FGF7, SFRP4, and VCAM1 emerged as genes associated with single-suture craniosynostosis due to their significantly large changes in gene expression compared to the control population. Pathway analysis implicated focal adhesion and extracellular matrix (ECM)-receptor interaction as differentially regulated gene networks when comparing all cases of single-suture synostosis and controls. Lastly, overall gene expression was found to be highly conserved between coronal and metopic cases, as evidenced by the fact that WNT2 and IGFBP2 were the only genes differentially regulated to a significantly large extent in a direct comparison. The identification of genes and gene networks associated with Fgf/Igf/Wnt signaling and ECM-mediated focal adhesion not only support the involvement of biomarkers previously reported to be related to craniosynostosis, but also introduce novel transcripts and pathways that may play critical roles in its pathogenesis.Craniosynostosis is a disease defined by premature fusion of one or more cranial sutures. The mechanistic pathology of single-suture craniosynostosis is complex and while a number of genetic biomarkers and environmental predispositions have been identified, in many cases the causes remain controversial and inconclusive. In this study, gene expression data from 199 patients with isolated sagittal (n = 100), unilateral coronal (n = 50), and metopic (n = 49) synostosis are compared against both a control population (n = 50), as well as each other. After controlling for variables contributing to potential bias,  Craniosynostosis is the pathologic fusion of calvarial bones that is associated with abnormal skull growth and increased intracranial pressure. While the pathogenesis of single-suture craniosynostosis (which occurs in approximately 1/2500 live births) is poorly understood, genetic causes are likely given a 7–10% recurrence rate FGFR1-3, TWIST1, EFNB1, FBN1, MSX2, RAB23, RECQL4, and TGFBR1-2Craniosynostosis can be further categorized into syndromic and non-syndromic forms. Mutations in a number of different genes have been associated with syndromic craniosynostosis such as The fact that a number of environmental and genetic risk factors have been associated with developing craniosynostosis suggests that there is no single gene, factor, or pathway responsible for causing single-suture craniosynostosis. Rather, several independent mechanisms likely lead to the occurrence of several different forms of craniosynostosis, thus complicating the elucidation of these mechanisms The transcriptomic study presented here is the largest of its kind, and the first to analyze gene expression changes in calvaria osteoblasts as they relate solely to nonsyndromic craniosynostosis. A rich set of transcriptomic data from a panel of well-characterized clinical samples was generated (199 synostosis cases and 50 controls), from which potentially pathogenic changes in gene expression among different forms of single-suture craniosynostosis were identified. In addition, subsequent pathway analysis on the dataset suggested that transcriptomic regulation of genes associated with extracellular matrix (ECM)-mediated focal adhesion play an important role in differentiating patients with craniosynostosis from unaffected individuals.FGF7), vascular cell adhesion molecule 1 (VCAM1), and secreted frizzled-related protein 4 (SFRP4) were considered to be significant and large in all three cases of single-suture synostosis when compared to controls  scores, which was defined as the percent variance explained by the first eigengene obtained from a decomposition of the probe-level data for each gene. In other words, high information content genes have consistent probe level expression, meaning that multiple probes within the same gene are changing in a uniform manner. The two thousand genes with the highest GIC scores are listed in controls .1 and m2) were consistent among metopic and coronal cases comprising approximately 79% (for metopic) and 54%  of the expression changes considered to be significantly large. Taken together, these results highlight the fact that there are consistent hallmarks of gene expression among osteoblasts derived from cases of synostosis, especially among coronal and metopic cases; however each form of the disease also possesses its own unique expression pattern.Of the 49 gene expression changes considered to be significant and large in at least one or more of the forms of single-suture synostosis , 36 wereWNT2 (wingless-type MMTV integration site family member 2) expression was found to be greater in coronal cases compared to metopic cases; however, WNT2 expression was significantly higher in both compared to controls (WNT2 expression was considered neither large (9% increase) nor significant (p>0.05) compared to controls. Decreased IGFBP2 (insulin-like growth factor binding protein 2) expression was specific to coronal cases as no significant expression differences were observed between metopic cases and control . Perhaps coronal and metopic synostosis share very similar gene expression profiles because these forms of single-suture craniosynostosis are rarer than sagittal synostosis and have fewer root causes. In contrast, sagittal craniosynostosis cases may appear more divergent because there are more root causes, which may or may not be related to its higher incidence in the general population compared to other forms of the disease. It is also possible that differences in the embryonic origin of the calvaria may explain some of the changes in gene expression that were observed, as the frontal and parietal bones are derived from neural crest and paraxial mesoderm, respectively The existing literature suggests that there is no single pathway that causes craniosynostosis; rather, several independent mechanisms likely lead to craniosynostotic endpoints. While genetic and environmental factors have been implicated in craniosynostosis, the goal of this manuscript was to identify key transcripts associated with single-suture craniosynostosis. While the expression for many genes with high GIC scores changed unilaterally, the clustering dendrograms suggested that sagittal cases were distinct from metopic and coronal cases . The higFGF7, VCAM1, and SFRP4  cause a number of craniosynostosis syndromes, including Apert, Crouzon, Muenke, and Pfeiffer syndromes FGF7 is expressed in loose mesenchyme surrounding the mesenchymal condensation FGF7 during mesenchymal condensation may lead to inappropriate ligand-receptor binding, increased mitogenic activity, and thus contribute to skeletal abnormalities related to craniosynostosis.Even though gene expression in sagittal cases appeared divergent from that of coronal and metopic cases, changes in the expression of three genes were found to be significant and large in all osteoblasts derived from cases of synostosis, nd SFRP4 . InitialFGF7, SFRP4 was identified as a significantly upregulated gene in all osteoblasts derived from cases of synostosis  were identified in these experiments is not surprising due to the fact that Wnt signaling has been implicated not only in genetic disease states related to bone, but also in bone and craniofacial development SFRP4 and WNT2 upregulation may appear counter-intuitive considering SFRP4 has been shown to antagonize Wnt activation SFRP4, is a counter-regulatory response to increased WNT2 expression or vice versa. In fact, simultaneous upregulation in the expression of WNT2 and SFRP4 has been previously reported in mouse skin and skeletal muscle WNT2 and SFRP1 upregulation in the tissues derived from syndromic craniosynostosis cases WNT2 has been shown to act via noncanonical pathways SFRP4 has been shown to inhibit canonical Wnt signaling in bone WNT2 and SFRP4 need to be performed in order to elucidate whether concurrent upregulation of these two genes in metopic and coronal cases is related to a compensatory cellular response, canonical/noncanonical Wnt signaling, or crosstalk with unidentified signaling cascades.Like nostosis . SFRP4 he extent . The facThe fact that transcripts associated with Fgf and Wnt signaling were identified as highly differentially regulated in synostosis cases compared to controls, suggests that investigating potential crosstalk mechanisms between these pathways may identify key aspects relating to the pathogenesis of craniosynostosis. Both Fgf and Wnt signaling have been implicated in the determination of mesenchymal cell fate and ossification mechanisms FGF7, SFRP4, and WNT2. To this end, pathway analysis was performed to elucidate gene sets in which individual gene expression changes may be smaller in magnitude, however, en masse these genes may heavily implicate specific pathways. When the list consisting of genes with high GIC scores was interrogated using DAVID, two pathways were significantly implicated to a greater degree than all the rest, focal adhesion and ECM-receptor interactions  (FLT1 is a receptor tyrosine kinase (RTK) that plays a key role in focal adhesion-mediated vascular development (IGF1R (insulin-like growth factor 1 receptor), another focal adhesion-related RTK, have been identified as potential causes of single-suture craniosynostosis IGF1), a high affinity ligand for IGF1R, was found to be upregulated in all osteoblasts derived from cases of synostosis, albeit only to a significant extent in coronal cases , vascular invasion , or other RTK cascades, should be considered potential candidate biomarkers for single-suture craniosynostosis.Finally, identification of ECM-mediated focal adhesion as a candidate network biomarker also substantiates the identification of on VCAM1  and FLT1eptor 1)  as diffeelopment . Furtheral cases . IGFBP2,nditions , is capaFGF7, SFRP4, and VCAM1 as novel genetic candidates for the cause of single-suture craniosynostosis like, but also confirmed the involvement of ECM-mediated focal adhesion and Ffg/Wnt/Igf signaling pathways that may contribute its pathogenesis. Furthermore, analysis of transcriptome changes suggest that while the expression of certain genes are consistent among all cases of craniosynostosis, expression patterns for coronal and metopic synostosis are quite similar, whereas gene expression in sagittal cases is more divergent. Future investigations into the regulation of these individual transcripts and gene networks related to the various forms of single-suture craniosynostosis must account for the fact that the mechanistic pathology of this disease is highly complex, likely resulting from a wide array of root causes, both genetic and environmental.This transcriptomic study has identified a number of potential transcripts and one network biomarker related to craniosynostosis from a rich set of whole genome gene expression data from calvarial osteoblasts derived from a large panel of clinical samples. The results from this study not only identified Written informed consent was obtained from all participants with single-suture craniosynostosis, whereas a waiver of consent was obtained from the Seattle Children's Hospital institutional review board (IRB) for the anonymous control samples used in this study. This study is HIPAA compliant, and we obtained independent prospective IRB approval from each participating center, including Seattle Children's Hospital, Northwestern University in Chicago, Children's Heath Care of Atlanta, and St. Louis Children's Hospital.Participants were enrolled as described previously in a prospective, four-center investigation of neurodevelopment among children with single-suture craniosynostosis –5mm diameter pieces and placed in 12-well plates (2 pieces per well) containing 2 mL of Waymouth's media supplemented with 2X antibiotic  and 10% FBS . Upon reaching confluence, the contents of each 12-well place were trypsinized using 0.05% Trypsin  and passaged into T75 flasks. Again, cells were grown to confluence and passaged into cryogenic vials