Our study demonstrates that a transcriptomic analysis of mRNA isolated from human whole blood can delineate both the temporal response and the key transcriptional differences following TLR2 and TLR4 ligation.The use of human whole blood for transcriptomic analysis has potential advantages over the use of isolated immune cells for studying the transcriptional response to pathogens and their products. Whole blood stimulation can be carried out in a laboratory without the expertise or equipment to isolate immune cells from blood, with the added advantage of being able to undertake experiments using very small volumes of blood. Toll like receptors (TLRs) are a family of pattern recognition receptors which recognise highly conserved microbial products. Using the TLR2 ligand (Pam3CSK4) and the TLR4 ligand (LPS), human whole blood was stimulated for 0, 1, 3, 6, 12 or 24 hours at which times mRNA was isolated and a comparative microarray was undertaken. A common NFκB transcriptional programme was identified following both TLR2 and TLR4 ligation which peaked at between 3 to 6 hours including upregulation of many of the NFκB family members. In contrast an interferon transcriptional response was observed following TLR4 but not TLR2 ligation as early as 1 hour post stimulation and peaking at 6 hours. These results recapitulate the findings observed in previously published studies using isolated murine and human myeloid cells indicating that  Microarray analysis is increasingly being used to advance our understanding of the complex transcriptional responses generated downstream of an experimental perturbation or during a disease state Toll-like receptors (TLR) are a family of pattern recognition receptors (PRR) which recognise highly conserved microbial products. In humans 10 functional TLRs have been described including TLR2 and TLR4 which are expressed on the cell surface and recognise conserved bacterial products A study collectively analysing data from multiple published papers from both human and murine macrophage transcriptional studies identified a group of genes upregulated following both TLR2 and TLR4 stimulation, which were predicted to be regulated by NFκB. Additionally a separate IFN-sensitive response element (ISRE) regulated set of genes expression was seen to be upregulated in the TLR4 (and TLR3) stimulations but not the TLR2 stimulation in vivo TLR4 ligand administration have also previously been undertaken in vivo administration of LPS, it was shown that mRNA expression of proinflammatory chemokines and cytokines  peaked at 2 to 4 hours after LPS administration, whereas the cytokine IL10 was maximal at 6 hours. In this study the transcription factors NFKB1, NFKB2, RELA and RELB were significantly expressed and seen to peak after the cytokines and chemokines. The peak time for transcription factors including the STAT  and IRF genes was at 4 to 6 hours in vivo experiments, studies are limited by ethical and practical considerations.Transcriptomic studies following in vitro studies has the potential advantage over isolated cell populations as these different components may have a differential response to stimulation. Autocrine and paracrine signalling between the differing cell populations may result in a response of the whole system that potentially better reflects the in-vivo response. Additionally whole blood potentially has advantages over PBMC, DCs or monocyte derived macrophages since it can be used in situations where it is not possible to obtain large volumes of blood to derive the isolated cell populations. Previously, In-vitro human whole blood has been used as a model to study TLR ligation, predominantly with measurement of specific cytokine protein levels Whole blood comprises cells of both the innate and adaptive immune system. Therefore the use of whole blood for in vitro global temporal transcriptional response to TLR4 and TLR2 ligation in human whole blood. To better understand this gene transcriptional response we used a variety of bioinformatics approaches to delineate both the temporal response and the key transcriptional differences resulting from TLR2 and TLR4 ligation and demonstrate that in a whole blood system that the response to TLR stimulation can resemble that previously identified in isolated immune cells.The objective of our study was to undertake a detailed comparative analysis of the This study was approved by the Central London 3 Research Ethics Committee (09/H0716/41). All participants gave written informed consent.Six healthy volunteers (self-reported questionnaire); three male, three female; aged 25–50 years old; of similar ethnic background were recruited into the study. Sixty ml of whole blood from each volunteer was taken at 9 am into 10 ml Vacutainers with sodium heparin 17 international units/ml (BD Vacutainer).Measured by Celltac Automated Hematology Analyzer  at 0 hour, volunteer’s results listed 2 for 0, 1, 3, 6, 12 or 24 hours at which point the contents of the well were thoroughly mixed with 2 mls Tempus Solution (Applied Biosystems/Ambion) to lyse the cells and stabilise the RNA. Samples stored at −80°C until RNA processing.In 24 well plates , 1 ml of heparinised whole blood was stimulated either in the presence of a final concentration of 200 ng/ml of Pam3CSK4 (Invivogen), 1 ng/ml of LPS  added in a volume of 100 µl with RPMI-1640 with GlutaMAX (Life Technologies). Media control samples were cultured with the addition of 100 µl of RPMI-1640 with GlutaMAX. Samples were incubated at 37°C, 5% COReagents (excepting LPS and Pam3CSK4) including Sodium heparin Vacutainers were tested for endotoxin contamination by Limulus assay and were found to be endotoxin free (<0.03 EU/ml). Pam3CSK4 was tested by the manufacturer and confirmed to be endotoxin free (<0.001 EU/µg).RNA was isolated using the PerfectPure RNA Blood kit (5-PRIME) according to manufacturer’s instructions. 2.5 µg of isolated RNA was globin RNA reduced using the GLOBINclear 96-well format kit (applied Biosystems/Ambion) according to the manufacturer’s instructions. Isolated and globin reduced RNA quantity was assessed using either Nanodrop 1000 or Nanodrop 8000 spectrophotometer , RNA quality was assessed using an Agilent 2100 Bioanalyser (Range 6.5–9.5) (Agilent technologies). 200 ng of globin reduced RNA was amplified to generate biotinylated amplified antisense cRNA using the Illumina CustomPrep RNA amplification kit (Applied Biosystems/Ambion). 750 ng of cRNA was hybridized to Illumina Human HT-12 V4 BeadChip arrays (Illumina) and scanned on Illumina iScan. GenomeStudio (Illumina) was used to perform quality control and generate signal intensity. Two samples were excluded from further analysis at this stage as they failed quality control measures .th percentile shift algorithm. Per-transcript normalisation was undertaken by normalisation to the median of a defined control group. Transcripts were then filtered out if they were not significantly (p<0.01) different in intensity value compared to the background in at least 10% of all the samples.Raw background subtracted data was processed using Genespring V12.6 (Agilent Technologies) and the following principles were applied to all analyses. After background subtraction low signal values (<10) were then set to a threshold of 10, log2 transformed and per chip normalised using 75p<0.01), followed by a further filtering of transcripts by fold change (FC) in which transcripts were filtered if less than 1.8 FC different between variables of interest. Expression heatmaps were generated within Genespring V12.6. Heatmap clustering was undertaken using Differential distance metric and Wards linkage rule, unless otherwise stated.The resulting transcripts were then subjected to statistical filtering (either One-way ANOVA or 2-way ANOVA) with multiple testing correction (Benjamini-Hochberg Media controls from 2 volunteers had evidence of activation of inflammatory genes by 3 hours of culture . This acThe data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE55375.k-means clustering into 9 clusters using Euclidian distance metric. Number of clusters was chosen by the number of 3rd order branches in the dendrogram from the LPS expression heatmap. Clusters were compared across stimulations using Pearsons Correlation (within Graphpad Prism V6).Within Genespring 12.6 the normalised significant transcript lists were separately clustered by www.ingenuity.com). Significant transcripts identified from GX microarray analysis were uploaded into IPA. For time point analyses these lists were filtered by mean FC (>1.8) compared to media control at the time point.The canonical pathway, gene function annotation and upstream analyses were generated through the use of IPA  From the globin reduced RNA cDNA was synthesised using High Capacity cDNA Reverse Transcription kit (Applied Biosystems), according to the manufacturer’s instructions followed by RNase H (Promega) treatment for 30 min at 37°C. IFNB1, IL1A, IL6, NFKB1, NFKB2, STAT1, STAT2 and IRF7 gene expression were quantified by real-time PCR  using the TaqMan system, and normalised to GAPDH mRNA.Primer probes used were IFNB1 (Hs01077958_s1); IL1A (Hs00174092_m1); IL6 (Hs00985639_m1); NFKB1 (Hs00765730_m1); NFKB2 (Hs010208901); STAT1 (Hs01013996_m1); STAT2 (Hs01013123_m1); IRF7 (Hs01014809_g1); GAPDH (Hs02758991_g1) .Analysis of the media controls alone over time revealed 377 significantly expressed transcripts over time. The peak of this difference was at 24 hours with 281 transcripts more than 1.8FC different compared to the 0 hour samples . These dTo gain insight into the differential temporal gene expression in response to TLR4 and TLR2 ligation we performed a comparative microarray analysis of LPS and Pam3CSK4 stimulated human whole blood and accompanying media controls over a time course. From four healthy human volunteers 1 ml of heparinised whole blood was stimulated with either the TLR4 ligand LPS (1 ng/ml), the TLR2 ligand Pam3CSK4 (200 ng/ml), (concentrations shown to result in a plateau in previous studies within our laboratory), or incubated only with media as a control. RNA was isolated at 0, 1, 3, 6, 12 and 24 hours for each stimulus or control.LPS (TLR4) stimulation induced a greater number of differentially regulated transcripts as compared to Pam3CSK4 (TLR2) and had a higher magnitude of response. For this reason the two stimulations were first analysed independently to generate the significant transcript lists compared to media control over the time course after stimulation. LPS stimulation resulted in the differential expression of 4777 transcripts (mapping to 3571 unique genes in IPA) whereas Pam3CSK4 stimulation resulted in only 1202 differentially regulated transcripts (mapping to 922 unique genes in IPA) . Expressk-means clustering was applied to cluster the significant transcript lists based on their similarity in expression over time for each stimulation separately  were small in terms of numbers of genes which were highly expressed by 1 hour after stimulation with both LPS and Pam3CSK4 and peaked in expression between 3 to 6 hours. 80% of the genes in the P2 cluster were found within the L7 cluster. These common genes were predominantly chemokines and cytokines: C15orf48, CCL2, CCL20, CCL3, CCL3L1, CCL3L3, CCL4L1, CCL4L2, CCL8, CCRL2, CXCL2, EBI3, IL1A, IL6 and TNF.The “Acute Phase response signalling” clusters (L4 and P8) were characterised by genes involved with inflammatory response. Approximately 60% of the genes in the P8 cluster were also found within the L4 cluster and these common genes included those involved with the inflammatory response: ORM1, ORM2, HAMP, IRAK2, PI3, PTGES, TNFAIP6, TNIP3. In addition, within the L4 cluster but not the P8 cluster were genes involved with IFN regulation: IFI44L, IFIT1, IFIT3, IFNG, OAS3, OASL.The “Protein Ubiquitination pathway” clusters (L1 and P1) had 104 common genes between stimulations which included heat shock protein genes  and Proteasome PA700/20S genes .The “Complement system” clusters (in both L8 and P5) were characterised by genes that were upregulated, had their highest expression at 24 hours and included the metallothionein genes  which were amongst the highest expressed genes at 24 hours following both LPS and Pam3CSK4 stimulations . ClusterCluster L2, which peaked at 6 hours and was characterised by “Activation of IRF by cytosolic pattern recognition receptors” as the top pathway did not have a similar pathway in the Pam3CSK4 clusters. Examination of the genes within this pathway revealed that there were detected a number of IFN regulated/regulatory genes .Clusters L3, L0, L6 and P4, P7 and P0 were all enriched for immune function pathways although there were no similarities between the stimulations in terms of top significant pathway and kinetic profile. Cluster P3 was enriched for the pathway “UDP-N-acetyl-D-galactosamine Biosythesis II” and there was no similar cluster following LPS stimulation.in vitro LPS stimulation of whole blood.To better understand the temporal response we undertook analysis at each time point using the significant transcript lists. Significantly expressed transcripts at each