. SSB to catalyze local hopping events is a third advantage that can be exploited to generate insertional mutants of genes near the donor locus. In mice, approximately 75% of remobilized SB transposons re-integrate within 3 Mb of the donor locus [SB transposition in X. tropicalis. Transgenic SB transposon frogs that have integrations in gene-dense regions of the genome can be used as donors for insertional mutagenesis strategies, as re-integration within a nearby gene may disrupt the normal activity of that locus. In the example presented here, the 8F transposon donor is located on scaffold 57, which maps to X. tropicalis chromosome 6 and is syntenic with human chromosome 7  to generate pBS-CAGGS-SB10. The 2.2 kb X. laevis γ1 crystallin promoter driving dsRed construct was a gift from Dr Robert Grainger  as described previously [SB10;γcRFP 2M produced progeny that expressed RFP robustly in the lens and was selected for further analysis.The generation of the eviously . The pCAgaespada . A 3,613lin-RFP; ). An appeviously . RFP-posX. tropicalis tadpoles were maintained at 28°C in static tanks and were staged according to Nieuwkoop and Faber [X. tropicalis animals were pre-primed with a 1:5 dilution of human chorionic gonadotropin (hCG) overnight, and primed the day of injection with 200 U of hCG. Fertilized eggs were obtained by natural matings. Injected eggs were allowed to heal at 28°C and transferred to tanks for growth at 28°C [nd Faber . Adult and Faber . The uni at 28°C . This prSB10;γcRFP 2M. First strand cDNA was synthesized and used as template for 32P-labelled PCR reactions as described previously [SB10 amplification (SB3 5'-GCCGCTCAGCAAGGAAGA-3' and SB4 5'-GAAGACCCATTTGCGACCAAG-3') annealed at 56°C and produced a 383 bp fragment. Primers to Xenopus α-actin were used as a control for RNA recovery.Total cellular RNA was isolated using the RNAeasy kit  from individual RFP-positive and RFP-negative stage 40 embryos generated by outcross of CAGGS-eviously . Primersg for 15 minutes at 4°C. The upper phase was transferred to a fresh tube and the protein concentration was measured using the Bradford assay . Aliquots of each sample were diluted with an equal volume of 2× Laemmli Sample Buffer containing β-mercaptoethanol (Bio-Rad) and denatured by heating at 100°C for 5 minutes. Proteins were separated by electrophoresis on 4% to 15% (w/v) Criterion precast polyacrylamide gels (Bio-Rad); pre-stained SDS-PAGE standards (Bio-Rad) were used as molecular weight markers. Proteins were transferred to Hybond-P polyvinylidene difluoride  membranes at 50 V for 1.5 hours at 4°C. Protein transfer was verified by staining the membrane with Ponceau S. Monoclonal anti-SB transposase antibody  was resuspended at a final concentration of 500 μg/mL and diluted 1:500 to probe the membranes blocked with Superblock  containing 0.05% (v/v) Tween 20. A secondary goat anti-mouse horseradish peroxidase-conjugated antibody was diluted to 1:10,000 and developed using the Chemiluminescent Detection System (Pierce). Membranes were stripped with Restore Western Blot Stripping Solution (Pierce) and re-probed with a mouse monoclonal antibody specific for Xenopus β-actin  as a control for protein recovery.Whole embryo or adult tissue samples were snap frozen in a dry ice and ethanol bath and stored at -80°C. 100 μl of RIPA buffer aminomethane with hydrogen chloride pH 7.5, 1% (v/v) nonyl phenoxypolyethoxylethanol (IPEGAL), 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate, 1X Complete Protease Inhibitor Cocktail ) was added to the frozen samples, mixed by vortex, extracted with Freon ) and centrifuged at 16,100 × A Leica FLIII fluorescent dissecting microscope was used to analyze GFP and RFP expression. Digital images were captured using a Nikon Ri1 color digital camera and the Nikon Elements Basic Research software package . Tadpoles were immobilized for photography by brief anesthesia in 0.015% (w/v) tricaine methanesulphonate.BglII, separated on a 0.7% (w/v) agarose gel and transferred to Hybond N+ hybridization transfer membranes . The hybridization