containing freezing media consisting of 90% fetal bovine serum and 10% DMSO and placed in a liquid nitrogen storage tank. Once ready to use, each osteoblast line was thawed and grown in T25 flasks containing Waymouth's media supplemented with 2X antibiotic (100X Pen/Strep/Fungizone) and 10% FBS. Subsets of the 249 cell lines included 50 controls and 100 sagittal, 50 coronal, and 49 metopic cases with craniosynostosis. Upon reaching 75% confluence, cells were trypsinized using 0.05% Trypsin, counted and passaged at a cell density of 175,000 cells per 25cm2. All cells were cultured at 37°C, 5% CO2, and 99% humidity. All cell lines were characterized as osteoblasts by alkaline phosphatase staining in 12-well plates. Briefly, one BCIP/NBT tablet (Sigma B5655) was dissolved in 10 mL deionized water, and 500 µL of this solution was added for 30 minutes to each cell line. Representative staining of osteoblasts is shown in Calvaria samples from craniosynostosis cases were obtained from discarded tissues during surgical reconstructive procedures, whereas control calvaria samples were obtained from discarded tissues from anonymous surgical or autopsy specimens. Harvested calvaria samples were then washed with Waymouth's media (Sigma W1625 lot 097K8303) and cleaned of all soft tissue. Calvarial were then sliced into thin 32, each osteoblast cell line was once again grown to 75% confluence, photographed for quality control purposes, washed twice with 1X PBS, and trypsinized. An equal volume of media containing FBS was added after trypsin exposure, and cells were centrifuged twice at 200 x g for 10 minutes at 4°C in nuclease free 15ml conical tubes (Corning 430791). Between centrifugation steps, cells were washed once with 1X PBS. Cell pellets were then kept on ice until RNA extraction. For RNA extraction, Roche High Pure miRNA Isolation Kit was used with accordance to the manufacturer's protocol (Roche 050080576001). RNA was stored immediately in −80°C and submitted for microarray processing on dry ice.Following the plating of 175,000 cells per 25cmst singular value was investigated. This value is referred to as the Gene Information Content (GIC). A cutoff for significant GIC scores was defined by permuting the probe-to-probe set map and calculating the percent variance explained for each permuted probe set. This was repeated one thousand times and the cutoff was defined as the 99th percentile of the permuted statistics. Furthermore, any probe set whose observed GIC was less than this value was removed from downstream analyses. All microarray data are MIAME compliant and the raw dataset has been deposited in the MIAME compliant Gene Expression Omnibus (GEO) database under accession number GSE27976 (http://www.ncbi.nlm.nih.gov/geo/).RNA integrity was assessed using the Agilent 2100 Bioanalyzer, and only samples passing quality control were analyzed for transcriptomic changes using Affymetrix Human Gene 1.0 ST arrays, on which 28,869 genes are represented. Raw microarray data was processed and analyzed with Bioconductor (KGFLP1) was identified as significant and large in all three cases of single-suture synostosis. KGFLP1 has been characterized as the likely product of a pseudogene with high sequence homology to the C-terminus region of FGF7 (UniProtKB: Q2TVT4). Because FGF7 and KGFLP1 share a high degree of nucleotide sequence identity and several probes that comprise the probe sets corresponding to these transcripts can cross-hybridize, the microarray data was also normalized at the individual probe level and summarized at the exon level using Affymetrix Expression Console software (http://www.affymetrix.com). This approach allowed us to assess the fluorescent signal associated with probes that do not cross-hybridize. For these results it was determined that FGF7 was in fact cross-hybridizing with the 3′ end probes of KGFLP1, and that all probes specific to KGFLP1 contained in the 5′ end were not differentially expressed.Upregulation of keratinocyte growth factor-like protein 1 http://david.abcc.ncifcrf.gov/) Homo sapiens as the background, the functional annotation tool was utilized to identify pathways heavily implicated in regards to the enriched dataset.The initial step in this process was to identify genes that were robustly expressed across the population of samples, which generated a list of two thousand genes ranked by gene information content (GIC) score . (GIC) wFrom the normalized data, genes with significant evidence for differential expression were identified using the limma package Figure S1Characterization of primary osteoblast lines. Representative alkaline phosphatase staining of primary osteoblast lines (10× magnification).(TIF)Click here for additional data file.Table S1Top 2000 genes with high information content.(DOC)Click here for additional data file.Table S2Changes in gene expression considered to be significant and large in at least one form of single-suture craniosynostosis compared to controls.(DOC)Click here for additional data file.Table S3Identification of significant KEGG pathways associated with craniosynostosis-related gene expression.(DOC)Click here for additional data file.Table S4Genes identified in the dataset related to ECM-mediated focal adhesion with significant changes in expression between cases and controls.(DOC)Click here for additional data file.


Filoviruses cause sporadic but highly lethal outbreaks of hemorrhagic fever in Africa in the human population. Currently, no drug or vaccine is available for treatment or prevention. A previous study with a vaccine candidate based on the low seroprevalent adenoviruses 26 and 35 (Ad26 and Ad35) was shown to provide protection against homologous Ebola Zaire challenge in non human primates (NHP) if applied in a prime-boost regimen. Here we have aimed to expand this principle to construct and evaluate Ad26 and Ad35 vectors for development of a vaccine to provide universal filovirus protection against all highly lethal strains that have caused major outbreaks in the past. We have therefore performed a phylogenetic analysis of filovirus glycoproteins to select the glycoproteins from two Ebola species , two Marburg strains (Marburg Angola and Marburg Ravn) and added the more distant non-lethal Ebola Ivory Coast species for broadest coverage. Ad26 and Ad35 vectors expressing these five filovirus glycoproteins were evaluated to induce a potent cellular and humoral immune response in mice. All adenoviral vectors induced a humoral immune response after single vaccination in a dose dependent manner that was cross-reactive within the Ebola and Marburg lineages. In addition, both strain-specific as well as cross-reactive T cell responses could be detected. A heterologous Ad26–Ad35 prime-boost regime enhanced mainly the humoral and to a lower extend the cellular immune response against the transgene. Combination of the five selected filovirus glycoproteins in one multivalent vaccine potentially elicits protective immunity in man against all major filovirus strains that have caused lethal outbreaks in the last 20 years. Unpredictable reoccurring sporadic outbreaks of lethal filovirus associated hemorrhagic fever pose a major risk in sub Saharan Africa as they have a high human case fatality rate of 25–90% Current promising vaccine candidates against Ebola or Marburg viruses are based on adenovirus (Ad) 5 11 vp. More recently we demonstrated protection after immunization in a heterologous Ad26–Ad35 prime-boost regimen in NHP against homologous challenge with Ebola Zaire Although these vaccine candidates have elicited protective immunity in NHP, their use as vaccine in humans may be limited. VSV and hPIV-type 3 are both replication competent vaccines and therefore pose a potential safety concern Most filovirus vaccine candidate efficacy studies in NHP evaluate protection, but rarely demonstrate a correlative immune response. So far the only correlate of protection against Ebola Zaire infection established in NHP studies are serum IgG levels In our present study we aimed to first assess and fully characterize the immune responses elicited by Ad35 and Ad26 vectors as vaccine platform against multiple filoviruses. To this end we constructed rAd26 and rAd35 vectors expressing the glycoproteins of five selected filovirus strains  and evaluated these vectors for their ability to induce strain specific and cross-reactive cellular and humoral immune responses in a mouse model.All animal work was performed according to the Dutch law  and Guidelines on the Protection of Experimental Animals by the Council of the European Committee (EU Dir. 86/609) after approval by the Dier Experimenten Commissie (DEC) of Crucell under permit numbers CRH0148 and CRH0159.The protein sequence of the filovirus glycoprotein was derived from the GenBank database . Nucleotide sequences  of the Ebola glycoproteins collected during an Ebola Zaire outbreak from 2001–2003 in Congo and Gabon Replication-incompetent, E1/E3-deleted recombinant adenoviral vectors based on adenovirus type 26 and 35 were engineered using the AdVac® system. Rescue and manufacturing of the replication deficient adenoviral vectors were performed in the complementing cell line PER.C6® The transgene sequences encoding the different glycoproteins (GP) of the Ebola and Marburg Filoviridae, were inserted in the E1-position of the Ad genome and the coding sequences were optimized for efficient expression in mammalian cells and were placed under transcriptional control of the human CMV promoter and the SV-40 polyadenylation sequence. This expression cassette was cloned into an E1-deleted-Adapter plasmid containing the left portion of the adenoviral genome including the left inverted terminal repeat (ITR) and packaging signal. Co-transfection of this Adapter plasmid with a cosmid containing the remainder of the Ad26/35 adenoviral sequence (lacking the E3-gene and including the Ad5 E4orf6) yielded the recombinant, E1/E3-deleted, replication-deficient vaccine vectors.Once transfected, a single plaque for each of the rAd EBOV vectors was purified and expanded up to a production scale. A two step cesium chloride gradient ultracentrifugation procedure was used to purify the rAd vectors, which were stored as single use aliquots below −65°C. Virus particle titers were quantified by measurement of optical density at 260 nm 10 vp was used as highest dose in this animal model to avoid potential toxicity that may also interference with the immune response. Serum was obtained by heart puncture under isoflorane anesthesia and spleens were removed aseptically after cervical dislocation.Six- to eight-weeks-old specific pathogen-free female BALB/c (H-2D) mice were purchased from Harlan  and kept at the institutional animal facility under specified pathogen-free conditions during the experiments. For the immunological studies, mice were vaccinated intramuscularly (i.m.) in the quadriceps of both hind legs (50 µl/leg) with the indicated vector particle (vp) dose. 10For production of the GP spike protein, the transmembrane domain and flanking cytoplasmic tail of the GP was deleted to ensure secretion of the glycoproteins into the supernatant and eliminate membrane insertion. The GP-ΔTm sequences were cloned into the pcDNA3.1 expression plasmids (Invitrogen) and subsequently used for transient transfection of human embryonic kidney 293T (Hek293T) cells in either T175 or T175III format. Culture supernatant was harvested and replenished with fresh medium at 4 to 8 days post transfection to obtain the GP protein, which was stored below −65°C. The glycoprotein yield was analyzed by western blot analysis.The humanized GP sequences chosen for the adenoviral transgenes stemmed from sequences of Ebola Zaire (NP_066246), Sudan Gulu (YP_138523) and Ivory Coast (YP_003815426) strains. For the Marburg transgenes the Ravn (ACD13005) and Angola (ADM72984) strain sequences were chosen.Galanthus Nivalis