Recent efforts toward HIV vaccine development include the design of immunogens that can engage B cell receptors with the potential to affinity mature into broadly neutralizing antibodies (bnAbs). V2-apex bnAbs, which bind a protein-glycan region on HIV envelope glycoprotein (Env) trimer, are among the most broad and potent described. We show here that a rare “glycan hole” at the V2 apex is enriched in HIV isolates neutralized by inferred precursors of prototype V2-apex bnAbs. To investigate whether this feature could focus neutralizing responses onto the apex bnAb region, we immunized wild-type rabbits with soluble trimers adapted from these Envs. Potent autologous tier 2 neutralizing responses targeting basic residues in strand C of the V2 region, which forms the core epitope for V2-apex bnAbs, were observed. Neutralizing monoclonal antibodies (mAbs) derived from these animals display features promising for subsequent broadening of the response.  •Precursor V2-apex bnAb neutralization-sensitive Envs possess a common glycan hole•Sensitive Envs can be adapted to SOSIP format as V2-apex-focusing immunogens•Immunogens elicit nAbs targeting critical components of the bnAb epitope in rabbits•Select nAbs elicited show dependence on V2 lysines and glycans also important for bnAbs Voss et al. show that select V2-apex-focusing immunogens derived from bnAb precursor neutralization-sensitive HIV isolates can reproducibly elicit autologous neutralizing responses to components of the bnAb epitope, including K169/K171 and N156, in a wild-type animal model. It is lTo assess the ability of our immunogens to focus responses to the V2 apex region, we immunized rabbits using all four V2-apex bnAb iP-binding SOSIPs given individually, as a mixture, or sequentially. Marked differences were found among the trimers in their ability to elicit autologous nAbs. The dominant target of these responses was the same glycan hole at the V2 apex that was required for neutralization by the iP versions of prototype V2-apex bnAbs. One trimer immunogen consistently elicited responses involving strand-C basic patch residues, which are also key contacts for prototype bnAbs. Therefore, it appears that this hole in the glycan shield may indeed improve exposure of the V2-apex bnAb region to neutralizing responses. Encouragingly, we also report the elicitation of neutralizing responses targeting several heterologous tier 2 viruses in one animal that was immunized with a SOSIP trimer cocktail.HIV isolates screened for sensitivity to neutralization by iP versions of PG9, CH01, and PGT145 antibodies  and the The V2-apex glycan hole is modeled onto the BG505 SOSIP structure , in FiguTo investigate whether the iP-sensitive Envs could be used as immunogens to guide a V2-apex-specific neutralizing response, we first incorporated SOSIP.664-trimer-stabilizing modifications as previously described . Four ofNext, we assessed the binding of the PG9, CH01 iPs, and CAP256 UCA Abs to the soluble SOSIP trimers by biolayer interferometry (BLI). Consistent with the neutralization data, the iP Abs displayed significant binding to the soluble trimers D. Among 8–9GlcNAc2 (Man8–9) and a smaller fraction of mannose Man5–7GlcNAc2 (Man5–7) and complex glycans  A, which  glycans B.Figure m) of 68.1°, 70.8°, and 65.1°, respectively. These thermostabilities are similar to that of BG505 SOSIP, which has a Tm of 68.1°C (m) at 57.7° and 66.9°, indicating the presence of an uncharacterized heterogeneous mixture of trimer populations  was used to assess the thermal stability of the trimers and showed single unfolding peaks for CRF250, C108, and MGRM8 trimers with thermal denaturation midpoints , V3-glycan (n = 16), CD4-binding site (CD4bs) (n = 14), gp120-gp41 interface (n = 10), non-neutralizing V3 (n = 11), and CD4-induced (CD4i) (n = 3) and gp41 cluster-1 (n = 1) D and S1COryctolagus cuniculus) as an animal model. This is because rabbits have previously induced well-characterized high-titer tier 2 neutralization in response to other SOSIP immunogens  also developed neutralizing activity against two other immunogen-matched autologous viruses (CRF250 and MGRM8) B. IntereTier 1 viruses expose a number of epitopes that are hidden on tier 2 viruses and can be neutralized by antibodies that target relatively conserved regions and are therefore cross-reactive but not broadly neutralizing against primary isolates . AnimalsAll animals that showed autologous neutralizing activity after the second boost were screened for neutralizing breadth using a number of tier 2 pseudoviruses selected based on similarity with component immunogen Envs  values given in micrograms per milliliter of polyclonal purified IgG. We assessed binding to SOSIP trimers, monomeric gp120s, and V3 peptides  and MGRM8 (HXB2 234) immunogens