membranes were probed with a 32P-radiolabeled fragment of the GFP open reading frame (approximately 700 bp) and exposed onto a GE Healthcare Life Sciences phosphorimager screen for detection.Genomic DNA was harvested from individual tadpoles by overnight proteinase K digestion at 56°C and phenol/chloroform/isoamyl alcohol extraction using standard protocols . GenomicSB transposon as described previously [SB transposon. All genomic PCR products were cloned into either pGEM-T Easy (Promega) or TOPO-TA  and sequenced. Novel sequences were queried against the Joint Genome Institute X. tropicalis genome  and scaffolds were assigned to chromosomes and linkage groups based on the genetic map developed at the University of Houston [Integration site analysis was performed EPTS LM-PCR to the right arm (IR/DR) of the eviously ,8. The i Houston .in situ hybridization; GFP: green fluorescent protein; hCG, human chorionic gonadotropin; IR/DR: indirect repeat/direct repeats; PCR: polymerase chain reaction; RFP: red fluorescent protein; RT: reverse transcriptase; SB: Sleeping Beauty; UTR: untranslated region.bp: base pair; CAGGS: chicken β-actin promoter coupled with a cytomegalovirus enhancer; EPTS LM-PCR: extension primer tag selection linker-mediated polymerase chain reaction; FISH: fluorescence The authors declare that they have no competing interests.SB10 transposase transgenic line. AKS and DEW provided mapping data to assign sequence scaffolds to the X. tropicalis linkage groups and/or chromosomes. PEM conceived the study, directed the project and wrote the manuscript. All authors read and approved the final manuscript.DAY carried out embryo injections, scored tadpoles, performed molecular analysis of transposon integration sites and helped prepare the manuscript. CMK performed molecular analyses of transposon integration sites, scored tadpoles and helped prepare the manuscript. EK performed embryo injections, scored progeny, assisted with molecular analyses and helped with general husbandry. HZ performed embryo injections and helped score progeny. MRJH generated the


Plain pneumococcal polysaccharide (PPS) booster administered during second year of life has been shown to cause hyporesponsiveness. We assessed the effects of PPS booster on splenic memory B cell responses and persistence of PPS-specific long-lived plasma cells in the bone marrow (BM).+, IgG+ follicles and PPS-specific antibody secreting cells (AbSC) in spleen and BM enumerated.Neonatal mice were primed subcutanously (s.c.) or intranasally (i.n.) with pneumococcal conjugate (Pnc1-TT) and the adjuvant LT-K63, and boosted with PPS+LT-K63 or saline 1, 2 or 3 times with 16 day intervals. Seven days after each booster, spleens were removed, germinal centers (GC), IgM+ AbSCs in BM.PPS booster s.c., but not i.n., compromised the Pnc1-TT-induced PPS-specific Abs by abrogating the Pnc1-TT-induced GC reaction and depleting PPS-specific AbSCs in spleen and limiting their homing to the BM. There was no difference in the frequency of PPS-specific AbSCs in spleen and BM between mice that received 1, 2 or 3 PPS boosters s.c.. Repeated PPS+LT-K63 booster i.n. reduced the frequency of PPS-specific IgG+ AbSCs resulting in no homing of new PPS-specific long-lived plasma cells to the BM or survival. These results should be taken into account in design of vaccination schedules where polysaccharides are being considered.PPS booster-induced hyporesponsiveness is caused by abrogation of conjugate-induced GC reaction and depletion of PPS-specific IgG Streptococcus pneumoniae characterizes the neonatal and infant period mainly due to the inability of the neonates and infants to elicit immune response to the PS capsule, a T cell independent type 2 (TI-2) antigen High susceptibility to infectious diseases by polysaccharide (PS) encapsulated bacteria like Hyporesponsiveness, defined as a lower antibody (Ab) level after the second immunization than after the first, has been observed after repeated immunizations with plain pneumococcal PS vaccines (PPV) in infants and toddlers for many of the