Lectin  diluted in phosphate buffered saline  to a concentration of 10 µg/ml. Remaining lectin solution was removed and 200 µl PBS/10% Fetal bovine serum (FBS) was added for blocking at RT for 2 hours. The plates were washed 2 times with PBS/0.2% Tween20 (PBS-T). Plates were either coated with an Ebola or a Marburg strain specific GP supernatant for 1 hour at RT and then washed 6 times with PBS-T. Mouse serum samples were diluted in sample buffer 1∶25 (PBS/0.2% Tween/1% FBS) and then in a 2-fold dilution series, added to the plates and incubated at RT for 1 hour. Plates were washed 6 times with PBS-T. Bound IgG was detected with goat-anti-mouse IgG conjugated to HRP (Cayman), diluted 1∶2000 in sample buffer and incubated for 1 hour at RT. Plates were washed 6 times with PBS-T. OPD (Sigma Aldrich) was added and incubated in the dark fo 10 minutes. The reaction was stopped and measured at 492 nm. Relative serum titers were calculated against a filovirus glycoprotein strain specific reference serum.Filovirus-specific humoral response was determined by a modified enzyme-linked immunosorbent assay (ELISA) to a previously described one The number of filovirus glycoprotein-specific, interferon gamma-secreting T cells splenocytes of immunized mice was determined with an ELISPOT assay as previously described Data are presented as (geo) means. Statistical analyses were performed with SPSS version 17.0 . Immune responses  among groups of animals were assessed with ANOVA and post-hoc Tukey test. For the cross reactivity of the humoral immune response an ANOVA and Dunnett post-hoc was used. Differences were considered significant when p≤0.05.Figure S1). Ebola can be subdivided into Ebola Zaire, Sudan, Ivory Coast, and Bundibugyo species whereas Marburg virus glycoprotein is more homologous in sequence and cannot be grouped in distinct virus species. The phylogenetic analysis of the glycoprotein resembles the filovirus taxonomy based on full-length nucleotide sequence diversity Filovirus outbreaks are unpredictable in nature and the potential use of filoviruses in a bioterrorism act stresses the need for a vaccine that can provide protection against all major strains. In order to obtain optimal coverage by a filovirus glycoprotein based vaccine we performed a phylogenetic analysis of all available glycoprotein amino acid sequence of all strains that caused outbreaks of hemorrhagic fever since the identification of filovirus as the causative agent in 1967 . To control for transgene expression and processing of the inserted glycoprotein, rAd26 and rAd35 were used to transduce the susceptible human lung carcinoma epithelial cell line A549 at different doses as a model for in vivo expression. A western blot analysis showed a dose dependent expression of glycoprotein for all filovirus proteins expressed by rAd26 and rAd35 vectors .We then generated and quality controlled the rAd26 and rAd35 adenoviral vectors expressing the selected glycoproteins (7 to 1010 vector particles (vp)) of each of the rAd26 and rAd35 vectors with the glycoprotein of Ebola Zaire, Ebola Sudan/Gulu, Ebola Ivory coast, Marburg Angola, or Marburg Ravn as a transgene. The humoral immune response against the homologous antigen was measured four weeks after immunization using a mouse IgG specific sandwich ELISA that was developed for each filovirus strain glycoprotein. At a dose of 109 and 1010 vp, a dose dependent glycoprotein specific humoral immune response was elicited by all different vectors .Serum IgG titers against filovirus glycoprotein after vaccination are the best correlate of protection against filovirus in NHP identified thus far  vectors Figure 110 vp rAd26 or rAd35 expressing the different filovirus glycoproteins under study were tested for reactivity against each of the five filovirus glycoproteins in an ELISA . Similar to the high reactivity in the ELISPOT with peptide pool 1, Ebola Sudan/Gulu reactive splenocytes were also highly reactive against two peptides that allowed mapping of the dominant epitope to the amino acid sequence DRPHTPQFLFQ of an H-2LD MHC1. Interestingly, a similar sequence in potential 9-mer MHC1 binding sequence or MHC1 anchoring motifes did not necessarily elicit a similar reactivity within the three Ebola strains . In a second immunization experiment, splenocytes from vaccinated mice were stimulated in the IFN-γ ELISPOT with single peptides from the reactive peptide pools. The response against a peptide was considered positive when it was higher than the average plus three times standard deviation of the response against medium alone. For each filovirus strain we identified specific peptides that contain a T cell epitope Table 1.For both Marburg strains, one peptide that is identical in sequence between the Marburg Angola and Ravn strains was mapped to contain a T cell epitope. Another peptide, p103, shares 77.7% amino acid identity of the 9-mer MHC1 binding motife between the Marburg Angola and Ravn, however only Marburg Angola was responsive to this peptide. An additional reactive peptide was identified for Marburg Ravn, but not for Marburg Angola. These results were confirmed for all peptides in a separate experiment (data not shown). For none of the filovirus antigens a significant difference between rAd26 and rAd35 to induce peptide reactive T cells was observed other than in the strength of the response, similar to the peptide pool reactivity.10 vp rAd26 and rAd35 elicits a potent humoral and cellular immune response against filovirus glycoproteins in mice. Encouraged by these results we evaluated if a heterologous prime-boost using rAd26 and rAd35 could further improve the filovirus glycoprotein specific humoral and cellular immune responses. We did not test a homologous prime-boost, since we have shown before for adenoviral vectors that compared to a heterologous regimen such a vaccination regimen only modestly increases the immune response in mice and NHP due to an anti-vector immune response 10 vp of rAd26.Ebo(Z), a rAd26 coding for Ebola Zaire glycoprotein, and four weeks later boosted with 1010 vp of heterologous rAd35.Ebo(Z). Another group was immunized first with rAd35.Ebo(Z) and four weeks later with rAd26.Ebo(Z) at the same dose. As a single vector immunization control, mice were immunized with rAd26.Ebo(Z) or rAd35.Ebo(Z) and boosted with rAd35.empty or rAd26.empty, respectively. Negative control groups received either rAd26.empty in combination with a boost of rAd35.empty or vice versa. For Marburg Angola a similar experimental strategy was used. The vaccine induced response was evaluated either at two or eight weeks post vaccination, to capture the acute and memory phase of the induced humoral and cellular immune response.Several studies have shown that rAd26 and rAd35 as components of a heterologous prime-boost regimen elicit a sustained T cell and humoral immune response against various antigens The humoral immune response elicited by the prime-boost regimen was significantly higher than the immune response elicited by single filovirus antigen expressing rAd26 or rAd35  and rAd35.Ebo(Z) is cross-reactive with Ebola Ivory Coast and Sudan/Gulu glycoprotein, but to a lower extend than homologous serum. This is in agreement with an earlier study that has shown that Ebola Zaire glycoprotein expressed by recombinant VSV is not able to protect against Ebola Sudan challenge in a NHP filovirus challenge model Sera from mice that were immunized with rAd26.Mar(A) or rAd35.Mar(A) were cross reactive with Marburg Ravn glycoptrotein and vice versa while the cellular immune response in these mice was mainly directed against the consensus peptide pool of the Angola and Ravn strains. Based on the cross reactivity between Marburg Angola and Ravn of both the humoral and cellular immune responses it is likely that one vaccine strain will be sufficient to protect against all Marburg strains as has been shown in a NHP filovirus cross-protection study It has been reported that the rAd26 and rAd35 utilize distinct cellular receptors for viral entry D MHC1. As the cellular immune response is biased in breadth in inbred mice we expect a higher breath of the response to Ebola Sudan/Gulu in NHP and in humans, which have a more diverse and complex MHC1 repertoire.The degree of protection from filovirus challenge that can be provided by the cellular immune response has so far not been studied in great detail and the magnitude and quality of responses that are needed for protection are mostly unknown. Furthermore, data on the cellular immune response after outbreaks in humans is very limited We failed to see a clear dose dependency for the cellular immune response which was different from the humoral immune response to insert and vector which were both dose dependent. It cannot be excluded that the strength of the cellular immune response depends on the antigen specific naïve T cell precursor frequency In this manuscript we have shown the high immunogenicty of a rAd26 and rAd35 based filovirus vaccine candidate for five filovirus strains and the enhanced immunogenicty of these recombinant adenoviral vectors when combined in a heterlogous prime-boost. Such a pentavalent adenoviral vector based vaccine would potentially protect against a wide range of filovirus strains. So far, the combination of glycoproteins of different filovirus strains did not interfere with optimal immune responses against each individual component, nor did it affect protection of NHP against filovirus challenge, as compared to the protection achieved with a monovalent vaccine Table S1Characterization of adenoviral vectors. All adenoviral vectors were characterized by determination of vector particle (VP) to infectious units (IU) ratio, transgene sequencing, and expression by western blot. All vectors were purified by Caesium chloride (CsCL) density purification.(PDF)Click here for additional data file.Figure S1Phylogenetic relationship of the major filovirus strains that have caused outbreaks. Phylogenetic tree of the filovirus glycoprotein of all major filovirus strains using the UPGMA method. Confidence values are displayed at internal branches as percent of 1000 bootstraps. The branch length represents the phylogenetic distance. For better visualization the Zaire and Sudan strains are underlayed in grey. The tree was constructed with the Muscle algorithm for protein sequence alignment and the CLC workbench software for drawing of the tree. Accession numbers of the sequences used are described in material and methods. The numbers behind the strain names are the last two digits of year of strain isolation. Ebola Reston was not included as so far no human cases have been reported.(PDF)Click here for additional data file.Figure S2Filovirus glycoprotein expression by rAd26 and rAd35 vectors. Western blot analysis of A549 cells cultured to 70% confluency in 24-well plates infected with either rAd26 or rAd35 coding for either Ebola Zaire, Ebola Sudan/Gulu, Ebola Ivory Coast, Marburg Angola, or Marburg Ravn (lane 1–3 at an multiplicity of infection (MOI) of 10000, 25000, or 50000 for rAd26 and at an MOI of 1000, 2500, or 5000 for rAd35 vectors). MOI were based on vp/cell and in vitro transduction efficacy of Ad26 is lower than for Ad35 for A549 cells which was adjusted for by using a 10-fold higher MOI. The positive controls are cells infected with rAd5 (MOI 5000) coding for the same antigen (lane 7). The murine serum to detect the antigens is isolated out of Balb/c mice i.m. injected with rAd5 vectors four weeks before. The in this way generated Ebola specific sera were predominantly reactive against the GP1 (or/and GP0), whereas the Marburg specific sera specifically reacted with sera against GP2 in the western blot. Negative controls are untreated (lane 6) and rAd35.empty or rAd26.empty vector  infected A549 cells. Lane 4 is loaded with molecular weight marker.(PDF)Click here for additional data file.