time point were identified by comparing the mean expression of the differentially regulated transcripts in response to LPS and Pam3CSK4 stimulation at each time point to the media control at that time point and filtering those transcripts which were less than 1.8 FC different to the media control  are shown  from 3 hours onwards and was most significant at 6 hours (p<0.01) at any time point following Pam3CSK4 stimulation.The “IFN signalling” pathway in response to LPS was only significant  combined accounted for approximately 20% of the significantly expressed genes at this time point. There was a large degree of overlap between stimulations of the significant cytokines/chemokines at 1 hour, with all of the 17 significant cytokines/chemokines genes from the Pam3CSK4 list also being identified in the LPS list . FollowiIn order to identify which transcriptional regulators may be responsible for the differences observed in gene expression between LPS and Pam3CSK4 stimulation we undertook upstream analysis of the gene lists at each time point within IPA. Upstream analysis attempts to predict which transcriptional regulator may be responsible for the observed differential gene expression by comparing the genes known to be regulated by a transcriptional regulator (derived from the literature) to those significantly differentially expressed genes identified at each time point from this analysis. It can be seen that the top predicted transcription regulator for both stimulations was the NFκB complex and that it was predicted to be activated from 1 hour onwards .By comparison of the mean mRNA expression of the predicted transcriptional regulators compared to media control it can be seen that the magnitude and temporal response was similar for LPS and Pam3CSK4 stimulation for PPRC1 and SP1 . The preThe NFκB genes REL, RELA, RELB, NFκB1 and NFκB2 were all significantly expressed and present in both LPS 4777 transcript and Pam3CSK4 1202 transcript lists . TranscrIn order to attempt to identify the cytokines that were potentially involved in autocrine/paracrine signalling and subsequent gene expression regulation in response to LPS or Pam3CSK4 stimulation, we again undertook per-time point upstream analysis within IPA. TNF, IL1B and IL1A were predicted as potentially activated cytokines, and this predicted activation was early (by 1 hour) and sustained . The mRNIL12B was predicted to be activated upon LPS stimulation but not Pam3CSK4 and this difference was observed in the mean mRNA expression of the IL12B gene (encoding for IL12p40) which was upregulated in response to LPS compared to media control and not following Pam3CSK4 stimulation . IFN cytk-means clustering, canonical pathway analysis and upstream analysis of both potential transcriptional regulators and cytokines highlighting a difference in IFN signalling following LPS and Pam3CSK4 stimulation.Our data show that as early as 1 hour post stimulation IFN gene expression is seen to be upregulated following LPS but not Pam3CSK4 stimulation. This difference in IFN signalling was further emphasised by the k-means clustering, predicted upstream analysis, and canonical pathway analysis as being differentially activated between LPS and Pam3CSK4 stimulations.IRF and STAT genes had been identified by To test if this difference in expression of IRF and STAT genes resulted in differential expression of IFN regulated genes we used a list of human type 1 IFN regulated genes generated from the Interferome database v2.0. We compared the expression of these type 1 IFN regulated genes between LPS and Pam3CSK4 stimulations. From this it can be seen that LPS stimulation results in a greater number of differentially regulated type 1 IFN genes compared to Pam3CSK4. In addition the magnitude of this differential regulation was much higher in LPS stimulated whole blood compared to Pam3CSK4 stimulated whole blood and the peak of this transcriptional response following both LPS and Pam3CSK4 was at 6 hours. .The similarity in NFκB signalling and difference in IFN signalling following Pam3CSK4 and LPS stimulation is reflected in the IPA canonical pathways. There is similarity in the IPA canonical pathway “NFκB signalling” at 3 hours (the peak of significance for this pathway following LPS and Pam3CSK4 stimulations) following LPS and Pam3CSK4 stimulation. However there is clear difference IFN Signalling canonical pathway at 6 hours  where there is seen to be difference following LPS and Pam3CSK4 stimulations .In vivo LPS stimulated human whole blood data was obtained from the Gene Expression Omnibus  and IRF and STAT transcriptional regulators   . Analysiin vitro upstream analysis and seen to be differentially expressed in-vitro and were also significantly differentially expressed following in vivo LPS administration  seen mainly following LPS stimulation. This significant difference in the IFN response between TLR4 and TLR2 ligation could be seen as early as 1 hour post stimulation in our study, and reinforced in the later time points despite the potentially complex autocrine and paracrine signalling in the human whole blood system.The common early transcriptional response following both LPS and Pam3CSK4 stimulations at 1 hour was characterised by highly expressed cytokines and chemokines  as well as the significant upregulation of transcriptional regulators including the NFκB family  and AP-1/CREB . The gene ZFP36 which encodes for the protein Tristetraprolin was also identified as significantly up regulated by 1 hour which has been shown to act in a post-transcriptional regulatory role by binding to the mRNA of some cytokines and promoting their degradation ex-vivo human blood leukocytes. Slight differences in the timing of peaks observed in our study and these other studies could potentially be explained by the difference in time points sampled or differences in the systems used Following LPS stimulation a differential transcriptional response of IFN regulated/regulatory genes was observed as compared to Pam3CSK4. This difference was detectable as early as 1 hour post LPS stimulation with the significant upregulation of the cytokine genes IFNB1 and IFNG and the transcriptional regulator IRF8 following LPS and not Pam3CSK4 stimulation. The IRF and STAT  genes were seen to be differentially regulated following LPS stimulation and the peak of their expression was at 6 hours. This difference in induction of IRF and STAT transcription regulators following TLR4 and not TLR2 ligation is consistent with previous studies which have compared the temporal transcriptional response following TLR2 and TLR4 ligation in murine DCs and macrophages in vitro transcriptional studies in-vivo stimulation where transcriptional difference peaked between 4 to 6 hours and by 24 hours the transcriptional signature had returned to baseline in vivo compared to in vitro and these differences in expression and persistence of differential expression observed in vitro compared to in vivo could be explained by trafficking/removal of activated immune cells out of the circulation in vivo, which is not possible to be represented in an in vitro whole blood system.Following LPS stimulation the peak of differential transcription compared to media control was at 6 hours in terms of the number of differentially regulated genes. At 24 hours 1443 genes were still significantly differentially regulated. This persistence of differential transcription at 24 hours is observed in human LPS stimulated in vitro stimulated human whole blood for transcriptomic analysis of the early innate immune response has potential advantages over the use of isolated immune cells as whole blood stimulation can be carried out in a laboratory where the expertise or equipment to isolate immune cells from blood is lacking. In addition the volume of blood needed for stimulation is much less than that required to isolate immune cells, meaning experiments can be performed in populations where access to larger volumes of blood is not possible e.g. paediatrics or it could be possible to undertake more stimulations/time points with a given volume. The results however must be interpreted in the context of the complex autocrine/paracrine interactions which occur in a mixed cell culture and therefore stimulation of isolated immune cells will remain advantageous to interrogate in detail a specific transcriptional response.The ability to utilise We have shown that human whole blood can be used to study the early temporal transcriptional response following TLR2 and TLR4 ligation and that the results obtained are comparable to those from isolated murine and human immune cells.Figure S1Activation of samples by culture conditions. (A) Heatmap of expression, clustered by transcripts, shows that 2 individuals (out of 6) media controls show activation (marked with arrows), the genes differentially expressed in these 2 media control samples are similar to LPS, but lower in magnitude. Transcripts identified by normalisation to 0 hour samples, filtering by detection from background, statistical filtering (2 way ANOVA with Benjamini Hochberg p<0.01) and then transcripts retained whose expression was >1.8 FC from another condition. (B) Activation is not due to length of time in transport conditions, nor is it individual specific. The single individual shown here had not previously activated, and was included in final dataset. Blood was collected at time point 0 and left in sealed vacutainers, the vacutainers were opened at one hour intervals and 1 ml human whole blood was either immediately mixed with Tempus solution (labelled as Direct from vacutainer) or plated for 3 hours with either media control (RPMI-1640 with GlutaMAX) or LPS (1 ng/ml) and then mixed with Tempus solution. Reagents and containers (including vacutainers) are endotoxin free (undetectable by Limulus assay - sensitivity <0.03 EU/ml). Heatmap of expression , clustered by transcripts shows that regardless of length of time in vacutainer activation occurred in all media control samples, and is not observed in the direct from vacutainer samples, implying that activation is dependent on culture conditions and is not a function of length of time ex-vivo or length of time spent in the vacutainers. Transcripts identified by normalisation to median of “Direct from vacutainer” samples, filtering by detection from background, statistical filtering (ANOVA with Benjamini Hochberg p<0.01) and then transcripts retained whose expression was >1.8 FC from another condition.(TIF)Click here for additional data file.Figure S2Transcriptional changes in media controls over time.(A) Heatmap of normalised expression values of 377 transcripts which were identified to be significantly differentially expressed overtime  and then transcripts retained whose expression was >1.8 FC from the 0 hour samples. Plotted above the heatmaps is the number of significantly expressed genes (mapped in IPA) that were differentially expressed at each time point by more than 1.8 FC compared to the 0 hour samples. (B) Top ten IPA canonical pathways of the significantly expressed genes at 24 hours, with the –log p value for the pathway and the significantly differentially expressed genes listed for each pathway.(TIF)Click here for additional data file.Figure S3Metallothionein gene expression. (A) Heatmap of averaged Metallothionein mRNA expression over time following LPS or Pam3CSK4 stimulation, values normalised to the median of the 0 hour. Note asynchronous scale.(TIF)Click here for additional data file.Figure S4Interferon regulated genes. Heatmap of averaged expression values of Type 1 Interferon regulated genes (List obtained from Interferome v2.0), normalised to the median of the 0 hour, genes retained if they were expressed greater than 1.8 FC from media control in at least one stimulation in one or more time points (resulting in 1105 genes). Graphed above heatmap is the mean absolute fold change of these Type 1 interferon regulated genes.(TIF)Click here for additional data file.Figure S5Real time PCR. Real time PCR of selected genes following LPS and Pam3CSK4 stimulations and media controls, normalised to GAPDH expression. Mean fold change calculated between media controls and stimulations.(TIF)Click here for additional data file.Table S1Volunteer whole blood composition measured by Celltac Automated Hematology Analyzer  at time point 0 hour.(TIF)Click here for additional data file.Table S2Pearson correlations for k-means derived clusters from .(TIF)Click here for additional data file.File S1Listings of transcripts, LPS k-means clusters from .(XLSX)Click here for additional data file.File S2Listings of transcripts, Pam3CSK4 k-means clusters from.(XLSX)Click here for additional data file.File S3LPS time course data. Listings of transcripts from 4777 significant transcript list whose mean expression was 1.8 FC different to media control at each time point from (XLSX)Click here for additional data file.File S4Pam3CSK4 time course data. Listings of transcripts from 1202 significant transcript list whose mean expression was 1.8 FC different to media control at each time point from (XLSX)Click here for additional data file.