B. These H) and light-chain (VL) transcripts from antigen-specific B cells were recovered by single-cell PCR amplification as previously described  The presence of a glycan hole may be important, as the only immunogen that failed to elicit V2-apex-directed neutralizing responses was MGRM8 SOSIP trimer, which has the least extensive glycan hole. This trimer lacks N130 but it does possess a PNG site within the V2′ region. The V2-apex glycan hole in C108, CRF250, and WITO immunogens should be similar in size, as these isolates possess identical PNG sites in this region of the trimer. Other factors may therefore contribute to differences in immunogenicity here. (2) Differing immunogen stability. The most successful trimer, C108, showed the highest proportion of trimers in a tightly closed native conformation by negative-stain EM and was the most thermostable. In addition, this immunogen had the lowest reactivity to non-neutralizing human mAbs and elicited the lowest levels of tier 1 neutralizing, V3-binding, and gp120 cross-reactive antibodies in rabbits. These data suggest that in addition to being a tightly packed trimer with minimal exposure of non-neutralizing epitopes, this immunogen is stable enough to maintain conformation throughout the immunization process, potentially contributing to its ability to focus responses onto neutralizing epitopes like the apex glycan hole. In general, trimer thermostability was associated with an improved ability to reproducibly elicit autologous neutralization, as has been demonstrated previously . (3) DifIn addition to comparing four iP targeting SOSIPs as immunogens individually, 2 out of 4 rabbits immunized with the SOSIP mixture developed high-titer nAbs to the C108 virus, and one of these (P5672) neutralized viruses corresponding to two of the other immunogens (CRF250 and MGRM8). The mixture was uniquely able to elicit potent neutralizing responses to MGRM8, while 4 out of 4 animals immunized with MGRM8 alone failed to do the same. CRF250 neutralizing activity in P5672 was trimer dependent, whereas all responses elicited using CRF250 immunogen alone were gp120 specific, suggesting a modulation of the neutralizing response to CRF250 in the context of the SOSIP mixture. Animal P5672 also uniquely elicited nAbs to five heterologous tier 2 viruses, and it is of interest to gain a better understanding of how this was accomplished.The range of trimer dependencies in C108- and WITO-immunized animals is interesting given the dominant epitope targeted was mapped to the V2 apex. This epitope is generally trimer dependent for prototype bnAbs. While rare, some strains of HIV, including C108, can bind to PG9 and PG16 bnAbs as monomeric gp120s C. This sWe isolated neutralizing mAbs to evaluate whether they possessed lineage properties that show promise for eventual affinity maturation into bnAbs. Like human V2-apex prototype bnAbs, all neutralizing rabbit mAbs recognize the V2 basic region of strand-C lysines (K169/K171) to some degree. Because CDRL3s lengths are longer in the rabbit antibody repertoire than in the human , contactIn summary, this study describes reproducible vaccine-elicited tier 2 autologous nAb responses directed toward an epitope that overlaps that of prototype V2-apex-directed bnAbs in an outbred animal model, and it provides a starting point from which to test boosting strategies designed to broaden these responses to generate V2-apex bnAbs capable of protecting against global HIV isolates.Mature, iP, and UCA antibody sequences used in this study are described in 50) in micrograms IgG per milliliter after fitting the curve of log[antibody] versus percent neutralization in Prism. For neutralization absorption assays, 0.1 mg/mL D368R gp120 or SOSIP was incubated with 200 μg/mL IgG for 1 hr at 37°C before adding virus and proceeding as above.Single-round infectious HIV-1 Env pseudoviruses were made as described previously . BrieflyJN944660 [C108.c03]; GenBank: EU13189 [CRF-T250-4]; GenBank: AY835451 [WITO.4130]; GenBank: MF510462 [MGRM-002-8]) were used to generate codon-optimized SOSIP.664 gp140 genes  for expression of soluble native trimers as described in Gallanthus nivalis lectin (GNL) (Vector Labs). Affinity-purified proteins were purified by size exclusion chromatography using a Superdex 200 10-300 GL column  in PBS.HIV-1 Env amino acid sequences .This study was approved and carried out in accordance with protocols provided to the Institutional Animal Care and Use Committee (IACUC) at The Scripps Research Institute  under approval number 14-0002. The rabbits were kept, immunized, and bled at TSRI in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and in adherence to the 20 female 12-week-old New Zealand white rabbits were primed and boosted with 50 μg protein and 