Mesothelia, which cover all coelomic organs and body cavities in vertebrates, perform diverse functions in embryonic and adult life. Yet, mesothelia are traditionally viewed as simple, uniform epithelia. Here we demonstrate distinct differences between visceral and parietal mesothelia, the most basic subdivision of this tissue type, in terms of gene expression, adhesion, migration, and invasion. Gene profiling determined that autotaxin, a secreted lysophospholipase D originally discovered as a tumor cell-motility-stimulating factor, was expressed exclusively in the more motile and invasive visceral mesothelia and at abnormally high levels in mesotheliomas. Gain and loss of function studies demonstrate that autotaxin signaling is indeed a critical factor responsible for phenotypic differences within mesothelia. Furthermore, we demonstrate that known and novel small molecule inhibitors of the autotaxin signaling pathway dramatically blunt migratory and invasive behaviors of aggressive mesotheliomas. Taken together, this study reveals distinct phenotypes within the mesothelial cell lineage, demonstrates that differential autotaxin expression is the molecular underpinning for these differences, and provides a novel target and lead compounds to intervene in invasive mesotheliomas. Mesothelia cover all coelomic organs and line all body cavities of the vertebrate providing a smooth, lubricated surface on which organs move. While healthy mesothelia in the adult appear uniform and innocuous, this cell type is actually highly plastic in nature performing multiple and varied functions in the embryo and adult. During organogenesis, mesothelia undergo epithelial mesenchymal transition (EMT) to provide all or nearly all vasculogenic cells in the generation of blood vessels as well as producing diverse stromal cells to internal organs Given the diversity of function in these various settings, it is interesting to note that nothing is known about potential heterogeneity within this cellular phenotype. As exemplified by a recent report Here, using the most basic subdivision of this cell type, visceral and parietal mesothelia, we reveal that mesothelia are not “similar”. In fact, gene profiling and protein expression analyses determined that these two mesothelia have fundamentally different patterns of gene expression. Visceral and parietal cells also exhibit dramatically different phenotypes in terms of such basic properties as movement, adhesion, invasion, and differentiation which define this cell type. Interestingly, autotaxin, a secreted lysophospholipase D that converts lysophosphatidylcholine into the lipid signaling molecule lysophosphatidic acid (LPA) and known to influence the aforementioned properties in other cell types eYFP mice. Striking differences in gene expression profiles were immediately obvious between the two phenotypes. Statistical analysis of 1,728 differentially-hybridized probes successfully separated the two cell types based on gene expression patterns  and parietal  mesothelia were isolated and subjected to microarray analyses. Greater than 95% of input was from mesothelial cells as judged by the eYFP marker expressed in a mesothelial-specific manner in Wt1-cre; Rosa26Rpatterns . 796 unipatterns , the mospatterns .The most differentially expressed gene product was autotaxin . This was of particular interest as autotaxin has suggested roles in promoting the aforementioned cellular activities which fundamental to mesothelial function. Detailed IF analysis demonstrated that autotaxin was specifically localized to visceral mesothelium on the surface of the small intestine and the omentum , a G-protein coupled receptor for LPA. Since LPA is a key downstream mediator of autotaxin pathway, this discovery, together with the available direct autotaxin lysophospholipase D inhibitors, expand the list of chemical probes to dissect the roles of the autotaxin pathway in mesothelial biology.To explore this signaling pathway further, a chemical genetic screen was conducted to discover novel small molecules that target different points in the autotaxin pathway . Interestingly, two of the autotaxin pathway inhibitors identified, 2440 and 8437, were found to selectively target Lpar1 . Intriguingly, autotaxin signaling has not been linked to any mesothelioma or manipulated in efforts to subdue their aggressive properties.When human pleural (MSTO) and peritoneal (ROB) mesotheliomas were probed for activity, exceedingly high levels of autotaxin were observed . FurtherIn this study, we report striking and fundamental differences in cellular behaviors and gene expression profiles between mesothelial cell types. Importantly, our data reveal that autotaxin signaling is an underlying mechanism that regulates divergent cell migration, invasion, and differentiation within mesothelial phenotypes. This finding in turn provides a heretofore-unidentified target to attack in mesotheliomas and pathological conditions involving mesothelia. Additionally, we provide novel reagents to inhibit and/or alter diverse mesothelial behaviors in organogenesis or pathological settings. Taken together, these data advance our fundamental understanding of mesothelial biology and lay the groundwork for future intervention and analysis of mesothelia in development, tissue repair, and disease.Heterogeneity within the mesothelial phenotype had not been explored. In fact, until now, all mesothelia have been regarded as a single cell type “hardly varying from site to site” The discovery that levels of autotaxin signaling are predictive of a mesothelial cell's migratory and invasive behavior is of particular interest. First, the highly restricted expression of this secreted lysophospholipase D initially discovered as a tumor cell-motility-stimulating factor provides mechanistic insight into the regulation of mesothelial cell behaviors Here for the first time, we demonstrate that autotaxin is found at abnormally high levels in human pleural and peritoneal mesotheliomas and that this signaling pathway regulates migratory and invasive of these cancerous cells. Autotaxin has also been implicated in chronic inflammatory disorders, diabetes, arthritis, and atherosclerosis All animal procedures were approved and performed in accordance with Vanderbilt University's Division of Animal Care and IACUC/OAWA.Small intestine, omentum, and body wall tissue samples were fixed in 4% paraformaldehyde for 2 h prior to sucrose infiltration and cryo-sectioning. Cultured cells were fixed in 4% paraformaldehyde for 30 min prior to staining. Blocking solution  was applied for 1 h at room temperature. Primary and secondary antibodies are listed in eYFP mice as previously reported 50  = 1.7 µM) and 8437 (IC50  = 6.4 µM).Visceral mesothelium was isolated from the omentum of adult Wt1-cre; Rosa26R3) Visceral and parietal mesothelial cells were suspended  in a rat tail collagen 1/PBS solution . Relative levels of gene expression were determined using TaqMan-based quantitative RT–PCR on a 7900HT platform. The TaqMan Gene Expression Assay (Applied Biosystems) for Autotaxin (Mm00516572_m1) was used. 18S (Hs99999901_s1) was used as an internal control. Triplicate DCT values were generated for each assay. The fold change in expression was determined by dividing experimental values by the control value, which was then set to 1.For each cell type, DIC images were recorded every minute for 2 h using a DeltaVision imaging system and softWoRx image acquisition software (Applied Precision). False colored images were generated for the position of each cell at 20 minute intervals. Cell migration tracks were quantified using the manual tracking plug-in of ImageJ. The position of each nucleus in DIC recordings was used as reference points. Displacement values were quantified as the total migration track. Velocity values were calculated by taking the displacement value divided by time (80 minutes). Directional persistence values were quantified as the final distance of cell relocation divided by total migration track.http://www.thefgsr.com/). Microarray images were scanned with an Affymetrix high resolution GenePix 4000B scanner. Raw.CEL files were subsequently uploaded into Partek Genomics Suite version 6.6  and processed using Robust Multi-chip Average (RMA) normalization p value of less than 0.05 were considered significantly altered. Statistical analyses (including B-H correction for multiple hypothesis testing) for identification of overrepresented ontologies, functions, and pathways were performed using DAVID (http://david.abcc.ncifcrf.gov), after initial statistical data analysis was performed to identify relevant gene sets. Using DAVID, genes were grouped based on functional similarity. All microarray data has been deposited in the Gene Expression Omnibus database, accession number GSE47161.Whole transcriptome expression analysis of total RNA was performed using Affymetrix mouse Gene 1.0 ST arrays, which cover 26,166 RefSeq transcripts. RNA quality was assessed using an Agilent 2100 before further processing and hybridization in the Vanderbilt Functional Shared Resource . ATX activity in the media was analyzed using the fluorogenic substrate FS-3 according to the manufacture protocol 5 cells/mL. Each cell suspension was added to the top chamber of a Boyden chamber culture plate insert  and placed in a 24-well culture plate containing supplemented media with or without ATX or one of the inhibitors. Cells were incubated for 6 h. Cells that migrated through the pores and were adherent to the bottom of the insert were fixed in 100%MeOH and stained with Giemsa (Sigma-Aldrich). The percent of cell migration was calculated by dividing the number of migrated cells in each group by the number of migrated cells in the visceral mesothelium group and multiplying by 100. The percent of untreated visceral cells that migrated was set to 100.Cell invasion was measured as previously reported Figure S1Autotaxin signaling regulates mesothelial and mesothelioma cell migration. Visceral mesothelial, parietal mesothelial, pleural mesothelioma (MSTO), and peritoneal mesothelioma (ROB) cells were cultured with and without the addition of autotaxin (ATX), the autotaxin inhibitor S32826, Lpar1 inhibitor 1 (2440), or Lpar1 inhibitor 2 (8437) and subjected to time-lapse imaging to monitor cell migration. The displacement (A) and velocity (B) for each cell type was calculated and quantified. The displacement and velocity of untreated visceral cells was set to 1 and fold changes were calculated for each subsequent cell type. Error bars were calculated using standard error of the mean (n = 15).(TIF)Click here for additional data file.Figure S2Autotaxin signaling regulates smooth muscle differentiation in mesothelia. Visceral and parietal mesothelial cells were cultured for 3 days with or without addition of autotaxin (ATX), the autotaxin inhibitor S32826, Lpar1 inhibitor 1 (2440), or Lpar1 inhibitor 2 (8437). Cells were analyzed for the expression of the smooth muscle α-actin (αsma) and smooth muscle myosin heavy chain (smMHC) by immunofluorescence. Visceral cells readily differentiated into smooth muscle and expressed both markers (A). Addition of ATX to visceral cells further promoted smooth muscle differentiation and increased the number of cells staining positive for smMHC (C), while addition of the S32826 (E) or the Lpar1 inhibitors  significantly decreased the number of smooth muscle cells. In contrast, untreated parietal cells rarely expressed αsma and were devoid of smMHC expression (B). However, addition of ATX to parietal cells promoted smooth muscle marker expression (D). Addition of S32826 or the Lpar1 inhibitors to parietal cells extinguished both smooth muscle markers . Nuclei are marked with DAPI.(TIF)Click here for additional data file.Figure S3Visceral mesothelial cells are more contractile compared to parietal mesothelial cells. Visceral and parietal mesothelial cells were seeded in collagen gel and cultured for 8 days. The area of each gel (white outline) was measured and compared to its original area (green outline). The visceral gel (A) was significantly smaller than the parietal gel (B). Areas are quantified in panel C. The asterisk represents a statistically significant difference . Error bars were calculated using standard error of the mean.(TIF)Click here for additional data file.Figure S4Isolation of visceral and parietal mesothelia. Visceral and parietal mesothelia were isolated from omentum (A) and the body wall (B) of adult Wt1-cre; Rosa26ReYFP mice. Cells were dissociated, cultured for 7 days, and stained for the eYFP marker to indicate a pure population of isolated cells.(TIF)Click here for additional data file.Table S1Examples of genes differentially expressed in visceral and parietal mesothelia. Positive fold differences indicate higher expression in visceral mesothelium and negative fold differences indicate lower expression in visceral mesothelium compared to parietal mesothelium.(DOCX)Click here for additional data file.Table S2Primary and Secondary Antibodies.(DOC)Click here for additional data file.