Type 2 diabetes is a major health problem in the Australian Indigenous population. Aboriginal Community Controlled Health Organisations (ACCHOs) are a primary care setting where there is opportunity to partner with health services to reduce the current evidence-practice gap in the provision of health care for Type 2 diabetes. The aim is to examine the effectiveness of a tailored ecologically-based collaborative model in achieving adherence to best practice clinical guidelines for Type 2 diabetes in ACCHOs. This study will examine whether the model results in improvements in diagnostic testing, monitoring and control of diabetes using reliable objective clinical indicators.ACCHOs across Australia who use the Communicare data management system, have at least one doctor providing health care and use an electronic system for pathology results will be eligible for the study. A cluster randomised wait -control design will be used in 18 ACCHOs (9 intervention and 9 wait control). Cross-sectional measurement of the proportion of eligible patients receiving National Health and Medical Research Council (NHMRC) recommended diabetes diagnostic testing, monitoring and control at each ACCHO will be completed at baseline and follow-up.At baseline the mean proportion of patients from clinics who: 1) received diagnostic testing was 58.5% (SD 23.4%) in intervention clinics and 71.4% (SD 14.5%) in control clinics; 2) appropriate monitoring for type 2 diabetes was 49.1% (SD 12.4%) in intervention clinics and 50.7% (SD 18.4%) in control clinics; and 3) appropriate control was 29.2% (SD 7.1 %) in intervention clinics and 34.2% (SD 8.4%) in control clinics.A significant evidence practice gap exists in this setting with a vulnerable population for type 2 diabetes care. The study has a number of potentially significant outcomes including the provision of a model for engaging ACCHOs in examining performance and improving their implementation of best evidence-practice; the development of resources such as staff orientation manuals; the use of naturally occurring reliable data sets for monitoring and feedback and as a tool for intervention delivery and outcome evaluation which may be generalisable to other populations.


Spns2-deficient mice rapidly lost auditory sensitivity and endocochlear potential (EP) from 2 to 3 weeks old. We found progressive degeneration of sensory hair cells in the organ of Corti, but the earliest defect was a decline in the EP, suggesting that dysfunction of the lateral wall was the primary lesion. In the lateral wall of adult mutants, we observed structural changes of marginal cell boundaries and of strial capillaries, and reduced expression of several key proteins involved in the generation of the EP , but these changes were likely to be secondary. Permeability of the boundaries of the stria vascularis and of the strial capillaries appeared normal. We also found focal retinal degeneration and anomalies of retinal capillaries together with anterior eye defects in Spns2 mutant mice. Targeted inactivation of Spns2 in red blood cells, platelets, or lymphatic or vascular endothelial cells did not affect hearing, but targeted ablation of Spns2 in the cochlea using a Sox10-Cre allele produced a similar auditory phenotype to the original mutation, suggesting that local Spns2 expression is critical for hearing in mammals. These findings indicate that Spns2 is required for normal maintenance of the EP and hence for normal auditory function, and support a role for S1P signalling in hearing.Spinster homolog 2 (Spns2) acts as a Sphingosine-1-phosphate (S1P) transporter in zebrafish and mice, regulating heart development and lymphocyte trafficking respectively. S1P is a biologically active lysophospholipid with multiple roles in signalling. The mechanism of action of Spns2 is still elusive in mammals. Here, we report that  Spns2 gene, affecting the release of a lipid called sphingosine-1-phosphate, which has an important role in several processes in the body. For the first time, we report that this molecular pathway is required for normal hearing through a role in generating a voltage difference that acts like a battery, allowing the sensory hair cells of the cochlea to detect sounds at extremely low levels. Without the normal function of the Spns2 gene and release of sphingosine-1-phosphate locally in the inner ear, the voltage in the cochlea declines, leading to rapid loss of sensitivity to sound and ultimately to complete deafness. The human version of this gene, SPNS2, may be involved in human deafness, and understanding the underlying mechanism presents an opportunity to develop potential treatments for this form of hearing loss.Progressive hearing loss is common in the human population but we know very little about the molecular mechanisms involved. Mutant mice are useful for investigating these mechanisms and have revealed a wide range of different abnormalities that can all lead to the same outcome: deafness. We report here our findings of a new mouse line with a mutation in the  Spinster homolog 2 (Spns2) is a multi-pass membrane protein belonging to the Spns family. Though the functions of Spns1 and Spns3 are largely unknown, Spns2 is known to act as a sphingosine-1-phosphate (S1P) transporter, based upon previous studies in zebrafish and mouse Spns2-deficient mice were initially discovered to be deaf during a large-scale screen of new mouse mutants carried out by the Sanger Institute's Mouse Genetics Project (MGP). The MGP uses the KOMP/EUCOMM resource of over 15,000 genes targeted in embryonic stem (ES) cells and aims to generate new mutants and screen them for a wide range of diseases and traits to reveal the function of 160 mutant genes each year Spns2-deficient mice showed early onset of hearing loss that progressed rapidly to profound deafness. This was associated with declining endocochlear potential (EP), which appeared to be the primary physiological defect. At later stages we observed degeneration of sensory hair cells and decreased expression of several key genes required for normal generation of the EP in the lateral wall of the cochlea, but these appeared to be secondary effects. By producing and analysing different conditional knockouts, we established that Spns2 expression was required locally in the inner ear rather than systemically. Our study suggests a vital role for Spns2 and S1P signalling in hearing.Spns2 gene . Quantitative real-time PCR revealed that residual transcript of Spns2 in cochleae, eyes and livers of the homozygous mice was substantially reduced compared to that of the heterozygous and wildtype mice . In other aspects of their phenotype, tm1b/tm1bSpns2 mice were broadly similar to tm1a/tm1aSpns2 mice . tm1a/tm1aSpns2 mice were the first to be available and were used for most experiments in this study, and may be more relevant to human disease because most disease-causing mutations reduce rather than eliminate gene activity.The introduction of a cassette with an additional splice acceptor site is predicted to interrupt normal transcription of the ns2 gene  and geneype mice . In ordetm1a/tm1aSpns2 mice had profound hearing impairment during screening at 14 weeks old by auditory brainstem response (ABR) measurement  suggesting normal vestibular function. ABR measurements were recorded at younger ages to find out the time of onset of hearing loss. Hearing impairment in tm1a/tm1aSpns2 mice can be detected as early as 2 weeks of age at high frequencies .The increase in ABR thresholds preceded degeneration of the organ of Corti suggesting that these were secondary changes rather than the primary cause of the hearing impairment.+ into the endolymph and generation of the endocochlear potential (EP) +-rich extracellular fluid, into the sensory hair cells through mechanoelectrical transduction channels. The lateral wall of the cochlea is composed of the stria vascularis, spiral prominence and the spiral ligament. A defect in the function of any of these components could interfere with the generation of the EP. Therefore, we measured the EP to evaluate the function of the lateral wall. The EP values of the control mice were normal, around 99 to 120 mV, which matched their normal hearing. tm1a/tm1aSpns2 mice had abnormally low EP values of 2 to 41 mV at both P21 and P28 when they were profoundly deaf , associated with irregular layout of marginal cell boundaries  in patches along the length of the cochlear duct, and apparently increased branching , Kv7.1 (Kcnq1), Cx26 (Gjb2), Cx30 (Gjb6), Na+, K+-ATPase (Atp1a1), NKCC1 (Slc12a2) and ZO-1 (Tjp1). In homozygotes aged P14  by crossing the tm1aSpns2 allele to a line expressing Flp recombinase to excise the inserted cassette  mice have profound hearing loss and propose an underlying mechanism: a rapid decline in EP paralleling loss of auditory sensitivity and preceding degeneration of hair cells, suggesting that the primary lesion is in the cochlear lateral wall, the site of EP production and maintenance Spns2 is expressed in hair cells as well as in the lateral wall, we cannot exclude the possibility that disruption of Spns2 function in the organ of Corti also contributed to raised ABR thresholds and hair cell degeneration. Analysis of mice with conditional knockout of Spns2 in hair cells and other cochlear cell types will be useful in dissecting the role of Spns2 further.Here, we report that in vitroSpns2 was expressed in blood vessels of the inner ear including spiral modiolar vessels. Any reduction in local S1P level due to Spns2 dysfunction may weaken vasoconstriction and explain the dilation of strial capillaries in tm1a/tm1aSpns2 mice. The relationship between capillary size and EP value is not unidirectional; both smaller and larger strial capillaries have been reported in different mouse mutants with low EP Spns2-deficient and control animals tm1a/tm1aSpns2 mice. BSA is a medium molecular mass tracer (66.4 kDa), so tracers of different sizes and properties such as Evans blue and cadaverine Spns2 acts as a transporter of S1P Spns2-deficient mice. However, the expression of most of these proteins appeared normal at the time when the EP has already started to decline at P14, suggesting that these are likely to be secondary effects. The morphological changes of marginal cell boundaries and reduction in marginal cell density together with a lack of expression of Kcnq1 in affected cells are also likely to be secondary changes because these features were normal when hearing started to deteriorate at P14 and they did not affect strial permeability. Loss of Kcnq1 expression in marginal cells with expanded luminal surfaces may be a common consequence of strial dysfunction because it has been reported in several different mutants with reduced EP Spns2-deficient mice needs further investigation.We found decreased expression of several proteins critical for normal EP production at 5–6 weeks of age in Spns2 expression was consistently in the hair cells and spiral prominence. The function of the spiral prominence is unclear. Two types of voltage-dependent K+ currents are expressed in spiral prominence epithelial cells, which may play a role in the homeostasis of inner ear fluids Pds), and the Pds mutant mouse also shows reduced EP Pds mutant shows a severe early developmental defect of the inner ear with extensive hydrops Spns2 mutants. This indicates that Spns2-deficient and Pds-deficient mice may have different mechanisms underlying the reduced EP and hearing impairment. Dysfunction of the spiral prominence in Spns2-deficient mice may be the main trigger of reduction of the EP and a series of pathological changes in inner ears.The most robust labeling for + recycling and are considered to mediate K+ translocation between the epithelial cell network of the organ of Corti and the fibrocyte network of the lateral wall, and to facilitate ion flow directed towards the stria vascularis One later change we saw in the lateral wall was a localised decrease of Gjb2 and Gjb6 expression in the type II fibrocytes of the spiral ligament located adjacent to