serotypes Our early life murine model reproduces the main features of infant responses to both plain PPS of serotype 1 (PPS-1) and PCV (PPS-1 conjugated to tetanus toxoid (TT); Pnc1-TT) The aim of this study was to determine the effects of single and repeated PPS-1 boosters on conjugate-induced PPS-1- and TT-carrier-specific B cell memory responses in the spleen, and homing of long-lived plasma cells  to the bone marrow (BM) in mice primed with Pnc1-TT as neonates.The results show that a single dose of plain PPS-1 s.c. is sufficient to completely deplete the PPS-1-specific memory cell and AbSC pool established by pneumococcal conjugate priming in neonatal mice, since PPS-1 abrogates the conjugate-induced germinal center reaction, thus causing hyporesponsiveness.The animal experiments were approved by the Experimental Animal Committee of Iceland (ref. YDL05020034/023/BE), according to the Act on Animal protection nr. 15/1994 (revised Dec. 2008) and Regulations on Animal experimentation nr. 279/2002.NMRI mice  were kept with free access to food and water, with regulated daylight, humidity and temperature. Breeding cages were checked daily and pups kept with the mothers until weaning.PPS of serotypes 1 (PPS-1) and 19F (PPS-19F) was provided and conjugated to tetanus-toxoid  + AbSCs by ELISPOT. The other half of each spleen was frozen in Tissue-Tek OCT compound  and stored at −70°C until cryosections were cut for immunohistological staining. Serum samples for Ab measurements were obtained 2 days before and 7 days after the booster. In the second set of experiments the long-term effect of PPS-1 booster on PPS-1- and TT-specific IgG+ AbSCs in spleen and BM from 7 days to 6 weeks was assessed as presented in the third result chapter. Neonatal (7 days) mice (8 per group and time point) were immunized s.c. with 0.5 µg Pnc1-TT w/wo 5.0 µg of LT-K63 in 50 µl saline or i.n. in 2× 3.0 µl saline (as described above). Mice were boosted 16 days later by the same route with saline or 5.0 µg of PPS-1 with 5.0 µg LT-K63 or with 0.5 µg Pnc1-TT with 5.0 µg LT-K63. Control mice received saline at all time points. Eight mice per group and time point were sacrificed on days 7, 23 and 39 after booster and cells from spleen and BM isolated to numerate PPS-1- and TT-specific IgG+ AbSCs by ELISPOT. Serum samples for Ab measurements were obtained 2 days before and 7, 23 and 39 days after booster. In the third set of experiments the effects of repeated PPS-1 boosters s.c. or i.n. on Pnc1-TT-induced PPS-1-specific memory cells in spleen and persistence of long-lived plasma cells in BM was assessed as described in the fourth and fifth results chapters. Neonatal mice (8 per group and time point) were primed with 0.5 µg Pnc1-TT with 5.0 µg LT-K63 in 50 µl saline s.c. or in 2× 3.0 µl saline i.n. (as described above) and boosters of PPS-1+LT-K63 or saline administered by the same routes 1, 2 or 3 times with 16 days interval. Seven days after each administration, at days 7, 23 or 39 after the first booster, PPS-1- and TT-specific IgG+ AbSCs were enumerated in spleen and BM. Serum samples for Ab measurements were obtained 2 days before and 7, 23 and 39 days after booster.Three sets of experiments were performed. In the first set of experiments, presented in the first and second results chapters, neonatal (7 days) mice (8 per group) were immunized s.c. with 0.5 µg Pnc1-TT or Pnc19F-TT (human dose in the PCV Prevenar is 2 µg) w/wo 5.0 µg of LT-K63 in 50 µl saline into the scapular girdle or i.n. in 2× 3.0 µl saline with 30 min interval into the nares. Mice were boosted 16 days later by the same route with saline or 5.0 µg of PPS-1 or PPS-19F (1/5 of a human dose) with 5.0 µg LT-K63 or 0.5 µg conjugate of serotype 1 or 19F with 5.0 µg LT-K63. Control mice received saline at all time-points. The mice were sacrificed 7 days after booster, cells isolated from BM and half of each spleen to numerate PPS-1- and TT-specific IgG8 cells/ml) were serially diluted in complete RPMI 1640 and incubated for 5 h at 37°C. Alkaline phosphate (ALP)-goat-anti-mouse IgG  was incubated