Gap junctions are involved in vascular growth and their expression pattern is modulated in response to hemodynamic conditions. They are clusters of intercellular channels formed by connexins (Cx) of which four subtypes are expressed in the cardiovascular system, namely Cx37, Cx40, Cx43 and Cx45. We hypothesize that high flow conditions affect vascular expression of Cx in vivo. To test this hypothesis, flow hemodynamics and subsequent changes in vascular expression of Cx were studied in an angioinductive rat arteriovenous (AV) loop model. Fifteen days after interposition of a femoral vein graft between femoral artery and vein encased in a fibrin-filled chamber strong neovascularization was evident that emerged predominantly from the graft. Blood flow through the grafted vessel was enhanced ∼4.5-fold accompanied by increased pulsatility exceeding arterial levels. Whereas Cx43 protein expression in the femoral vein is negligible at physiologic flow conditions as judged by immunostaining its expression was enhanced in the endothelium of the venous graft exposed to these hemodynamic changes for 5 days. This was most likely due to enhanced transcription since Cx43 mRNA increased likewise, whereas Cx37 mRNA expression remained unaffected and Cx40 mRNA was reduced. Although enhanced Cx43 expression in regions of high flow in vivo has already been demonstrated, the arteriovenous graft used in the present study provides a reliable model to verify an association between Cx43 expression and high flow conditions in vivo that was selective for this Cx. We conclude that enhancement of blood flow and its oscillation possibly associated with the transition from laminar to more turbulent flow induces Cx43 expression in a vein serving as an AV loop. It is tempting to speculate that this upregulation is involved in the vessel formation occuring in this model as Cx43 was suggested to be involved in angiogenesis. Blood vessels enable oxygen supply and delivery of nutrients for multicellular organisms. To fulfil these tasks and meet required tissue demands the vascular system is able to adapt by distinct mechanisms that act on different time scales. Acutely, vessels constrict or dilate thereby adjusting vascular resistance and thus regulating local blood flow to tissues in a wide dynamic range. Herein, mechanical forces exerted by the flowing blood onto the vessel wall in conjunction with signals derived from the tissue are key players. Importantly, vasomotor signals are transmitted along the vessel length through intercellular channels which are provided by vascular gap junctions, thus coordinating the cellular behaviour in the microcirculation Recent findings suggest that gap junctional communication is also critically involved in vascular growth and angiogenesis in vivo which is distantly related to the early work of Erol and Spira Previously, we have established a unique model of angiogenesis We hypothesize, that changes in mechanical forces exerted by alterations in blood flow pattern modulate the expression of connexins within the vascular wall in vivo and may change the vascular identity of a vessel (artery vs. vein). To test this hypothesis, blood flow was characterized in the rat AV loop model and vascular expression of Cx was assessed at the mRNA and protein level in a vein used as interpositioned vessel.Experiments were performed in accordance with the German Animal Welfare Act and approved by the Institutional Animal Care and Use Committee of the Regierungspräsidium Mittelfranken (54–2532.1–34/09). 39 male Lewis rats (Charles River Laboratories) at the age of 2–4 month weighting 280–385 g were anaesthetized using isoflurane (Baxter). An arteriovenous (AV) loop was constructed between the left femoral artery and vein by interposition of a venous graft from the contralateral thigh. The AVL was either placed subcutaneously or embedded within a fibringel-filled chamber to induce three-dimensional angiogenesis. Angiogenesis was investigated 15 days after surgery at the previously estimated time of new vessel formation The used cylindrical chamber exhibited an inner diameter of 10 mm and a height of 6 mm . Four centrally placed tubes prevented displacement of the loop. The chamber was filled with 1000 µL fibrin sealant  containing fibrinogen (10 mg/mL), thrombin (2 IU/mL) and aprotinin (1500 KIE/mL) to prolong fibrinolysis All operations were performed using a surgical microscope  and by the same investigator (J.C.). After induction of anaesthesia , animals received tramadol , benzylpenicillin-streptomycin  and heparine . After midventral incision of the medial left thigh, femoral artery and vein were dissected and separated from the pelvic artery downstream to the bifurcation of the femoral artery at knee level. A 20 mm femoral vein graft was harvested from the contralateral thigh and interposed between the left femoral artery and vein by microsurgical techniques using 11/0 sutures . Thereafter, the entire AV loop was either placed subcutaneously or embedded into the isolation chamber by placing the AV loop carefully around the four plastic tubes. The chamber was filled (see above), closed and fixed onto the underlying adductor fascia . Finally, the skin was closed using Vicryl 3/0 and 4/0 (Ethicon).The animals were housed in the animal facility of the University of Erlangen Medical Centre and kept at a 12 h dark/light cycle with free access to standard chow (Sniff) and water. At the end of the experiment animals were sacrified by intracardial injection of a combination of embutramid, mebezonium and tetracain (15 ml/kg: T61®: Intervet) under deep general anaesthesia (5% isoflurane).For conventional vessel visualization the distal descending aorta was cannulated (24-gauge catheter) after abdominal midline incision. The vasculature was flushed using 100 mL isotonic salt solution containing heparin (100 IE/mL) before 30 mL warmed (37°C) India ink solution [50% v/v India ink (Rohrer&Klinger) in 5% gelatin and 4% mannitol] were injected into the aorta to visualize perfused vessels around the constructed AV loop. For micro-CT analyses, 20 mL yellow Microfil (MV-122) containing 5% of MV Curing Agent (both from Flowtech) was applied instead.After explantation, constructs were fixed using formaldehyde , dehydrated and embedded in paraffin. Histological slices with a thickness of 3 µm were obtained at two standardized planes  perpendicular to the longitudinal AV loop axis using a microtome (Leica Microsystems). Slices were stained by hematoxylin and eosin according to standard protocols and visualized using conventional microscopy . Subsequent processing of the images was performed using Olympus cellSens dimension (Olympus).Micro-CT scans were acquired on a high resolution, cone-beam micro-CT scanner developed at the Institute of Medical Physics, University Erlangen, Germany (ForBild). The image-enhancing algorithms and technical specifications are described in detail elsewhere For whole-mount immunostaining of Cx the femoral artery and vein were carefully removed after sacrificing the animal 5 days post-surgery. All vessels were opened longitudinally and pinned flat, luminal-side up. After fixation  and washing (PBS), specimens were blocked and permeabilized . The preparation was then incubated with the primary antibody  in blocking solution overnight at 4°C. After washing , immunocomplexes were visualized by goat anti-rabbit or donkey anti-mouse IgG . After final washing with PBS (3×30 min) the vessels were mounted flat on a slide using Mowiol . Staining was visualized by means of confocal laser scanning microscopy .By means of a microcirculatory flow probe  blood flow was monitored continuously at the femoral vessels (artery and vein) and at the AV loop after surgery before the transfer into the fibrin filled chamber for at least 10 min. This procedure was repeated 15 days after implantation to assess blood flow in the AV loop before and after passing the chamber. Additionally, blood flow was recorded in femoral vessels not subjected to surgical interposition during topical superfusion of papaverin  to achieve maximal vasodilation and acquire physiological flow values in these vessels. Data were collected at 240 Hz and stored using commercial data acquisition software .Subcutaneously implanted venous AV loop grafts were harvested 15 days after surgery during isoflurane anesthesia, instantaneously frozen in liquid nitrogen and stored at −70°C until later analysis. A part of the femoral vein, which was dissected and reintegrated into the venous limb of the circulation by microsurgical techniques served as control [sham operated group]. This vessel was treated otherwise similar to those vessels interpositioned between artery and vein.Frozen tissue was disrupted using a mortar and pestle, total RNA extracted by TRIzol (Invitrogen), and purified with RNeasy kits . After reverse transcription , quantitaive PCR (qPCR) was performed using iQ SYBR Green Supermix (BioRad). Connexins and GAPDH as reference gene were detected using the following primers:5′-TGACAGCAGGTGGGGTGCTCTT-3′ (forward) and 5′-AGCCAGCAGCCTACATCAGTGC-3′ (reverse),Cx37: 5′- GGGCTACTCCTCAAATCCCC-3′ (forward) and 5′- TTGCCAAGGTTCCCATTCCT-5′ (reverse),Cx40: 5′-CGTGCCGCAATTACAACAAGCA-3′ (forward) and 5′-TGGAGTTCATGTCCAGCAGCAA-3′ (reverse),Cx43: 5′-ACCACCCAGCCCAGCAAGGATA-3′ (forward) and 5′-GCCCCTCCTGTTGTTATGGGGTCT-3′ (reverse).GAPDH: The comparative CT method was used for quantification of gene expression.t test. For more than 2 groups analysis of variance (one-way ANOVA) followed by the Bonferroni post hoc test was used. Differences were considered significant at a corrected error probability of P<0.05.Data are presented as means ±SEM and compared using unpaired High resolution blood flow data was recorded by means of microvascular flow probes in 26 rats. In femoral vessels without arteriovenous (AV) loop, mean blood flow amounted to about 0.67 mL/min in artery and 0.51 mL/min in vein. However, flow was substantially more pulsatile in arteries than in veins which is reflected by the difference between maximal and minimal flow during a cardiac cycle. This flow amplitude was approximately 7 fold larger in the artery . The intAngiogenesis was monitored by placing the AV loop into an isolated subcutaneous chamber filled with a fibrin matrix. After 15 days without addition of angiogenic compounds, a strong neovascularization was evident within the 3-dimensional construct. The newly formed vessels were perfused as they could be visualized after ink injection  and arosThe expression of connexins (Cx) was studied in the femoral artery and vein. Vascular cells can be differentiated by simultaneous staining of the nuclei, which are broader in endothelial cells and oriented longitudinally to the vessel axis whereas nuclei in smooth muscle cells that are longitudinally wrapped around the vessel lumen are thinner and oriented perpendicularly As outlined above, the venous graft was exposed to pronounced alterations in mechanical forces, which were even larger than in the femoral artery. Interestingly, these hemodynamic changes modified the expression of Cx at the transcriptional level. Two weeks after implantation of the graft, levels of Cx37 mRNA in the graft remained unchanged compared to the sham operated control group, whereas the levels of mRNA Cx40 decreased substantially . In contThe protein expression of Cx37 and Cx40, which was hardly detectable in untreated veins, was similarly weak in grafted veins. Alterations of these Cx isoforms were thus not detected by whole mount immunolabeling after 5 days of AV loop flow exposure . In contUsing an established model to modulate hemodynamic forces acting on a venous graft interpositioned to create an arteriovenous loop we demonstrate that the imposed substantial hemodynamic changes result in considerable modulation of connexin expression in the femoral vein. A striking finding is the strong upregulation of Cx43 expression that precedes the subsequently ensuing angiogenesis which was described in this model previously We characterized herein the underlying hemodynamic forces acting on the venous graft, which was previously identified as the effector of the early AV loop neovascularisation After 15 days, a large network of new vessels was observed within the 3-dimensional construct by means of micro-CT which arose from the venous graft and was perfused as evidenced by staining following ink injection. During this period of neovascularization blood flow through the graft decreased and reattained the level of non-treated vessels. The substantial decrease of flow reflects a pronounced increase of resistance along the grafted vessel and was associated with a reduction of flow amplitude. Across the loop, the flow amplitude decreased and thus was significantly lower at its outlet suggesting that the blood perfused at least partially a microcirculatory bed, most likely the newly formed vessels.The detailed evaluation of Cx expression in the grafted vessels revealed substantial changes at the transcriptional and protein level. In untreated vessels, Cx37 and Cx40 were found to be expressed in the endothelium of the femoral artery, whereas these Cx were only merely detectable in the femoral vein which is in line with previous studies In veins subjected to enhanced flow and pulsatility, Cx37 mRNA remained unchanged and Cx40 mRNA decreased. Immunostaining did not reveal obvious modifications in protein levels of Cx37 and Cx40, which were rather weak in these grafted and also in control veins. In marked contrast, Cx43 expression was enhanced at the mRNA as well as the protein level in grafted vessels and Cx43 was localized in endothelial cell membranes. Since angiogenic factors were not added and ischemic tissue is not present in the current model, we suggest that the hemodynamic alteration is causal for the upregulation of Cx43 in endothelial cells in grafted veins and its integration into the membrane. Previously, it was suggested that pulsatility of flow is central for arterial identity and related to Cx40 expression The increase of Cx43 was observed already on day 5, a time point at which angiogenesis is still lacking In summary, we present evidence that Cx43 expression is strongly upregulated in a grafted vein exposed to large oscillatory flow and demonstrate a phenotypic change of a vessel in vivo with respect to Cx expression. The subsequently ensueing angiogenesis that is observed independent of angiogenic factors may be a consequence of this phenotypic change. This observation may have implications in diseases in which angiogenesis is a therapeutical measure.