the spiral prominence. Type II fibrocytes are important for KS1pr2-null mice are deaf and share some pathological changes with Spns2-deficient mice, such as disorganized cell boundaries of marginal cells, dilated capillaries in the stria vascularis, and degeneration of the organ of Corti S1pr2-null or S1pr2/S1pr3 double null mice, no overt vestibular defects were found in Spns2-deficient mice. Thus, we propose that the Spns2-S1P-S1PR2 signalling axis is important for normal hearing function. A similar Spns2-S1P-S1PR2 signalling axis may exist in bones as both S1pr2-deficient Spns2-deficient S1P is a bioactive lipid and acts as a second messenger intracellularly and as a ligand for cell surface G protein-coupled receptors extracellularly Spns2 function in blood vessel or lymphatic endothelial cells, platelets or red blood cells did not affect hearing, suggesting that systemic loss of Spns2 activity in these tissues does not mediate the hearing loss we see in the tm1aSpns2 mutants. However, when we deleted Spns2 locally in the inner ear using the Sox10-Cre recombinase, the resulting mutants were deaf. Sox10 is expressed throughout the otic epithelium from an early stage of development as well as in cranial neural crest-derived cells, so can effectively drive deletion of exon 3 of the tm1cSpns2 allele in the entire inner ear tm1a/tm1aSpns2 mice is due to local loss of Spns2 function in the inner ear.Systemic disruption of tm1apns2 and Stm1bpns2 homozygous mutants. We did not see anterior eye defects in any of the 5 conditional alleles, consistent with normal anterior eye development in another conditional Spns2;Tie2-Cre mutant mouse Spns2 also plays a role in retinal blood vessels. Our results showed that global Spns2 knockout resulted in a mild phenotype of the retinal vasculature (thin and irregular veins) with decreased pericyte coverage in the central retina which may be related to the widely known role of S1P signalling in angiogenesis Spns2 in these tissues. We also found focal retinal degeneration in these mutant eyes suggesting a role for Spns2 in the photoreceptor and/or retinal pigment epithelium. Taken together, these findings suggest that SPNS2 is not only a candidate gene for involvement in deafness, but also for deaf-blind syndromes.Defects of the anterior eye were only seen in the SSpns2-deficient mice displayed rapidly progressive hearing impairment associated with a rapid decline in the EP between P14 and P21. The mechanism by which Spns2 deficiency leads to decreased EP merits further investigation, but it most likely involves local S1P signalling. Following the early drop in the EP, later changes include reduced expression of key proteins involved in cochlear homeostasis and ultimately sensory hair cell loss. Our findings suggest that Spns2 is a promising candidate gene for human deafness. Furthermore, Spns2-deficient mice may serve as a model to learn more about the role of S1P signalling in auditory function and the mechanism underlying at least one form of strial hearing loss.In summary, we report here that Mouse studies were carried out in accordance with UK Home Office regulations and the UK Animals (Scientific Procedures) Act of 1986 (ASPA) under a UK Home Office licence, and the study was approved by the Wellcome Trust Sanger Institute's Ethical Review Committee. Mice were culled using methods approved under this licence to minimize any possibility of suffering. The mice were maintained in individually-ventilated cages at a standard temperature and humidity and in specific pathogen-free conditions. Either sex was used for this study.Spns2 mutant allele we used carries a promoter-driven cassette designed to interrupt normal gene transcription but flanked by Frt sites to enable its removal and conversion to a conditional allele with a critical exon surrounded by LoxP sites WtsiSpns2, abbreviated to tm1aSpns2 in this study. A schematic of the knockout-first design of tm1aSpns2 allele is shown in tm1aSpns2 was confirmed by a series of genotyping PCR analyses The tm1aSpns2 colony was maintained on a mixed genetic C57BL/6BrdTyrc-Brd;C57BL/6Dnk;C57BL/6N background. tm1a/tm1aSpns2 mice were crossed to Tg(CMV-Cre)Brd/WtsiHprt transgenic mice (on a C57BL/6NTac background) with systemic expression of Cre recombinase to remove the cassette and produce mice carrying the tm1bSpns2 allele /WtsiGt(ROSA)26Sor mice expressing Flp recombinase ubiquitously in which the promoter-driven cassette was excised and exon 3 was retained flanked by LoxP sites 1Wdr) Spns2 in the inner ear and craniofacial neural crest-derived tissues tm1dSpns2 allele was confirmed by co-presence of tm1cSpns2 and Sox10-Cre allele PCR bands. Since S1P is released from different blood cells and endothelial cells, we used mice expressing Tie1-Cre tm1c/tm1cSpns2 mice to produce the conditional knockouts.Spns2 probe was designed to cover the 3′ untranslated region (Applied Biosystem). Hypoxanthine-guanine phospharibosyltransferase (Hprt) was amplified simultaneously  as an internal reference. The relative quantity of Spns2 was calculated using the 2−ΔΔCt method The organ of Corti, lateral wall , eyes and livers of postnatal day (P)4 homozygous, heterozygous and wildtype littermate mice were dissected in RNAlater (n = 3 for each genotype). Total RNA was isolated with QIAshredder columns  and the RNAeasy mini kit . RNA was normalized to the same concentration for cDNA synthesis using oligo dT and SuperScrip II (Invitrogen). Real-time PCR was performed in triplicate for each sample using a CFX connect real time PCR machine (BIO-RAD). The Spns2 due to the LacZ gene inserted in the cassette of tm1aSpns2 allele (Spns2 promotor. Inner ears of P10 and P14 heterozygous and homozygous mice (at least three mice of each age group) were dissected out and fixed in 4% paraformaldehyde (PFA) for 45 minutes to 2 hrs. These were washed twice with PBS and decalcified in 10% EDTA until soft. After a PBS wash and immersing in 30% sucrose, inner ears were embedded in Agarose type VII , then mounted using OCT compound ready for cryosectioning at 14 µm. Sections were treated with Solution A  for 15 mins, then incubated with Solution B (Solution A plus 5 mM K3Fe(CN)6; 5 mM K4Fe(CN)6; 1 mg/ml X-Gal in DMSO) over night at 37°C. Sections were rinsed in water then counterstained in Fast Red to label nuclei, mounted and examined.X-gal staining can be used to visualise the expression of a allele , downstrtm1aSpns2 mice were used at the standard maximum intensity of 95 dB SPL with free field delivery as shown in Mice were anaesthetised by ketamine hydrochloride  and xylazine hydrochloride  and subcutaneous needle electrodes were inserted on the vertex (active), and over the left (reference) and right (ground) bullae. A calibrated sound system was used to deliver free-field click (0.01 ms duration) and tone pip  stimuli at a range of intensity levels in 5 dB steps. Averaged responses to 256 stimuli, presented at 42.2 per second, were analysed and thresholds established as the lowest sound intensity giving a visually-detectable ABR response The temporal bones were isolated. The inner ears were dissected out and fixed by 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer with 3 mM calcium chloride at room temperature for 3 hours. Cochleae were finely dissected in PBS. This was followed by further processing using an osmium-thiocarbohydrazide-osmium (OTOTO) method Inner ears  were dissected out and gently perfused with 2.5% glutaraldehyde, 1% paraformaldehyde in 0.1M sodium phosphate buffer with 0.8 mM calcium chloride through the round and oval windows and a small hole in the apex then fixed at room temperature for 2 hours. Secondary fixation was in 1% osmium tetroxide in sodium phosphate buffer for 1 hour. After 5 washes in 0.1 M sodium phosphate buffer, inner ears were decalcified in 0.1M EDTA at 4°C until soft. Then the samples were dehydrated through an ethanol series, staining in 2% uranyl acetate at the 30% ethanol stage, embedded in Epon resin mixed 1∶1 and then 3∶1 in propylene oxide for 30 min and infiltrated overnight under vacuum in neat resin. The samples were embedded at 60°C for 24–48 hours. 1 µm sections were cut through the modiolar plane and stained with toluidine blue for light microscope observation. 60 nm sections were cut on a Leica EM UC6 ultramicrotome, stained in 2% uranyl acetate and aqueous lead citrate and imaged on an FEI Spirit Biotwin 120 kV transmission electron microscope using a Tietz F4.15 CCD.Mice were anaesthetized with 0.01 ml/g body weight of 20% urethane, a tracheal cannula was inserted and the bulla was opened to reveal the cochlea while the body temperature was kept at 37°C by a feedback-controlled heating pad. A small hole was made in the bony wall of the cochlea over the basal turn of scala media, and a micropipette electrode filled with 150 mM potassium chloride was advanced through the hole and through the lateral wall into the scala media. The potential difference between the scala media and a reference silver/silver chloride pellet under the dorsal skin was recorded The inner ears were rapidly dissected out and fixed in 4% paraformaldehyde at room temperature for 2 hours. The lateral walls were dissected out in PBS for surface preparation. Filamentous actin was visualized by rhodamine phalloidin  at room temperature for 2 hours. Strial capillaries were visualized by Isolectin B4  at 4°C, overnight in PBS with 10% sheep serum). Samples were mounted with Vectashield Mounting medium  and imaged by confocal microscopy . The numbers of capillary branch points per field (220×220-µm fields) in the middle turn  of the stria vascularis  was quantified using image J. Data were presented as a density in a 100×100 µm field and statistics analysis was conducted using Mann-Whitney Rank Sum Test, SigmaPlot v12.0. Surface preparations were also used for analysis of Kcnq1 expression, using overnight incubation at 4°C with goat anti-Kcnq1 polyclonal antibody  followed by washing with PBS and incubation with donkey anti-goat secondary antibody  prior to analysis by confocal microscopy. At least three homozygotes and three controls were used at P14, P28 and 6 months for phalloidin labeling to show marginal cell boundaries, and P14 and 5–6 weeks for Kcnq1 expression. The density of marginal cells was measured in the phalloidin-labelled whole mount preparations by counting the number of cells defined by their labeled boundaries in two areas each 100×100 µm from the middle turn (40–70%) of each cochlea .