100 U Iscomatrix in 600 μL PBS (300 μL IM injection into each hind leg). Blood was drawn from the marginal ear vein into EDTA or untreated blood collection tubes. All procedures were performed in anesthetized animals. EDTA plasmas were diluted three times with PBS and PBMCs purified on a Lymphoprep (STEMCELL Technology) density gradient. PBMCs were cryopreserved in FBS plus 10% DMSO.Clotted, filtered serum samples were mixed with protein A/G and incubated overnight at 4°C with agitation. Beads were washed with PBS and eluted using 0.1 M citric acid (pH 3.0) and neutralized with 1 M Tris (pH 9.0). Purified IgG was buffer exchanged into PBS and stored at 200 μg/mL at 4°C for neutralization and ELISAs.4-Biotin kit  MaxiSorp plates (Thermo Fisher Scientific) were coated overnight at 4°C with 2 μg/mL gp120 protein or 2 μg/mL streptavidin (Thermo Fisher Scientific). Plates were blocked for 1 hr with 3% BSA and washed three times with 0.05% Tween 20-PBS (PBS-T) (pH 7.4). Streptavidin-coated plates were incubated with randomly biotinylated SOSIPs in 1% BSA plus PBS-T for 2 hr and washed three times with PBS-T. Purified rabbit serum IgG or human mAbs were added at a maximum concentration of 200 μg and 10 μg/mL, respectively, serially diluted 1:6 in 1% BSA plus PBS-T, and incubated at room temperature (RT) for 1 hr. Plates were washed three times with PBS-T. Alkaline-phosphatase-conjugated goat anti-human or anti-rabbit IgG Fc secondary antibody (Jackson ImmunoResearch Laboratories) was diluted 1:500 in 1% BSA PBS-T and added to plates for 1 hr at RT. Plates were washed three times with PBS-T and incubated with phosphatase (Sigma) for 60 min and the absorbance at 405 nm recorded. The bnAb reactivity with biotinylated SOSIPs proved that these reagents contained native epitopes. Direct coating of gp120 on plates showed similar results but better dynamic range than gp120 capture using an anti-C5 antibody.SOSIP ELISA reagents were randomly biotinylated using a 2:1 molar ratio of biotin reagent to trimer using the EZ-link-NHS-PEGFluorescence-activated cell sorting of cryopreserved PBMCs was performed as described in Correlations in data reported in J.E.V., R.A., and L.E.M. designed the experiments. J.E.V., R.A., L.E.M., N.d.V., R.P.F., T.M., C.-Y.S., D.S., S.N.K., F.G., and L.K.P. performed the experiments. R.T.W., A.B.W., M.C., and I.A.W. contributed critical reagents. J.E.V., R.A., and D.R.B. analyzed the data and wrote the paper.


Drosophila FoxA transcription factor homolog Fork Head (FKH). Intestinal FKH induction extends lifespan, highlighting a role for the gut. FKH binds to and is phosphorylated by AKT and Target of Rapamycin. Gut-specific FKH upregulation improves gut barrier function in aged flies. Additionally, it increases the expression of nutrient transporters, as does lowered IIS. Evolutionary conservation of this effect of lowered IIS is suggested by the upregulation of related nutrient transporters in insulin receptor substrate 1 knockout mouse intestine. Our study highlights a critical role played by FKH in the gut in mediating anti-aging effects of reduced IIS. Malnutrition caused by poor intestinal absorption is a major problem in the elderly, and a better understanding of the mechanisms involved will have important therapeutic implications for human aging.Reduced activity of nutrient-sensing signaling networks can extend organismal lifespan, yet the underlying biology remains unclear. We show that the anti-aging effects of rapamycin and reduced intestinal insulin/insulin growth factor (IGF) signaling (IIS) require the   •Drosophila FKH biochemically interacts with AKT and TOR•IIS- and rapamycin-induced longevity requires FKH•Gut tissue, specifically differentiated cells, mediates FKH’s pro-longevity effects•FKH activity in the gut upregulates intestinal nutrient transporters Drosophila, and they characterize FKH’s essential involvement in increased longevity via this network. They pinpoint FKH’s pro-longevity effect to the gut, and they show increased expression of nutrient transporters and improvement of barrier function by FKH activity.Bolukbasi et al. identify the transcription factor FKH as a mediator of nutrient-sensing pathway signaling in  Drosophila  and Target of Rapamycin (TOR) influences healthy lifespan in diverse eukaryotic organisms, including mammals . Specifiosophila .In addition to its effect on aging, the IIS/TOR network regulates growth, metabolism, stress responses, and fecundity, potentially resulting in undesired side effects of reduction of network activity. For example, at some doses, rapamycin is a strong immunosuppressant  and can Recent studies identified the intestinal tissue as pivotal in aging , and theC. elegans and Drosophila, the single Fork Head Box O (FoxO) TF is required for