overnight and the reaction developed by 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium in ALP development buffer . Spots were counted using a microscope  and analyzed with KS ELISPOT (Zeiss).PPS-1- and TT-specific AbSCs were enumerated by enzyme-linked immunosorbent spot (ELISPOT) PPS-1- and TT-specific IgG Abs were measured by enzyme-linked immunosorbent assay (ELISA) Avidity of IgG Abs was measured by ELISA with a potassium thiocyanate (KSCN) elution step Spleens frozen in Tissue-Tek OCT (Sakura) were cut into 7 µm cryosections at 4 levels separated by 210 µm, starting 700 µm into the tissue, fixed in acetone for 10 min and stored at −70°C. Four sections/spleen were stained with peanut agglutinin (PNA)-biotin  followed by ALP-conjugated avidin  or TexasRed-avidin  to enumerate GCs, where GCs with immature staining pattern were excluded. Double fluorescent staining was performed with PNA and MOMA-1-FITC  that identifies metallophilic marginal macrophages that form a continuous circular lining around the follicles. Sections were also stained with IgM-HRP or IgG-HRP (Southern Biotechnology Associates) to enumerate non-switched and switched B cells. The sections were photographed with a digital camera  using a microscope (Zeiss) equipped with ×10 and ×40 objectives and AxioImaging software . The average of GCs per follicle was calculated for each spleen.7 colony forming units (CFU) of S. pneumoniae serotype 1  in log-phase resuspended in 50 µl sterile saline, as previously described Six weeks after booster, mice were challenged i.n. with 2.3×10Comparison between groups and time points was performed by Mann Whitney Rank sum test (unpaired data) using GraphPad Prism . A P value of <0.05 was considered statistically significant.+ AbSCs in spleen and BM and PPS-1 specific antibodies in serum was studied 7 days after priming neonatal mice s.c. or i.n. with Pnc1-TT+LT-K63.The effects of PPS-1 booster on IgG+ AbSCs in spleen than mice that received saline or Pnc1-TT, reflected in lower serum IgG anti-PPS-1 levels. No difference was observed in the frequency of PPS-1-specific IgG+ AbSCs in the BM of mice that received PPS-1 booster compared with saline, but there was a difference compared with Pnc1-TT booster . PPS-1 booster s.c. also reduced unswitched IgM+ and switched IgG+ follicles compared with saline booster s.c.. In mice that received PPS-1 booster s.c., the PNA staining pattern in spleen differed from that in other immunized mice, as GCs that looked less mature were observed. Also, staining pattern of anti-IgG differed, with less IgG+ cells in the marginal zone (MZ) and hardly any in GCs  at day 7 in spleen compared with saline, but not in BM where the frequency tended to be higher (P = 0.090) . Furthermore, the avidity  . Twenty = 0.090) . The rede points . The enh avidity  and the  avidity  of the a avidity . The depaffected .Our results demonstrate that a PPS-1 booster given s.c., but not i.n., depleted the Pnc1-TT-induced PPS-1-specific memory cell and AbSC pool in spleen and the reduction persisted until 6 weeks after the booster. No homing of new PPS-1-specific long-lived plasma cells to the BM was detected.+ Abs in serum.We further investigated the effects of repeated  PPS-1 boosters s.c. on Pnc1-TT induced PPS-1-specific memory cells in spleen and persistence of long-lived plasma cells in BM and PPS-1-specific IgG+ Abs and IgG+ AbSCs were compromised in spleen, but not BM, 7 days after the 1st PPS-1 booster s.c. (st booster and 7 days after the 2nd booster) reduced the frequency of PPS-1-specific IgG+ AbSCs both in spleen and BM and serum IgG anti-PPS-1 levels. Three PPS-1 boosters  reduced the frequency of PPS-1-specific IgG+ AbSCs in spleen and BM and serum IgG anti-PPS-1 levels. There were no differences in the frequency of PPS-1-specific IgG+ AbSCs in spleen and BM between one, two or three PPS-1 boosters at days 23 and 39, reflected in comparable serum IgG anti-PPS-1 levels , two PPS-1 boosters i.n. yielded higher frequency of PPS-1-specific IgG+ AbSCs in spleen than saline, but lower frequency in BM. Serum