+, K+-ATPase (α1 subunit) monoclonal , mouse anti- NKCC1 monoclonal  and rabbit anti-ZO-1 polyclonal . Sections were blocked by incubation with 10% sheep serum (with 0.1% TritonX-100 in PBS) for 40 mins. Sections were incubated with appropriate primary antibodies overnight at 4°C, washed with PBS and incubated with corresponding secondary antibodies at room temperature for 2 hours . After washing with PBS, slides were imaged by confocal microscopy. Three mice of each genotype (tm1a/tm1aSpns2 and +/+Spns2) were used for each antibody at P14 and 5–6 weeks old.The cochleae were dissected out and fixed in 4% PFA at room temperature for 2 hours. Cryosections were obtained as described above for X-gal staining. We used the following antibodies: rabbit anti-Kcnj10 polyclonal , rabbit anti-Gjb2 polyclonal , rabbit anti-Gjb6 polyclonal , mouse anti- NaThe inner ears  were dissected and round and oval windows were opened in PBS containing 1 mM calcium chloride. A hole was made in the basal turn leading to the scala media. The membranous labyrinth was perfused for 5 minutes with 400 µl Sulfo-NHS-LC-Biotin  through the round and oval windows and the hole exposing the endolymphatic compartment. Following a PBS wash, the inner ears were fixed in 4% paraformaldehyde at room temperature for 2 hours, and processed for cryosectioning as described above. The biotin tracer was detected by fluorescein isothiocyanate (FITC)-conjugated streptavidin  incubating at room temperature for 30 min. Samples perfused with PBS alone were used as negative controls.2 inhalation and the auditory bullae dissected out. Whole cochleae were exposed and fixed by removing a small piece of bone at the apex and gently perfusing 4% PFA through the round and oval windows. Cochleae were then immersed in fixative and left on a rotator for 1.5 hours at room temperature. Whole-mounts of stria vascularis were dissected from fixed cochleae, covered with Vectashield Mounting Media  in glass bottom culture dishes (MatTek Corp.) and imaged using confocal laser-scanning microscopy .Mice  were warmed in a 39°C incubator for 5 minutes and then held in a mouse restrainer so that the tail was accessible and the tail vein visible. A 50 µl aliquot of 5% (w/v) FITC-conjugated bovine serum albumin , made up in sterile PBS, was injected into the tail vein. The mice were left at room temperature for 45–60 minutes to allow the BSA-FITC to permeate all capillaries and to allow for any vascular extravasation. The mice were sacrificed by COMice underwent ophthalmic screening at 15 weeks of age. They were assessed for gross morphological changes to the eye using a slit lamp (Zeiss SL130) and ophthalmoscope (Heine Omega 500). The eye was examined both undilated and dilated . Images using the slit lamp were collected using a Leica DFC420 camera. The mice were culled under terminal anaesthesia followed by cervical dislocation and both eyes from 3 male homozygous mutants and 3 wildtype mice were removed and fixed. Pupil-optic nerve sections were processed, stained with hematoxylin and eosin, and standard images were captured under light microscopy for review For whole mount retinal analysis, heterozygotes and homozygotes were used  and the eyes removed and fixed in 4% PFA. Retinae were prepared and stained as described Figure S1Spns2 expression in the vestibular system, normal gross structure of inner ears and normal organ of Corti at P4. A,B: X-gal staining showed expression of Spns2 in the vestibular system at P10. Labelling (blue) was detected in the cristae  and maculae (utricular macula shown here) . Scale bar: 20 µm. C,D: Cleared inner ears showed no apparent differences in gross structure between Spns2 homozygous mutants and controls at 4 weeks old. Scale bar: 1 mm. E,F: Scanning electron microscopy showed no abnormalities of the surface of the organ of Corti at P4 in the Spns2 homozygous mutants compared with littermate controls. Scale bar: 10 µm.(TIF)Click here for additional data file.Figure S2A,B: Confocal images focussed at the level of the basal cell boundaries, visualised by phalloidin staining (red). No obvious change was seen in Spns2 homozygous mutants at 4 weeks old. Scale bar: 10 µm. C,D: The stria vascularis showed dilated and tortuous capillaries with increased branch points in all five Spns2 homozygous mutants studied at 4 weeks old. Strial capillaries were visualised by isolectin B4 (green). Scale bar: 50 µm. E,F: Strial hyperpigmentation was pronounced in older mutants. A seven month old Spns2 homozygous mutant (F) had obvious accumulation of pigment in the stria vascularis. Scale bar: 20 µm.Whole-mount stria vascularis examination. (TIF)Click here for additional data file.Figure S3+/K+-ATPase, NKCC1 and ZO-1 in lateral wall and Kcnj10 in spiral ganglion at 5–6 weeks. Na+/K+-ATPase (red) labelling in stria vascularis and type II fibrocytes in Spns2 homozygous mutants (A) was comparable with that of controls (B). Notice absence of Gjb2 labelling in the type II fibrocytes in the mutants. NKCC1 (red) labelling in the Spns2 homozygous mutants was located in stria vascularis and type II fibrocytes and appeared similar in the controls . ZO-1 (green) labelling was present in the basal cells of the stria in both Spns2 homozygous mutants and controls . G,H: Acetylated α-tubulin (red) labelled spiral ganglion neurons and Kcnj10 (green) labelled satellite cells. Kcnj10 expression in Spns2 homozygous mutants was present and comparable with controls, suggesting that the reduced Kcnj10 labelling observed in the stria Click here for additional data file.Figure S4A: Analysis of the percentage of pericyte coverage of the retinal blood vessels revealed a significantly reduced coverage in the mutants in the central retina , but no significant difference in coverage of the peripheral vessels   between the Spns2 mutant homozygotes and heterozygous controls at P10. B: tm1aSpns2 homozygous mutants displayed open eyelids at birth.Pericyte coverage of retinal blood vessels and open eyelids at birth. (TIF)Click here for additional data file.


The rate and extent of CD4 cell recovery varies widely among HIV-infected patients with different baseline CD4 cell count strata. The objective of the study was to assess trends in CD4 cell counts in HIV-infected patients after initiation of antiretroviral therapy in Tigray, Northern Ethiopia.A retrospective cross-sectional study was conducted by reviewing medical records of HIV patients who received antiretroviral treatment at twenty health centers in Tigray region during 2008–2012. Multi-stage cluster sampling technique was employed to collect data, and the data were analyzed using SPSS version 20.0 software.The median change from baseline to the most recent CD4 cell count was +292 cells/μl. By 5 years, the overall median  CD4 cell count was 444(263-557) cells/μl while the median (IQR) CD4 cell count was 342(246-580) cells/μl among patients with baseline CD4 cell counts ≤200 cells/μl, 500(241-557) cells/μl among those with baseline CD4 cell counts of 201–350 cells/μl, and 652(537-767) cells/μl among those with baseline CD4 cell counts >350 cells/μl. Higher baseline CD4 cell counts and being male were independently associated with the risk of immunological non-response at 12 months. Furthermore, it was also investigated that these factors were significant predictors of subsequent CD4 cell recovery.Patients with higher baseline CD4 cell stratum returned to normal CD4 Cell counts though they had an increased risk of immunological non-response at 12 months compared to those with the least baseline CD4 cell stratum. The findings suggest that consideration be given to initiation of HAART at a CD4 cell count >350 cells/μl to achieve better immune recovery, and to HIV-infected male patients to improve their health seeking behavior. CD4 cell counts are commonly used markers of HIV disease progression and for starting and monitoring antiretroviral treatment in the absence of viral load ,2. SustaCommencement of ART at a baseline CD4 cell count <200 cells/μl may lead to a high proportion of patients with acquired immunodeficiency syndrome (AIDS) and a high case fatality rate ,7. A sigTo the best of our knowledge, there are no studies from the study area concerning rates of CD4 cell recovery and rates of immunological non-response to ART among HIV-infected patients. Therefore, we conducted a retrospective study in order to assess the trends of CD4 cell recovery among HIV patients after initiation of antiretroviral therapy in Tigray, Northern Ethiopia.The study was conducted in Tigray region, Northern Ethiopia. Twenty health centers in the six administrative zones of the region were included in the study. A retrospective study was conducted by reviewing medical records of HIV patients aged 18 years or greater who received antiretroviral treatment at twenty health centers in Tigray region, Northern Ethiopia during 2008–2012. ART delivery was started in September 2003 as fee service and the free ART program started in March 2005. Pregnant women, seriously ill and diabetic patients were also excluded.The required sample was calculated using a single population proportion formula. The following parameters were taken into account during the calculation of sample size: 50% rate of CD4 cell count recovery among HIV-infected patients (to achieve maximum representative sample size), 95% confidence interval and 5% margin of error. Then the determined sample size, after considering 10% non-response rate, was multiplied by 2 to consider the cluster effect and increase power. Thus, a required sample size of 844 medical records of HIV patients was required for this study.Multistage cluster sampling technique was used to select the different health centers. Hence, twenty health centers were randomly selected proportionally among the sixty health centers addressed by MSH/USAID care and support programme from the six administrative zones found in Tigray region. Study participants from the selected health centers were chosen by systematic random sampling method.A baseline and 6-monthly CD4 cell count, and basic information such as patients’ sex, age, weight and WHO clinical stage were collected from medical records.Data were entered and analyzed using SPSS for Windows, version 20.0. The median (IQR) in the absolute CD4 cell count at baseline and every six months thereafter was determined. Changes in CD4 cell count every six months were also examined and stratified on the basis of baseline CD4 cell count .Categorical variables were summarized as frequencies and percentages while numerical variables with non-normal distribution were summarized as median and IQR. Logistic regression was conducted to examine factors associated with the risks of immunological non-response (defined as failure to attain an absolute CD4 cell count increase from baseline of at least 50 cells/μl at 12 months of ART). A multivariate least-squares linear regression analysis was employed to assess changes in CD4 cell count on the basis of baseline CD4 cell count and these other covariates. An initial analysis included all covariates. A final multivariate model was computed using a backward stepwise approach, retaining only those variables that were statistically significant. All tests of significance were two-sided, with p<0.05 indicating statistical significance.The study was approved for ethical issues by the Health Research Ethics Review Committee of College of Health Sciences, Mekelle University. Data collection was conducted after further approval of the study by the Tigray regional health bureau and directors of each health centers. The purpose of the study was explained to the study participants and all study participants gave a written informed consent.A total of 841 medical records were reviewed; most (68.7%) of the patients were female. At baseline majority (79.5%) of the patients were less than 45 years old with a mean age (SD) of 36.34 (9.48) years. More than half (56.2%) of the patients were in WHO clinical stage III at baseline. About two-third (64.4%) of the patients were found to have baseline CD4 cell counts less than 200 cells/μl. The weight distribution shows that nearly three-forth (75.1%) of patients were found to have weight of 40–60 Kg .th and 60th months where the median CD4 cell count showed a fall. The median change from baseline to the most recent CD4 cell count was +292 cells/μl, with the overall median (IQR) CD4 cell count increased to 444(263–557) cells/μl by 5 years after the start of ART. The overall median (IQR) CD4 cell count in our HIV patients increased by about 2-fold and 3-fold from baseline 152 (98–224) cells/μl, reaching 300(201–398) and 460(240–704) cells/μl at 12 and 48 months after initiation of ART, respectively. By 5 years, the median (IQR) CD4 cell count increased to 342(246–580) cells/μl among patients with a baseline CD4 cell count of ≤200 cells/μl, to 500(241–557) cells/μl among those with a baseline CD4 cell count of 201–350 cells/μl, and to 652(537–767) cells/μl among those with a baseline CD4 cell count of >350 cells/μl. The median (IQR) CD4 cell count of patients with a baseline CD4 cell count of ≤200 cells/μl were about doubled and tripled by 12 and 36 months after initiation of ART, respectively.The six monthly changes in the median CD4 cell count after the commencement of ART and the number of patients having CD4 data is plotted in An analysis of the six monthly increase in CD4 cell count demonstrated that, the slope of the plotted overall CD4 cell count increased significantly  until 48 months except between 18 and 30 months, where it slightly increased. The proportions of