Anopheles gambiae, the principal vector of human malaria, naturally transmits very few arboviruses, the most important of which is O'nyong-nyong virus (ONNV). Here we have investigated the A. gambiae immune response to systemic ONNV infection using forward and reverse genetic approaches.Mosquito-borne viral diseases cause significant burden in much of the developing world. Although host-virus interactions have been studied extensively in the vertebrate host, little is known about mosquito responses to viral infection. In contrast to mosquitoes of the Aedes and Culex genera, A. gambiae inoculated with ONNV and investigate the antiviral function of candidate genes through RNAi gene silencing assays. Our results demonstrate that A. gambiae responses to systemic viral infection involve genes covering all aspects of innate immunity including pathogen recognition, modulation of immune signalling, complement-mediated lysis/opsonisation and other immune effector mechanisms. Patterns of transcriptional regulation and co-infections of A. gambiae with ONNV and the rodent malaria parasite Plasmodium berghei suggest that hemolymph immune responses to viral infection are diverted away from melanisation. We show that four viral responsive genes encoding two putative recognition receptors, a galectin and an MD2-like receptor, and two effector lysozymes, function in limiting viral load.We have used DNA microarrays to profile the transcriptional response of A. gambiae mosquitoes, and has revealed interesting differences between A. gambiae and other invertebrates. Our data suggest that mechanisms employed by A. gambiae are distinct from described invertebrate antiviral immunity to date, and involve the complement-like branch of the humoral immune response, supressing the melanisation response that is prominent in anti-parasitic immunity. The antiviral immune response in A. gambiae is thus composed of some key conserved mechanisms to target viral infection such as RNAi but includes other diverse and possibly species-specific mechanisms.This study is the first step in elucidating the antiviral mechanisms of  Anopheles gambiae mosquitoes and O'nyong-nyong virus to study mosquito immune responses to infection. We examined the global transcriptional responses of A. gambiae to viral infection of the mosquito blood equivalent (the hemolymph) identifying a number of genes with immune functions that are switched on or off in response to infection, including complement-like proteins that circulate in the mosquito hemolymph. The switching on of these genes combined with co-infection experiments with malaria parasites suggests that viral infection inhibits the melanisation pathway. Through silencing the function of a selection of viral responsive genes, we identified four genes that have roles in A. gambiae anti-viral immunity; two putative recognition receptors ; two effector lysozymes. These molecules have previously non-described roles in antiviral immunity, and suggest uncharacterised mechanisms for targeting viral infection in A. gambiae mosquitoes.Mosquito-borne viral diseases are found across the globe and are responsible for numerous severe human infections. In order to develop novel methods for prevention and treatment of these diseases, detailed understanding of the biology of viral infection and transmission is required. Little is known about invertebrate responses to infection in mosquito hosts. In this study we used a model system of  Arthropod-borne viruses (arboviruses) are a significant health burden across the world. They represent an emerging and resurgent group of pathogens Drosophila melanogaster as a model system, as the power of genetics and the extensive knowledgebase in Drosophila have been invaluable in establishing the foundations for insect antiviral immunity research. However, the biology of arboviruses is tightly linked to the physiology of haematophagous arthropods, and as such research in model organisms may not be fully relevant to the transmission of viruses and associated vector defence. A forward research approach is required to effectively study the vector responses to arboviruses, utilizing findings in Drosophila as guidance.Extensive research has been carried out into insect immune responses to bacterial, fungal and parasitic infections; however, it is only recently that invertebrate antiviral immunity has received analogous attention. Initial studies have used Aedes aegypti. JAK/STAT related genes are differentially regulated in response to DENV infection Ae. aegypti can be made more or less susceptible to DENV through silencing of DOME (receptor of the JAK/STAT pathway) and PIAS (negative regulator of the JAK/STAT pathway) respectively Ae. aegypti JAK/STAT pathway are regulated by DENV infection, two of which have been shown to be DENV antagonists AgAGO2 (a member of the RISC complex) is an antagonist of ONNV in A. gambiaeAeAGO2, AeDCR2 and AeTSN  are Sindbis virus (SINV) antagonists in Ae. aegyptiAeDCR2 had also been shown to be a DENV antagonist Ae. aegypti infected with SINV and DENV Ae. aegyptiAe. aegypti strains showing the importance of the Toll pathway in mosquito antiviral immunity Mosquitoes launch robust immune responses against a variety of pathogens: recognition of pathogen associated molecular patterns (PAMPS) leads to activation of immune signalling pathways associated with production of potent anti-microbial peptides (AMPs) or cascades that lead to pathogen lysis, phagocytosis, melanisation or cellular encapsulation by hemocytes, the white blood cell equivalents Aedes and Culex mosquitoes transmit numerous viruses, Anopheles mosquitoes  are known to be the primary vectors of only O'nyong-nyong virus (ONNV). ONNV is a positive (sense) strand single stranded RNA (+ssRNA) virus of the Alphavirus family, with reported epidemics in West Africa in the 1960s and 1990s A. gambiae is shown to be slow and restricted in tissue tropism compared to most vector-virus combinations A. gambiae to ONNV infection of the hemolymph to identify viral responsive genes, and then used RNAi silencing to test a selection of identified genes for antiviral function. Our results confirm that in A. gambiae the RNAi pathway is a key antiviral mechanism, however, the JAK/STAT and the Toll pathway do not have a significant role in regulating systemic ONNV infection. We further identify four viral responsive genes with novel functions in mosquito antiviral immunity. Patterns of immune gene expression coupled with co-infections of A. gambiae with the rodent malaria parasite P. berghei suggest that viral infection inhibits parasite melanisation. Overall, we demonstrate that A. gambiae uses a combination of conserved antiviral pathways, including RNAi, and novel uncharacterised mechanisms to target ONNV infections.Whereas the in vitro from the infectious clone template was purified using the RNeasy mini kit (Qiagen). RNA concentration and purity was ascertained using a Nanodrop . 2 µg of RNA were transfected into a confluent culture of VERO cells in a T75 flask using the Transmessenger transfection reagent (Qiagen). Cells were observed for cytopathic effects and GFP expression at 24 hours post transfection. At 72 hours post infection cells were scraped and filtered through a 0.22 µm filter, aliquoted and stored at −80°C. 250 µl of first passage 5′ONNVic-eGFP was used to infect a large culture of confluent VERO cells. At 72 hours post infection cells were scraped, filtered through a 0.22 µm filter, aliquoted and stored at −80°C. Second passage virus was used in all experiments.5′ONNVic-eGFP plasmid was kindly provided by Dr Brian Foy, Colorado State University. 5′ONNCiv-eGFP infectious clones were generated as described in Adult mosquitoes were maintained as described in detail by Sinden and co-workers Newly emerged female mosquitoes were inoculated with the required dilution of second passage 5′ONNVic-eGFP in MEM (Invitrogen), using a pulled capillary glass needle and a Nanoject (Drummond Scientific). Inoculated mosquitoes were kept in cohorts of 30–50 and maintained as described by nsP3 ONNV gene (Pools of ∼30 whole mosquitoes were homogenised in 200 µl of Drosophila Schneiders medium (Gibco). Homogenates were centrifuged at 3000 g for 30 minutes at 4°C to pellet debris. Supernatant was transferred to a new 1.5 ml eppendorf tube and centrifuged at 5000 rpm for a further 30 minutes at 4°C. The supernatant was filtered through a 0.2 um filter, and 140 µl of the filtrate was used for viral RNA extraction using the Qiagen viral RNA extraction kit according to the manufacturer's instructions. 10 µl of vRNA was used to generate cDNA using the Superscript II kit (Invitrogen). To ascertain the abundance of viral RNA, or viral genome copy number, an absolute quantification method was used. cDNA was generated from vRNA extracted from a sample with a known viral titre, calculated using standard plaque assay. A standard curve the sample was generated using neat, 1∶5, 1∶10. 1∶50, 1∶100 and 1∶500 dilutions of cDNA. Qrt-PCR was carried out using SybrGreen reagents (Applied Biosystems) and primers against the NNV gene . The staTotal RNA was extracted from pools of ∼10 mosquitoes in TRIzol (Invitrogen) 4 days after dsRNA treatment. cDNA was generated from total RNA using the Superscript II kit (Invitrogen). Primers were designed against GOIs such that there was no overlap with dsRNA probes  includinStandard plaque assays were carried out as described in A. gambiae mosquitoes was amplified and labelled using the Low RNA Input Amplification kit . In brief: 2 µg of total RNA was used in a random primed reverse transcription reaction to generate cDNA. After amplification by conversion to cDNA, cDNA was transcribed to copy messenger RNA (cmRNA) incorporating either Cy-3UTP (for the reference sample) or Cy-5UTP (for the test sample) fluorescent nucleotide analogs. cmRNA quality and labelling efficiency was assessed by spectrophotometry using a Nanodrop . If cmRNA yield was sufficient and Cy-3UTP or Cy-5UTP labelling was successful, 825 ng of RNA was hybridised to the Agilent 4X44K array in 2× GEx-hybridisation buffer HI-RPM at 60°C for 17 hours. Hybridised slides were washed with GE wash buffer 1 at RT for one minute and GE wash buffer 2 at 37°C for one minute, to remove excess labelled cmRNA prior to scanning.Total RNA extracted from whole homogenates of www.vectorbase.org).Microarrays were scanned using a GenePix semiconfocal microarray scanner  Gene Pix Pro 6.1 was used to record feature signal intensity, to eliminate local backgrounds, for grid alignment and manual inspection of feature quality. Average feature diameter was calculated and features lying outside three standard deviations of the mean were excluded from analysis. The ratio of feature intensity verses local and global backgrounds were calculated and features not exceeding background intensities were excluded from analysis. Features were normalised using Genespring 6.1 (Axon instruments) by locally weighted linear regression methods (Lowess). Feature intensities of the three biological replicates were averaged. T-test p-values were calculated, and normalised data was filtered to exclude data with p-values greater than 0.05. Data was further filtered to include only genes showing 2-fold and greater regulation. Candidate