IgG anti-PPS levels were comparable. Thirty nine days after the 1st booster (7 days after the 3rd booster), two or three PPS i.n. boosters resulted in comparable frequencies of PPS-1-specific IgG+ AbSCs in the spleen and serum IgG anti-PPS-1 levels as saline administration. However, in the BM the frequency of PPS-1-specific IgG+ AbSCs was reduced  in serum measured by ELISA, weekly from week 2 to 7 after immunization of neonatal mice with Pnc1-TT+LT-K63 s.c. (A and B) or Pnc19F-TT+LT-K63 s.c. (C) that received a booster with saline , 0.5 µg of PPS-1+5.0 µg LT-K63 , 1.0 µg of PPS-1+5.0 µg LT-K63 , 5.0 µg of PPS-1+5.0 µg LT-K63  or 5.0 µg of PPS-19F+5.0 µg LT-K63 , 0.5 µg of Pnc1-TT  or Pnc19F-TT+5.0 µg LT-K63  16 days later  or 4 weeks later (A right panel). Time of immunization is indicated by arrows.(TIF)Click here for additional data file.Figure S2The PPS-1 boosters s.c. had no detrimental effects on the frequency of carrier protein-specific AbSCs in spleen and bone marrow. TT-specific IgG+ AbSCs, shown as number of spots (mean±SD) per 106 cells, in spleen (A) and bone marrow (B) measured by ELISPOT, TT-IgG Abs (mean EU/ml±SD) in serum (C) measured by ELISA, at day 7, 23 and 39 after s.c. booster with saline, PPS-1+LT-K63 or unvaccinated control. Statistical difference between test groups and unvaccinated controls is indicated; * P<0.05; ** P≤0.001. The results shown are from one of two independent experiments (eight mice/group for each time point) showing comparable results.(TIF)Click here for additional data file.Figure S3The frequency of TT-specific AbSCs in spleen and bone marrow was not affected by the PPS-1 boosters i.n.. TT-specific IgG+ AbSCs, shown as number of spots (mean±SD) per 106 cells, in spleen (A) and bone marrow (B) measured by ELISPOT, TT-IgG Abs (mean EU/ml±SD) in serum (C) measured by ELISA, at day 7, 23 and 39 after i.n. booster with saline, PPS-1+LT-K63 or unvaccinated control. Statistical difference between test groups and controls is indicated; * P<0.05; ** P≤0.001.The results shown are from one of two independent experiments (eight mice/group for each time point) showing comparable results.(TIF)Click here for additional data file.


Many research studies in developmental neurosciences have been focused on neuroepithelial cellular events at the tissue, cellular, and molecular levels, in order to understand how the brain develops from a single structure, the neural tube. Although a couple of researchers  introduced the idea that the cavity of the neural tube and its content, the embryonic cerebrospinal fluid (CSF), plays a key role in the brain development, it is not until the last two decades, with the work of several research groups that embryonic CSF has been considered an essential part of the developing brain, which exerts a direct influence on basic neuroepithelial behavior such as cellular survival, replication, and neurogenic differentiation.The CSF composition during embryogenesis and fetal period is really complex as has been shown by proteomic analysis. However, we are yet to determine the final composition of this fluid. In this way the paper of Vera et al. is relevant because they report the presence of new components in the developing CSF, such as SCO-Spondin with a specific effect in neurogenesis. This paper also establishes a link between embryonic CSF research and the research focused in circumventricular organs  secretion and biological role in which Prof. Esteban Rodriguez is a relevant figure.In addition, this paper supports the idea that CSF composition and biological properties change and evolve across development with the addition of specific components in a temporally and spatially regulated manner, giving us the idea that there are several aspects of neural precursors behavior control, regulated by CSF which changes progressively from embryo to the adult.Finally this paper is relevant because it is focused on a specific component of CSF involved in a local regulation of neurogenesis suggesting that this fluid can play global but also local, regulatory roles in developing brain.