patients who achieved CD4 cell counts of ≥500 cells/μl after receiving 48 months of suppressive ART were 40.8%, 45% and 93.3% among patients with baseline CD4 cell counts of ≤200, 201–350 and >350 cells/μl, respectively. The percentages of patients who achieved CD4 cell counts of ≥750 cells/μl after receiving 48 months of ART were 19.8%, 25% and 60% among patients with baseline CD4 cell counts of ≤200, 201–350 and >350 cells/μl, respectively.Baseline CD4 cell count was evaluated whether it was a risk factor associated with immunological non-response (defined as a CD4 cell increase of <50 cells/μl at 12 months). The percentages of patients failed to attain an absolute CD4 cell count increase from baseline CD4 cell count of at least 50 cells/μl at 12 months was 22.7%. The proportions of patients who were immunological non-responders were 17.9%, 28% and 50% among patients with baseline CD4 cell counts of ≤200, 201–350 and >350 cells/μl, respectively. Furthermore, logistic regression analysis revealed that baseline CD4 cell count was a significant predictor of immunologic failure. An increased risk of immunological non-response was independently associated with higher baseline CD4 cell counts of 201–350  and > 350 cells/μl . An increment of <50 cells/μl was also independently associated with male patients. Male patients had about two times increased odds of risk of immunological non-response compared to female patients  .Multivariate regression analysis shown that baseline CD4 cell count was a significant determinant of subsequent CD4 cell recovery . Two anath and 60th months where the median CD4 cell count showed a slight decline [This retrospective study was carried out to assess the trends in CD4 cell recovery among HIV patients after initiation of ART and the effect of baseline characteristics on CD4 cell count response. Improvements in overall CD4 cell count among the patients was seen over time until year 4 though it declined thereafter. These findings are consistent with the retrospective longitudinal study conducted in eastern Ethiopia where the median CD4 lymphocyte count had improved over the five year period except at the 54 decline . Other s decline . Actuall decline .Immunological response rates were evaluated in patients with different baseline CD4 cell in this survey. These results indicated that patients with baseline CD4 cell counts of ≤200 cells/μ had greater rates of immunological response compared to those with higher baseline CD4 cell counts. These findings are in line with that of another study where patients with the lowest CD4 counts had similar or greater rates of CD4 cell recovery and a lower risk of immunological non-response . HoweverOn the other hand, the present work reported that patients with lower baseline CD4 cell counts had lower peaks CD4 cell counts. Patients with a baseline CD4 cell count of >350 cells/μl stratum had CD4 cell counts that returned to almost normal levels. These results are consistent with that of a longitudinal observational study among patients receiving primary HIV care in Baltimore, Maryland which shows only patients with a baseline CD4 cell count of >350 cells/μl had CD4 cell counts that returned to nearly normal levels though CD4 cell count was increased and maintained in all CD4 cell count strata to 6 years . Other sThese findings indicate that the highest CD4 cell counts were achieved when HAART was started at a baseline CD4 cell count >350 cells/μl. The CD4 cell count is less likely to return to normal when HAART is started at lower CD4 cell counts, this could be a reason to consider initiating HAART before the CD4 cell count decreases to <350 cells/μl.In this study, an increased risk of immunological non-response was significantly observed among male patients like another study . Other sIn conclusion, the present findings confirm the previous studies that the degree of CD4 depletion prior to ART initiation is the most consistent determinant of subsequent immune reconstitution. Low baseline CD4 count at entry to an ART programme was associated with increased risks of morbidity and mortality . A meta-


Immunosuppression is the hallmark HIV infection associated with gradual loss of CD4+Tcells. The effector immune response is regulated by CD4+CD25+Foxp3+Tcells (Tregs). Enumeration of Tregs frequency showed equivocal results before and here we showed clearly their frequency by flowcytometry.One hundred and eight individuals comprising of 71 HIV positive patients (44 patients undergoing HAART and 27 without HAART) and 37 HIV negative healthy controls. PBMCs were isolated and stained with CD4/FITC, CD25/APC, FoxP3/PE antibodies (BD Pharmingen) and analyzed for Tregs frequency by flowcytometer .Our study showed that healthy controls had a Tregs frequency of 4.1%. When Tregs frequency analyzed among HIV/AIDS patients who are yet to receive HAART we found a significant reduction in Tregs population i.e. 1.2%. Encouragingly this deep plunge in Tregs population was found to be restored and surged upon HAART up to 12.6%. However, these changes in percentages did not alter based on their gender or age or absolute CD4 count of the study population.HAART is known to decrease viral load and improve the patient’s CD4 cells count. Here we report that HAART has improved the Tregs frequency. It is yet to be known that the consequence of resurging Tregs during HIV/AIDS.


We explored the meaning of variations in tissue oxygen saturation downslope (StO2 target. The slope of the desaturation curve was assessed separately for the first part (StO2 down1) and the last part (StO2 down2) of the curve and the difference between Down2 - Down1 was calculated.In this prospective observational study, NIRS (thenar eminence) was applied every day in 93 patients admitted to the ICU. A VOT was performed using a 40% StO2 Down1 or Down2 between ICU survivors (n = 76) and ICU nonsurvivors (n = 17) over the first 10 days in the ICU, while Down2 - Down1 was higher in ICU nonsurvivors .No significant differences were seen in StOICU nonsurvivors tended to show a flattening in the last part of the desaturation curve during a VOT, suggesting a reduced tissue oxygen extraction. This may depend on microvascular dysfunction and/or cellular hypometabolic status.


The chronic pain sufferer is currently faced with a lack of objective tools to identify the source of their pain. The overarching goal is to develop clinical [18F]FDG PET/MRI methods to more accurately localize sites of increased neuronal and muscular metabolism or inflammation as it relates to neurogenic sources of pain and to ultimately improve outcomes of chronic pain sufferers. The aims are to 1) correlate imaging findings with location of pain symptomology, 2) predict location of symptoms based on imaging findings alone and 3) to determine whether the imaging results affect current management decisions. Six patients suffering from chronic lower extremity neuropathic pain  have been imaged with a PET/MRI system  from mid thorax through the feet. All patients underwent PET/MR imaging one hour after a injection of 10mCi [18F]FDG. Two radiologists evaluated PET/MR images (one blinded and the other unblinded to patient exam/history). ROI analysis showed focal increased [18F]FDG uptake in affected nerves and muscle (approx 2-4 times more) over background tissue in various regions of the body in 5 of 6 patients at the site of greatest pain symptoms and other areas of the body (SUVmax of Target 0.9-4.2 vs. Background 0.2-1.2). The radiologist blind to the patient history/exam was able to correctly identify side/location of the symptoms in 5 out of 6 patients. Imaging results were reviewed with the referring physician, who then determined whether a modification in the management plan was needed: 1/6 no change, 2/6 mild modification  and 3/6 significant modification.


To properly assess the development of Her2 therapeutics in an immune tolerant model, we previously generated a transgenic mouse model in which expression of the human Her2 protein was present in both the brain and mammary tissue. This mouse model has facilitated the development of Her2 targeted therapies in a clinically relevant and suitable model. While heterozygous Her2+/- mice appear to develop in a similar manner to wild type mice (Her2-/-), it has proven difficult to generate homozygous Her2+/+ mice, potentially due to embryonic lethality. In this study, we performed whole genome sequencing to determine if the integration site of the Her2 transgene was responsible for this lethality. Indeed, we report that the Her2 transgene had integrated into the Pds5b (precocious dissociation of sisters) gene on chromosome 5, as a 162 copy concatemer. Furthermore, our findings demonstrate that Her2+/+ mice, similar to Pds5b-/- mice, are embryonic lethal and confirm the necessity for Pds5b in embryonic development. This study confirms the value of whole genome sequencing in determining the integration site of transgenes to gain insight into associated phenotypes.The development of antigen-targeted therapeutics is dependent on the preferential expression of tumor-associated antigens (TAA) at targetable levels on the tumor. Tumor-associated antigens can be generated  Furthermore, the restricted expression of these TSAs on tumor cells allow for the generation of specific therapeutic agents with little on-target/off-tumor effects. However, many antigens are also expressed on normal tissues to varying degrees, and are termed tumor-associated antigens (TAA). Cellular dysregulation that occurs during the oncogenic process can also lead to upregulation of normal tissue proteins. Whilst still present on the tissue cells of origin, this increased level of protein expression can sometimes be correlated with patient prognosis, and therefore can be identified as a TAA [Tumor cells often display an altered array of proteins that distinguish them from the cells of origin. On occasion, these proteins arise from oncogenic mutations, and are termed tumor-specific antigens (TSA), with expression on the tumor cells and conversely absent on the ‘normal’ surrounding tissue. These TSAs are generated as a TAA . Such isas a TAA ,3. Moreoas a TAA –6.de novo [As Her2 is not exclusively present on breast tissue but is also found in the brain, the lower intestine and lung, immunotherapies directed against the Her2 antigen may also encounter on-target/off-tumor effects resulting in toxicity and autoimmunity . In addide novo .Multiple mouse models of the TAA Her2 have emerged and facilitated the study and development of Her2 therapeutics. Two of the most commonly used promoters used to drive the expression of the Her2 antigen are the mouse mammary tumor virus (MMTV) and the whey acidic protein (WAP) promoter. Whilst both are highly expressed in the mammary gland, expression of the MMTV promoter has also been found in other organs, including the kidneys, lungs, testes as well as in T cells ,10. Simiet al. therefore generated the human Her2 (Her2) transgenic mouse model driven by the whey acidic protein (WAP) promoter [In order to adequately assess potential therapies in which the Her2 antigen could be targeted specifically on tumor cells, leaving the surrounding normal tissues with basal expression unaffected, it was necessary to generate a clinically relevant mouse model. Piechocki promoter . In thispromoter ,19–21. T+ tumors. Conversely, Her2 tumors are often rejected in wildtype mice due to the high level of immunogenicity against the Her2 antigen. In addition, DNA vaccination with the Her2 antigen prior to Her2+ tumor challenge induced a robust anti-tumor response, with up to 33% survival of Her2+ mice , indicating these mice were able to overcome tolerance to the Her2 antigen, and successfully mount an immune response. No autoimmunity or off-target responses were recorded in this study [Primary analysis of the Her2 mouse model has demonstrated its tolerance to Her2is study .The tolerance to Her2 tumors in this mouse model has been critical in the development of immunotherapies against the Her2 antigen, particularly in solid established tumor models. The adoptive transfer of T cells genetically modified with a chimeric antigen receptor specific for the Her2 antigen into Her2 tumor bearing mice have demonstrated both tumor regression and prolonged survival in the absence of autoimmunity ,23.