genes were selected based on several criteria, including gene ontology, and known roles of orthologous genes. Microarray data has been submitted to the open access Vectorbase database (GAATTAATACGACTCACTATAGGGAGA) added to their 5′ ends. Polymerase chain reaction (PCR) was carried out using cDNA derived from A. gambiae mosquitoes and PCR products were sequenced to confirm correct amplification for each probe. PCR amplicons were used to synthesise dsRNA using the T7 MEGAscript kit (Ambion). Concentration of dsRNA was adjusted to 3 µg/µl and stored at −80°C until use.Primers were designed  for 200–P. berghei ANKA clone 259c12 was maintained in Theiler's original mice  as described in P. berghei infected blood. For mosquito infections, three days after passage with infected blood mice were terminally anaesthetised with an intramuscular (IM) injection of 0.05 ml/10 g body weight of Rompun , Ketastet  and PBS in a 1∶2∶3 ratio.P. berghei infected mouse, maintained at 19°C for 72 h post blood feeding to allow successful parasite development and were subsequent maintained at 27°C to allow for optimal viral replication. Unfed mosquitoes were removed between 24 and 48 h post blood feeding, when the blood bolus is clearly visible through the abdomen of the mosquito. Seven days post blood feeding, mosquito midguts were dissected and fixed in 4% PFA. Fixed midguts were mounted in Vectorshield (Vectorlabs) on glass slides with sealed coverslips. Live oocysts expressing GFP were counted using fluorescence, and melanised ookinetes were counted using light microscopy.Newly emerged adult G3 mosquitoes were intrathoracically inoculated with ∼1640 PFU 5′ONNVic-eGFP. Inoculated mosquitoes were maintained at 27°C for 48 h. Mosquitoes were starved of sugar for 4–5 hours prior to blood feeding. Mosquitoes were fed on a terminally anaesthetised P. berghei oocysts/ookinete quantification, results were subject to the Man Whitney U-test for statistical significance. Significance was accepted where P<0.001***, P<0.01**, P<0.05*. For analysis of changes in P. berghei melanisation prevalence, results were subject to the Chi Squared test for statistical significance, where P<0.001***. Statistical significance in microarray experiments was calculated using the T-test comparing normalised (Lowess) expression data. Differential regulation was considered were fold change in expression was greater than 2 and P<0.05 over three biological replicates.For plaque assay experiments and P. berghei by blood feeding on parasite-infected mice were approved and carried out under the UK Home Office License PLL70/6347 awarded in January 2008 and PPL70/7185 awarded in November 2010. The procedures are of mild severity and the numbers of animals used are minimized by incorporation of the most economical protocols. Opportunities for reduction, refinement and replacement of animal experiments are constantly monitored and new protocols are implemented following approval by the Imperial College Ethical Review Committee. The experimental procedures for the ONNV work were approved by the HSE and the Imperial College GM Safety Committee.This study was carried out in strict accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. The protocols for maintenance of mosquitoes by blood feeding and for infection of mosquitoes with A. gambiae mosquitoes. Adult mosquitoes were intrathoracically inoculated with ∼1640 PFU/mosquito. Viral RNA (vRNA) was extracted from 10 pooled mosquitoes every day over 9 days and quantitative real-time PCR (qrt-PCR) was used to calculate the viral genome copy number/mosquito (A. gambiae mosquitoes and the same infectious clone of ONNV Using an infectious clone of ONNV encoding enhanced GFP (5′ONNVic-eGFP) under the control of a duplicated viral subgenomic promoter , 4DPI  and 9DPI (where infection levels have significantly dropped). Transcriptional profiling of whole mosquito homogenates from infected versus mock-infected mosquitoes revealed a large number of viral responsive genes. Initial exposure to virus (1DPI) triggered the differential regulation of 66 genes (53 upregulated and 13 downregulated), increasing to 211 genes (119 upregulated and 92 downregulated) at 4DPI and dropping to 23 genes (20 upregulated and 3 downregulated) at 9DPI  demonstrated very little overlap in gene expression indicating that these pathways are not activated by ONNV infection. In fact at 4DPI genes involved in the RNAi, JAK/STAT and IMD pathways were downregulated suggesting inhibition of these signalling pathways. Downregulated genes encode the Tudor-SN (TSN), a component of the RNAi RISC complex, the janus kinase HOP of the JAK/STAT pathway, and IKKb, a positive regulator of the IMD pathway. In contrast to decreased IKKb transcripts, DPT and CEC3 (AMPs thought to be downstream of the IMD pathway) were upregulated at 1DPI and 4DPI.A surprisingly small number of genes from immune signalling pathways known to respond to viral infection in other invertebrates were differentially regulated; comparison of viral responsive genes with those downstream of the Toll and IMD pathway in ML1/9), three galectins (GALE6-8), one fibrinogen-like protein (FREP50) and GNBPB1 all increased in transcript abundance. Additionally a large number of genes encoding proteins implicated in humoral immunity were upregulated, consisting of LRIMs and complement-like Thioester-containing proteins (TEPs). Different LRIMs and TEPs were regulated during the different phases of infection; LRIM1/4 and TEP5 at 1DPI; LRIM7 and TEP4/9/10/12 at 1DPI and 4DPI; LRIM10 and TEP14 at 4DPI and LRRD7 at 4DPI and 9DPI.Genes encoding putative recognition receptors were upregulated at 1DPI and 4DPI; two MD2-like receptors (CTLMA2 and CLIPA2). The roles of the other regulated CLIPs and CTLs are not known, although they probably function in the modulation of signalling that regulates humoral responses. Genes encoding additional putative immune effectors were upregulated, the majority of which at 4DPI, including two hydrogen peroxidases, a glutathione peroxidase and three lysozymes. Additionally apoptosis related genes also responded to viral infection. Downregulation of the inhibitor of apoptosis-1 (IAP1) and upregulation of Caspase-6 (CASPS6) at 4DPI suggests that apoptosis may be triggered.A number of clip-domain serine proteases and their inactive homologs (CLIPs) and C-type lectins (CTLs) were upregulated including two known inhibitors of melanisation  on viral titres. Mosquitoes were co-inoculated with dsRNA corresponding to a gene of interest and ∼3000 PFU of 5′ONNVic-eGFP. The viral RNA genome copy number per mosquito was calculated 7DPI using qrt-PCR. 19 genes were selected from the viral responsive immune genes identified in our transcriptional analysis and from the classical immune signalling pathways. DsRNA corresponding to AgAGO2 and the ONNV nsP3 gene were included as positive and negative controls respectively, while dsRNA corresponding to the Escherichia coli LacZ gene was used as a reference to calculate percentage changes in viral infection loads. As expected KD of AgAGO2 resulted in increased 5′ONNVic-eGFP titres and nsP3 silencing resulted in decreased 5′ONNVic-eGFP titres, indicating that our screening method was accurate , which are known to be involved in antiviral immunity in Ae. aegypti and D. melanogaster, as well as of the IMD pathway (REL2) did not have an effect on 5′ONNVic-eGFP titres or yielded data that were highly variable and therefore inconclusive. However, 5 of the 10 tested viral responsive immune genes identified in our gene profiling experiments appeared to be viral antagonists and were selected for further investigation: the putative recognition receptors ML1 and GALE8, and the antimicrobial peptides LYSC4, LYSC6 and CEC3.Transcriptional profiling highlighted immune genes that respond to infection, however, whether these genes have genuine antiviral functions could not be inferred from expression profiling alone. To identify genes that have roles in accurate . InteresML1 KD resulted in a 6.2 fold increase (P<0.0001), LYSC4 KD resulted in a 6 fold increase (P<0.0001), LYSC6 KD resulted in a 5.4 fold increase (P<0.001) and GALE8 KD resulted in a 2 fold increase (P = 0.0163) in median viral titres at 7DPI. The prevalence of infection was similar for all genes tested . Qrt-PCR was used to confirm the reduction of ML1 (91%), GALE8 (80%) and LYSC4 (82%) transcripts in mosquitoes after dsRNA treatment and to confirm the transcriptional profile of these genes after systemic infection . Indeed through our gene silencing experiments we have observed that activating or inhibiting both pathways has little effect on ONNV titres. These data indicate that not only does systemic ONNV infection fail to trigger Toll and JAK/STAT signalling, but that genes downstream of these two pathways do not target ONNV infection.Our transcriptional profiling of ONNV infected mosquitoes has identified a large number of viral responsive genes. A significant proportion of these genes have no known function, indicating that Ae. aegypti and A. gambiae responses to viral infection may be the route of infection used in the experimental designs. The JAK/STAT and the Toll pathways have been shown to be important in regulating flavivirus DENV midgut infection after an infectious blood meal, which is the natural route of infection. In our study, intrathoracic inoculation was used to infect mosquitoes with ONNV in order to overcome the restricted tissue tropism and very limited dissemination rates observed following oral infection with ONNV, thus achieving higher levels and prevalence of systemic infection. Although a role of the JAK/STAT and the Toll pathway in midgut defense against ONNV is possible, our results demonstrate that once the virus enters the mosquito homocoel, immune responses other than the JAK/STAT and the Toll pathways are involved in regulating systemic antiviral immunity. Other studies investigating immune responses of insects to alphavirus infection have also suggested or demonstrated that the JAK/STAT and Toll signalling pathways do not target alphaviral infection. Sanders et al Ae. aegypti; despite suggesting a role for the Toll pathway during early infection, they found no differential regulation of JAK/STAT or Toll pathway components. Fragkoudis et al (2008) Ae. albopictus cells, and in fact infection inhibits activation of the JAK/STAT, Toll and IMD pathways, probably through reducing host cell gene expression. The difference observed between immune gene regulation and function using Ae. aegypti/DENV and A. gambiae/ONNV may also be a feature of flavivirus verses alphavirus infection respectively.A possible contributing factor to the surprising difference observed between Plasmodium parasites and bacteria IKKb, a positive regulator of the IMD pathway, is downregulated at 4DPI, suggesting that the pathway may be inhibited. In contrast, upregulation of LRIM1 and CEC3, which are downstream targets of the A. gambiae IMD pathway Diptericin that is downstream of the IMD pathway in DrosophilaREL2, the NF-κB factor of the IMD pathway, has no significant effect on ONNV titres, suggesting that the contribution of this pathway to ONNV infection, whether inhibited or activated, is minor.In addition to Toll signalling, a second NF-κB