+/-) mice display a normal phenotype and are similar to their wild type littermates. However, transgenic mice are often bred in a homozygous state, ensuring the progeny themselves are transgenic and thereby reducing the variability between breeding pairs of heterozygous matings. Attempts at generating homozygous Her2+/+ mice have been unsuccessful, potentially since the inheritance of the two Her2 transgenes in this model resulted in embryonic lethality. As no overt pathology or abnormalities were observed in heterozygous mice, we speculated that it would be unlikely that the inheritance of twice as many Her2 copies would result in this lethality. Rather, we hypothesised that perhaps the integration of the Her2 transgene had interrupted a gene essential for embryonic or fetal development. To answer this, we performed whole genome sequencing (WGS) on Her2+/- mice to determine the integration site of the transgene. Whole genome sequencing revealed the integration of the Her2 transgene had indeed interrupted a gene, Pds5b, whose function is known to be integral in the segregation of chromosomes in both meiosis and mitosis [+/+ mice display greater development defects than previously reported for some Pds5b-/- mice [The generation of transgenic mice often involves the random integration of the desired transgene into the genome. While this integration often has little effect on the normal phenotype of the mice, there may be occasions where transgene integration could potentially disrupt a gene integral for development and survival. Heterozygous Her2 mice  with 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/mL penicillin and 100 ug/mL streptomycin (Life technologies). The murine E0771 (LMC variant) breast adenocarcinoma line was kindly donated by Prof. Robin Anderson  and maintained at 37°C in 10% CO2 in Dulbecco's modified Eagle medium (DMEM), supplemented as above. Both cell lines were retrovirally transduced with the human Her-2 antigen (ERB) as previously described [The murine 24JK fibrosarcoma cell line was kindly donated by Dr. Patrick Hwu   and mainescribed .6 24JKERB or 1 x 105 E0771ERB were injected into Her2+/- mice subcutaneously or 5 x 105 E0771ERB injected into the mammary fat pad. Tumors were measured on days as stated and mice were sacrificed when tumor size reached ethical limit of 150 mm2.1 x 10+ mice were interbred to generate litters consisting of Her2-/-, Her2+/- and Her2+/+ mice. Embryos were harvested from pregnant Her2+/- females from E14 onwards and analysed for signs of malformation and abnormal development. Tail clippings were taken at this time point and analysed for the presence of the Her2 transgene via polymerase chain reaction (PCR).Heterozygous Her2+/- transgenic mice using the DNeasy Blood and Tissue Kit (Qiagen) according to manufacturers’ instructions or using phenol:chloroform extraction. The DNA was quantified and purity verified. 500 ng of DNA were fragmented using a focal acoustic device (Covaris S2) and used to prepare libraries with the KAPA Library Preparation Kit for Illumina platforms (KAPABIOSYSTEMS). Libraries were size selected to an average fragment size of 600 bp using the PippinPrep Instrument (SAGE Science). Three indexed libraries were pooled and sequenced across three lanes of an Illumina HiSeq2500 flowcell using High Output chemistry v3 (Illumina).Genomic DNA was extracted from the tails of Her2http://genome.ucsc.edu) and the putative Her2 transgene sequence . As a target for sequence alignment we concatenate the mm10 build of the mouse reference . The seei et al ). Bowtie+/+, Her2+/- and Her2-/- was isolated from tail clippings of E14.5 to E19 pups as previously mentioned. Briefly, genomic DNA was extracted from tissue samples (~1–2 mm2) using QuickExtract DNA extraction solution . After adding 20 μL QuickExtract solution to each tissue sample, the samples were incubated at 65°C for 20 minutes, then 95°C for a further 20 minutes. The samples were then diluted 1:10 in sterile deionised water, and 1 μL was used for PCR. PCR amplification was performed using the following primers; Pds5b Forward 5’ GGACTATTTACAGGAAACGTC 3’, Pds5b Reverse 5’ AGCAAGCCACCAGTAAACG 3’ and Her2 Forward 5’ GTCACAGGGGCCTCATCC 3’. The Her2 forward primer spanned the junction point of insertion of the transgene concatemer into the chromosome, with the first 14 bp annealing to the truncated 3’ end of the transgene, and the final 4 bp annealing to the Pds5b gene at insertion. DNA was amplified with the following conditions; 95°C for 5 minutes, 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds, repeat step 2 to 4 for 35 cycles, 72°C for 5 minutes. Amplified DNA was analysed on a 1% agarose gel.Genomic DNA of Her2+ tumors have been shown to grow poorly in wild type immunocompetent mice due to the highly immunogenic nature of the antigen. The expression of the human Her2 antigen from birth in the Her2 transgenic mouse models generated a functioning immune system tolerant to the Her2 antigen. As a result, Her2+ tumors were able to sufficiently evade the adaptive immune system and grow without hindrance. To validate tolerance of Her2+/- mice, we first demonstrated the ability of Her2+ tumors to grow in Her2 transgenic mice. The murine sarcoma cell line 24JKERB consistently grew subcutaneously in Her2 transgenic mice , instead of the expected 75%, suggesting perhaps a complete absence of homozygous mice. To further substantiate our hypothesis, we had previously attempted generating homozygous mice through intensive iterative breeding programs however were never able to achieve 100% transgenic progeny. As no overt pathology in terms of development or immune function was noted in heterozygous mice, we hypothesised that perhaps the transgene integration site had interrupted an integral gene. We decided to utilize whole genome sequencing (WGS) to determine the integration site, as it is a fast, efficient and reliable method.The generation of transgenic mouse models often involves the microinjection of the linearised DNA transgene into oocytes at the pro-nuclear stage. For most transgenes, this integration occurs in a non-homologous manner, as often these transgenes are preceded by a highly active promoter thus the transcriptional activity of the surrounding genome (with the exception of silencing epigenetic regulation) is irrelevant to the transgene expression. The Her2 mice were previously generated in such a manner, where the 2.5 kb whey acidic protein (WAP) promoter driving the 4.4kb c-ErbB2 transgene cDNA was digested to create a linearized transgene fragment, which was then microinjected into C57BL/6 oocytes  Fig 2)Fig 2). TCTGGGGATCCTCTAGAGTCGACCTGCAGGCA-TGC) at the 5’ end of the WAP promoter was found using WGS analysis (AGGGGAGGTAACCCTGGCCCCTTTGGTCGG-GGCCCC) between the WAP promoter and Her2 5’ untranslated region (UTR) during our WGS analysis was determined to be homologous to the 5’ UTR of a novel variant of human Her2 cDNA as reported by Yamamoto et al. ([AATAAAGACCCAGGGGGAGAAGCTGGGATCCTCTAGAGTCGACGCATGCAAGCTTNAATGA) at the 3’ region of the Her2 3’ UTR was absent in our WGS analysis. This 61 nucleotide sequence at the 3’ region of the Her2 3’ UTR contained a poly-adenylation sequence, AATAAA (bolded above). As such, the WAP-Her2 transgene found by WGS is lacking an endogenous 3’ poly-adenylation sequence, and it is likely the gene uses a poly-A sequence downstream of the integration site.We observed some non-Her2 sequences using WGS. A 34 nucleotide insert  of the transgenic material into the native DNA of the mice. We performed structural variation analysis on the alignments using Socrates  and usedThe breakpoint detection performed with Socrates predicted 173 fusions in the data. To establish the insertion point we searched for overlap with the transgene sequence within the breakpoint set. There were three fusions that overlapped with the transgene: one from the transgene position 1 to the transgene position 6850, another from chromosome 5 at position 150719804 to the transgene (WAP-Her2) at position 4025, and finally from WAP-Her2 at position 2972 to chromosome 5 at position 150719794. A 10 nt duplication (150719804 to 150719794) was observed at these breakpoints in the host genome. The first fusion was caused by the transgene arranged in a 162 copy concatemer and therefore looping back onto its own start. The latter two breakpoints corresponded to the insertion site of the transgene into the native genome. Using whole genome sequencing, we were able to establish the WAP-Her2 transgene had inserted just 19 nucleotides upstream of exon 3 (150719823) in the Pds5b gene on chromosome 5  or one band for Pds5b . A proportion of embryos produced a single Her2 DNA band, indicating their homozygosity . Her2+/-or Pds5b .+/- mice harboured a large number of copies of the Her2 transgene (162 copies), we hypothesised that a further increase in transgene copies in homozygous Her2+/+ mice (324 copies) was unlikely to produce an additive effect, and thus have very little effect on the overall phenotype in these mice. Instead, we proposed that two copies of the Her2 concatemer present in Her2+/+ mice would completely prohibit normal Pds5b production and function. In addition, we hypothesised that Her2+/+ mice would display a phenotype similar to that of Pds5b-/- mice.The insertion of the WAP-Her2 transgene concatemer was found to lie within an intron (19 nucleotides upstream of exon 3) of the Pds5b gene, presumably disrupting and inhibiting normal transcription and translation of Pds5b. Although the RNA splice acceptor site was retained, the insertion of 162 copies of the transgene, constituting approximately 1.1 Mb, likely prevented effective pre-mRNA processing. As heterozygous Her2-/- models also display a myriad of defects in multiple organs, it is speculated that Pds5b may also play a role in regulating the transcription of a number of genes essential for development and organogenesis [Pds5b  is a homolog of Pds5, a regulatory factor of the cohesin complex. The cohesin complex is essential in dictating accurate chromosomal segregation during sister chromatid cohesion for cell division, in both mitosis and meiosis ,32. Disrogenesis . Furtherogenesis .Pds5b-/- mice have yielded conflicting results, which may be due to varying levels of expression and subsequent penetrance observed in these models [Pds5b-/- mice were found to survive to birth but died shortly after. These mice displayed signs of labored breathing, exhibited respiratory distress and had cardiac abnormalities, all of which may have contributed to their early death [Pds5b-/- mice presented with signs of growth retardation, including facial dysmorphisms, smaller head and limb structures and abnormal cleft palates. However, other studies using Pds5b-/- have observed a more severe phenotype, with embryonic lethality occurring at the late post-implantation stages (E16.5 to E18.5), with no Pds5b-/- mice surviving until birth (0 out of 500) [Pds5b alleles greatly affected embryonic development and resulted in embryonic death, however no centromeric cohesion defects were observed in Pds5b+/- mouse embryonic fibroblasts (MEFs) [While highly conserved, the function and necessity of Pds5b greatly varies between species . Pds5b-e models . In one ly death . The rol of 500) . In thats (MEFs) .+/- x Her2+/- mice to generate litters of Her2+/+, Her2+/- and Her2-/- mice. As developmental defects observed in Pds5b-/- mice were only apparent at E14.5 to E16.5 [+/+ mice and Her2+/- or Her2-/- mice. Her2+/+ mice taken at E14-E15 displayed obvious developmental defects, were severely underdeveloped and appeared much smaller than either Her2+/- or Her2-/- mice  was the most efficient and accurate method for determining the transgene integration site in our studies. Prior to WGS, determining the integration point of a transgene required a combination of multiple assays, each laborious and time consuming. Fluorescent in situ hybridisation (FISH) requires the generation of a sequence specific probe, and can only reveal the location of the transgene on a global genome scale. To determine the exact nucleotide location, TAIL-PCR or primer walking would have to be applied [In the present study, homozygous Her2 applied . FinallyPds5b. The WAP-Her2 concatemer consisted of 162 copies of the transgene, each orientated in a ‘head to tail’ manner. Taking into account the length of the transgene and the high copy number present, we hypothesise the integration of the WAP-Her2 transgene would have isolated the Pds5b promoter from the coding sequence downstream of the integration site, and thus effectively disrupted accurate transcription of the Pds5b gene. We cannot conclude whether an insertion of this size may have also impacted transcription of the genes flanking the Pds5b gene, and thus potentially have contributed to the observed phenotype. However, the similarities between our mice and other models deficient in Pds5b lead us to hypothesize the main phenotypic characteristics of this model was due to the dysregulated expression of Pds5b.Our results from WGS revealed the WAP-Her2 transgene had formed a long unidirectional concatemer and had integrated at a single position in the genome, upstream of exon 3 of the protein-coding gene, It was interesting to note both the copy number and orientation of the WAP-Her2 transgene in the Her2 tg mice. Whole genome sequencing analysis revealed 162 copies (in heterozygous mice) were present and moreover, that all 162 copies were ligated together and had in fact integrated into one location. Furthermore, the orientation of each transgene in the transgene polymer was observed to be ligated in a unidirectional manner and always present in a 5’ to 3’ orientation. Other reports characterizing transgenic mice models have observed similar results, where the transgene has been found to concatenate into a long unidirectional concatemer, with each transgene present in a ‘head to tail’ orientation –42. ThisPds5b. This confirmed the gene disruption. While further downstream analysis of transcriptional and translational expression of Pds5b, such as RT-PCR or western blot analysis, were not performed, the complete absence of Pds5b at a genomic level in addition to the striking similarities observed between our Her+/+ and other published Pds5b-/- models rendered further analysis of downstream expression beyond the scope of the present study, whose aim was to demonstrate the ability of WGS to provide insight into the difficulty in generating homozygous transgenic mice.We further substantiated the WGS results by using PCR to detect the presence or absence of an intact genomic Pds5 models differs greatly between species. Pds5 null S.cerevisiae indicate it plays an integral role in maintaining cohesion and condensation, and is subsequently vital for accurate cell division [Pds5 in Schizosaccharomyces pombe has shown to have little effect on sister chromatid cohesion [Pds5b in mice show varying phenotypes, with some studies observing prenatal or embryonic lethality while others report survival of null mice up until birth but death shortly after. The discrepancies observed may be a result of the methodology and efficiency of the deletion. Indeed, we observed variance in the severity within the same litter in our study. Similar to findings observed by Losada et al. [Pds5b in our model resulted in embryonic lethality prior to E16.The necessity of Pds5b in organogenesis and development has been difficult to accurately assess. Despite the high level of conservation between species, the role of Pds5b and subsequently the phenotype generated from division ,44. In ccohesion . Furthera et al.  the inte+/+ mice, we observed no overt developmental abnormalities in Her2+/- mice compared to Her2-/- littermates. This suggests that Pds5b is required in a dose independent manner, and that one functional copy of Pds5b is sufficient for normal development. Similar results have been observed in mouse embryonic fibroblasts (MEFs) deficient in one or two alleles for Pds5b [Pds5b allele, however, consistent with what we observe in the Her2+/+ mice, loss of both Pds5b alleles resulted in defective embryonic development leading to prenatal lethality. In addition to the increased frequency of aneuploidy observed in null hepatocytes, Losada et al. hypothesise this early lethality may be due to the inability of null cells to maintain mitotic arrest, resulting in an early exit from mitosis and subsequently a reduction in their proliferative capacity [Pds5b gene, and a similar phenotype of lethality and deformities in homozygous embryos to previous studies of Pds5b-deficient mice. A full analysis of the Pds5b-disrupted mice and comparison to other mouse strains deficient in the Pds5b gene was considered beyond the scope of the current manuscript, and therefore in-depth analyses including centromeric cohesion defects and aneuploidy were not performed, but could be the focus of future studies for investigators of Pds5b biology.In comparison to the severe phenotype observed in Her2or Pds5b . Centromcapacity . The cenPds5b in a dose-independent manner, in addition to the high copy number of Her2 (162 copies) observed in phenotypically normal heterozygous Her2+/- mice lead us to conclude that the interruption of both alleles of Pds5b expression, rather than the increase in Her2 transgene copies (from 162 to 324), resulted in the embryonic lethality of Her2+/+ mice. Other published mouse models have observed the threshold for high transgene expression and subsequent phenotypic deformations to occur at much lower copy numbers (~10–30 copies), therefore we predict the additional 162 copies present in homozygous mice may not have contributed greatly to the embryonic lethality [Pds5b transgenic rescue in Her2+/+ mice in future studies.In considering whether twice the number of copies of Her2 could have contributed to the observed embryonic lethality, we suggest that the requirement of ethality ,47. HowePds5b-/- have shown contrasting results to that reported by Losada and what we observe in our study. Milbrandt et al. report no observable defects in chromosome cohesion in Pds5b-/- mice, with null mice surviving to birth [Pds5a and Pds5b, Milbrandt et al. observed early embryonic death, with no double homozygous embryos present at E9.5 [Pds5b-/- mice have shown the expression of Pds5a is independent of Pds5b expression [Interestingly, other models of to birth . These mto birth . Using c at E9.5 . This evpression , indicatde novo, in addition to minimising side effects associated with off-tumor targeting, Her2 immunotherapies that engage the hosts’ immune system must also overcome the issue of self-tolerance.The generation of the human Her2 model has facilitated the development of many Her2 based therapeutics and has proved to be an important tool in immunotherapy. The basal expression of tumor antigens on normal tissue is a major hurdle in immunotherapy, and designing therapeutics that specifically target tumor antigens whilst leaving surrounding tissue unscathed is challenging. The first and only clinical trial involving genetically modified chimeric antigen receptor (CAR) bearing T cells directed towards the Her2 antigen highlighted the importance of on-tumor effects. Low levels of Her2 present on lung epithelial cells were found to trigger the infused CAR T cells, resulting in a cytokine storm and eventuating in the patient’s death . Therefo+ tumor growth, and in addition, allowed for the assessment of autoimmune or on-target/off-tumor effects from therapy. As such, the Her2 model has been utilized extensively in the design and testing of Her2 therapeutics, including those designed to target Her2+ breast cancer stem cells [hi regulatory T cells) [Pds5b gene, a regulator of the cohesin complex and integral for both organogenesis and development. We demonstrate that similar to other Pds5b-/- models, Her2+/+ mice are severely underdeveloped, resulting perinatal death and ultimately their resorption from E16 onwards. Our results further contribute to the understanding behind the Her2 transgenic model.The expression of human Her2 from birth in Her2 mice ensured sufficient immune tolerance to permit Her2em cells , Her2 DNT cells) –51 as weT cells) ,23. In tS1 FigThe mouse WAP promoter is highlighted in blue, 5’ untranslated region (UTR) of the human ErbB2 cDNA in orange, open reading frame of the human ErbB2 cDNA in pink with the open reading frame (ORF) translated sequence underneath, followed by the 3’ UTR of the human ErbB2 cDNA in yellow. Underlined = truncation of final copy in inserted concatemer and 14 bp of genotyping primer.(PDF)Click here for additional data file.


Bromodomain inhibition of the transcriptional coactivators CBP/EP300 as a therapeutic strategy to target the IRF4 network in multiple myeloma. Published January 05, 2016Owing to a production error the published Supplementary files 1 and 2 were incorrect. Supplementary file 1 was published with the legend for Supplementary file 2 and a file from another eLife article was published with the legend for Supplementary file 1.The correct Supplementary files 1 and 2 are available here.Supplementary file 1. Includes qPCR primer sequences and UPL probe numbers for RT-qPCR experiments described in the manuscript.Supplementary file 2. Indicates the source of all cell lines used, as well as results of mycoplasma testing throughout the course of these studies.The article has now been corrected accordingly.


Until 2011, the main risk factor for the spread of HIV-1 in Romanian adult population was heterosexual contact. More recently, the number of HIV-1 new cases among IDUs significantly increased. This new subepidemic is characterized by circulation of particular forms of infective strains (CRF14_BG), high prevalence of HCV co-infections and infective endocarditis. Genotypic methods are currently used for testing viral tropism in HIV infected patients. Deep sequencing proved to have much higher sensitivity than population sequencing in detecting minority - CXCR4 tropic viruses. Previous studies suggested that the presence of CXCR4 phenotype at baseline is frequently associated with a faster disease progression. The aim of the study was to evaluate the viral tropism at the moment of HIV diagnostic in IDU patients.[coreceptor] bioinformatic algorithm and subtyping with REGA tool version 2.0.We have analyzed sequences from 19 IDUs that presented low CD4 counts (<200 cells/cmm) and/or CDC stage C when HIV-1 infection was diagnosed. They were compared with strains from 24 heterosexuals diagnosed at the same time with the IDUs were included in the study. RT PCR was performed to amplify the V3 loop. Population sequencing was done using BigDye chemistry and 3500 Genetic Analyzer. Deep-sequencing was performed on the GS Junior 454 sequencing platform and AVA software was used to analyze the output sequences. The tropism prediction was assessed by geno2phenoThe IDU group was mainly infected with recombinant forms: CRF14_BG and recombinants between F1 and CRF14_BG ; the heterosexuals had F1 subtype viruses . CXCR4 tropism was associated with IDUs and in particular with CRF14_BG (p=0.0027). All the CRF14_BG were X4 by population sequencing. Furthermore, when tested with deep sequencing the viral populations of CRF14_BG samples were exclusively X4 (no minority R5 populations). Dual tropic (CCR5 and CXCR4) populations were more frequent in F1 samples isolated from heterosexuals and predicted as X4 by population sequencing. We found concordance between the predictions of the two methods. In the heterosexual group both techniques predicted mainly CCR5 viruses.CXCR4 tropic CRF14_BG viruses were very common in IDUs at baseline. This may contribute to faster disease progression in this population than in heterosexuals infected with the F1 CCR5 tropic strains.