signalling pathway, the IMD pathway, regulates numerous genes that target D. melanogaster infected with DCV Ae. aegypti infected with DENV A. gambiae mosquitoes through silencing of the nsP3 viral gene and inhibition of RNAi by silencing AgAGO2. Interestingly transcriptional profiling revealed that TSN, a component of the RISC complex in the classical RNAi pathway, is downregulated at 4DPI. It is possible that this downregulation is mediated by ONNV, as inhibition of RNAi would be advantageous for infection. ONNV and other alphaviruses are thought not to encode a direct suppressor of RNAi B2 protein of the FHV (Flock House Virus) nsP2 protein localises to the nucleus of infected cells where its function is unknown but could modulate expression of host genes RNAi has been demonstrated in a number of invertebrates to target and limit viral infection TSN, two further genes involved in the miRNA and piRNA pathways, DCR1 and AgAGO5, and a group of DEAD-box helicases are downregulated at 4DPI. There is evidence that the piRNA pathway has antiviral functions in Drosophila, as Piwi mutant flies are more susceptible to WNV than wild-type flies RIG-I and the RIG-I-like receptors (RLRs) that have well defined roles in viral RNA recognition in mammalian systems DDX3X also has antiviral roles, and multiple viruses have been shown to interact with this protein and modulate its function DCR1, has no effect on ONNV titre.In addition to the downregulation of A. gambiae mosquitoes have a humoral branch of the immune system that recognises and eliminates invading pathogens. A ternary complex of two proteins of the LRIM family, LRIM1 and APL1C, and the complement-like TEP1 has been shown to target invading Plasmodium parasites for lysis or melanisation LRIM1 as well as several other members of the LRIM and TEP families are upregulated following infection, we investigated whether these humoral responses triggered by viral infection can interfere with Plasmodium infections. ONNV and P. berghei co-infections of A. gambiae were timed such that parasites traverse the midgut and enter the hemolymph 4 days after ONNV infection, thus encountering virus-induced humoral immune responses in the hemolymph. Our results reveal that parasite melanisation is significantly inhibited in the presence of ONNV. These results are consistent with our transcriptional analysis that shows upregulation of two important negative regulators of melanisation at 4DPI: CTLMA2 and CLIPA2LRIM1 alone is not sufficient to cause significant parasite lysis, and that parallel upregulation of APL1C and the complement effector protein TEP1 would be needed. However, among additional TEPs upregulated by ONNV infection are TEP4 and TEP9, which have been recently shown to also form complexes with LRIM1LRIM1/TEP complexes promote antiviral responses such as virus lysis or clearance of infected cells.In addition to the immune signalling pathways, ML1, GALE8, LYSC4 and LYSC6. ML1 is one of two MD-2 like receptors upregulated by ONNV infection and a known P. falciparum antagonist MD2 protein forms part of an LPS sensing mechanism in mammals MD2-TLR4 signalling triggers the expression of pro-inflammatory cytokines in response to ebola envelope protein TLR4 signalling has also been linked to several other viral infections including the vesicular stomatitus virus, respiratory syncytial virus, mouse mammary tumor virus MD2 in these interactions is not clear. A TLR binding partner for the MLs has yet to be identified in flies and mosquitoes. One hypothesis is that the ML proteins may act as an extracellular surveillance system that recognise viral PAMPs and lead to signalling via a Toll receptor. At least 10 Toll receptors have been identified to date in A. gambiae, but their role in immune responses is yet unclear Four novel ONNV antagonists have been identified through our RNAi screen; A. gambiaeGALE6-8. All three galectins are part of a mosquito specific expansion of the Galectin family, including also GALE4 and GALE5. This expansion maybe due to the haematophagous lifestyle of mosquitoes, and subsequent exposure to a disparate group of blood-borne pathogens compared to D. melanogaster, including viruses. Therefore, the upregulation of several galectins in response to ONNV infection suggests that this group of mosquito specific galectins have antiviral roles. Galectins are known to function at several levels of antiviral defence, from initial recognition and blocking of envelope and fusion glycoproteins to the activation and amplification of the innate and adaptive immune responses Galectin 1 cross-links the N-glycans displayed in the envelope proteins of Nipah and Hendra viruses (paramyxoviruses that induce syncytia in infected cells), directly blocking cell infection and cell-cell fusion Galectin 3 secretion and carbohydrate binding increase upon Herpesvirus 1 infection Beta-galactoside binding galectins are found in many organisms and display a complex repertoire: the multiple isoforms and their observed plasticity in sugar binding suggests substantial diversity in their glycan recognition properties LYSC4 and LYSC6, appear to have antagonistic effects against ONNV infections. Although lysozymes are classical antibacterial proteins that function through perturbation of cell membranes Three lysozymes are also induced by ONNV infection and two of those, A. gambiae mosquitoes, and has revealed interesting differences between A. gambiae and other invertebrates. The finding that two pathways with known antiviral roles in other invertebrate-virus systems do not significantly modulate systemic ONNV infection indicates that A. gambiae may use other immune mechanisms to recognise and fight viral infections. Our data suggest that these mechanisms involve the complement-like branch of the humoral immune response, and that the melanisation response that is prominent in anti-parasitic immunity is suppressed. The antiviral immune response in A. gambiae is thus composed of some key conserved mechanisms to target viral infection such as RNAi but includes other diverse and possibly species-specific mechanisms.In summary, this study is the first step in elucidating the antiviral mechanisms of Figure S1Qrt-PCR confirmation of virally responsive genes. The expression of 3 virally responsive genes ascertained by microarray analysis (white diamonds) was confirmed using qrt-PCR (black diamonds). cDNA was generated from RNA extracted from 5′ONNVic-eGFP infected and mock infected (LacZ control) mosquitoes. Transcript levels are expressed as the fold change of those observed in the LacZ control. Error bars represent standard deviation of 3 biological replicates.(TIF)Click here for additional data file.Table S1Genes differentially regulated by 5′ONNVic-eGFP infection. Two-fold or greater fold change ratios are shown in black text for 1DPI, 4DPI and 9DPI. Fold change ratios less than 2-fold regulated that have passed all filters outlines in the www.vectorbase.org).(DOC)Click here for additional data file.Table S2Immune genes regulated by viral infection at days 1,4 and 9 post infection. Two-fold or greater fold change ratios are shown in black text for 1DPI, 4DPI and 9DPI).. Fold change ratios less than 2-fold regulated that have passed all filters outlines in the www.vectorbase.org).(DOCX)Click here for additional data file.Table S3Primers used for A. qrt-PCR and B. RNAi probe generation.A. Primers were designed using Primer3 for 50–150 bp sections of genes of interest. B. Primers were designed for 200–600 bp sections of genes of interest, with a T7 promotor sequence (GAATTAATACGACTCACTATAGGGAGA) added to their 5′ ends, ensuring no overlap between sections used for designing qrt-PCR primers.(DOC)Click here for additional data file.


The recent results of the RV144 trial demonstrate that HIV-specific antibodies may provide protection from infection. DNA vaccines may represent an important HIV vaccine platform since they are safe, relatively inexpensive to manufacture, and stable at room temperature. However, the platform has been used largely as a prime modality limited to induce low level CD4 T cell responses in NHP and humans. In this study we sought to improve our current DNA vaccines to induce HIV-specific antibodies with plasmid vaccination alone.Groups of 5 Indian Rhesus Macaques were vaccinated with pHIV consensus gag, pol, and clade C envelopes delivered IM with in-vivo electroporation at weeks 0, 4 and 12. Immunogenicity was measured two weeks after each dose.Three doses of an HIV DNA vaccine alone induced robust cellular and antibody responses. Despite using clade C based enveloped, high binding titers were detectable against gp120s from multiple clades. Neutralizing titers were in the 100’s range to a panel of clade B and C tier 1 viruses. These data establish that designed DNA envelop antigens can drive functional immunity in NHP.Multiple improvements to DNA vaccine technology have significantly enhanced the immunogenicity of the platform. Just three doses of a plasmid based HIV vaccine induced robust binding and neutralizing antibodies as well as effector T-cell responses in NHP. We are currently expanding the immunity induced by these constructs through novel DNA adjuvants as well as in prime-boost combinations.


Placement of nasogastric tubes (NGTs) is commonplace in critical care. Misplacement of NGTs is rare and considered a never event . StrategThis small-scale study of 25 NGT placements during a 5-week period collated data supplied by questionnaire by healthcare workers responsible for NGT placements.Analysis of Adverse Incident Reports identified no never events of misplaced NGTs within the previous 10 years. This audit revealed that the commonest type of NGT was a radio-opaque tube with stylet (corflo) (92% of placements), with occasional use of the electromagnetic placement system (cortrak) (8% of placements). Sizes 10 (40%) and 12 (56%) were most common. Tube placement was confirmed by: X-ray (72%); pH of aspirates (35%); electromagnetic tube placement (one patient). The time taken from decision to place NGT to use varied (range 15 to 510 minutes). Little distinction was seen in the time taken to use and NGT confirmed by aspirate alone (205 minutes) or by X-ray (220 minutes), although the shortest interval was seen in electromagnetic NGT placement (15 minutes). The cost of NGTs confirmed by aspirate alone was low (approximately £10.00), higher with X-ray confirmation/electromagnetic placement (approximately £45.00).Despite the small dataset the results demonstrate a concerning delay in the application of enteral feeding and/or drug administration. Whilst reassuring in the steps taken to avoid never events, this study demonstrates that there may be delays in time-critical administration of enteral medicine or optimal nutritional practices. This study reveals a significant problem with aspirating gastric contents for pH testing, necessitating a large number of X-ray position confirmations. Even if the frequency and volume of gastric aspiration were greater, there is a belief that pH testing may not be sufficiently accurate . It is possible that new technologies such as electromagnetic NGT placement may allow faster/equally safe practices. Further study including cost/benefit analysis will be needed to confirm this.