extended lifespan from lowered IIS  . For instance, in ered IIS . In C. eTF HSF-1  and the TF HSF-1  are alsoen (AOP) . In Drosan dFOXO .Drosophila FoxA homolog FKH is the founding member and namesake, and it plays an essential role in embryonic development (The Fork Head box (FOX) family of TFs shares a conserved DNA-binding domain, whose sequence assigns them to subclasses from FoxA to FoxS . The Droelopment . FKH alselopment . Mammalielopment , and livelopment .Drosophila FKH in the IIS-and-TOR-signaling network and, in particular, its key role in intestinal aging. We find that FKH interacts with and is phosphorylated by both dAKT and dTOR, placing it as a central transcriptional regulator. Concordantly, we demonstrate an essential requirement for FKH for both IIS- and rapamycin-induced longevity, as well as IIS-induced starvation resistance, a phenotype previously shown to be dFOXO independent  driver to produce a graded increase in FKH expression, by varying the concentration of the activating drug RU486. Strong FKH induction led to short-lived flies (−6). Intermediate induction at 50 and 100 μM RU486 resulted in early life mortality, and upregulation at 200 μM RU486 resulted in significantly short-lived flies. To more precisely define the range of induction that maximized lifespan, we used 10 and 20 μM RU486, and we found significant extension at both concentrations (−6). Moderate, ubiquitous, adult-specific upregulation of FKH expression is thus sufficient to extend Drosophila lifespan in a dose-dependent manner.We used the inducible daughterless Gene Switch , a Gram-negative bacterium that has been used as a model of natural infection in Drosophila larvae and adults , suggesting a decline in immune response over age. Overexpression of FKH led to increased survival at both ages , and, thereby, it controls the expression of dFOXO target genes. We first determined whether IIS activity has similar effects on FKH, by searching for the appearance of a slow-migrating FKH protein band on polyacrylamide gels . However, RNAi against fkh both substantially reduced the lifespan of wild-type flies and blocked the extension of lifespan by InRDN , but they lost this lifespan extension with RNAi against fkh  that also prevented lifespan extension upon overexpression of UAS-InRDN and heterozygous chico1 mutation , which was abolished by ubiquitous, adult-specific RNAi against fkh (−4). The biochemical effect of rapamycin upon FKH RNAi was confirmed by assessing phosphorylation levels of S6K, which were decreased on both wild-type and FKH knockdown backgrounds following rapamycin treatment . Importantly, since ubiquitous RNAi again fkh shortened lifespan, the extension of lifespan in these flies by DR indicates that the failure to extend in response to reduced IIS and rapamycin was specific to these interventions.TOR kinase can play a role in the extension of lifespan by dietary restriction (DR) , and theongevity . We subjTogether these results indicate that FKH is required for rapamycin-induced, but not DR-induced, longevity.In addition to its effect on lifespan, lowered IIS is associated with resistance to starvation, oxidative stress, and xenobiotics. We therefore determined whether these types of stress resistance require FKH function.InRDN were resistant to paraquat . Knocking down fkh resulted in a loss of starvation resistance in flies expressing InRDN (−5). Resistance to starvation upon the downregulation of IIS thus requires FKH function.Resistance to starvation was assessed by exposing flies only to agar. Ubiquitous RNAi against GFP or arvation C. Flies −7), as did fkh RNAi alone (−5). Knocking down fkh in flies with lowered IIS neither restored nor exacerbated their fecundity deficit. These results suggest that FKH is not required for the reduced fecundity observed upon the downregulation of IIS. The decrease in fecundity observed upon reduced IIS and fkh RNAi alone was not additive, and, therefore, it was likely to be occurring through independent mechanisms.IIS mutant females often have a fecundity deficit , and we dfoxo or aop activity in the abdominal fat body and adult midgut results in longevity, whereas their upregulation in the gut alone does not  is not sufficient to extend lifespan, suggesting involvement of other gut cell types.To determine how FKH acts in the gut to extend lifespan, we examined the gut barrier function by feeding aged flies with a blue dye that does not normally leak into the body cavity , and we ISC proliferation increases with age, eventually leading to intestinal dysplasia . To deteThese results suggest that FKH overexpression in the gut maintains barrier function during aging, but it does not affect ISC proliferation.fkh in the gut by RNAi with the TiGS driver. Treatment of uninduced flies with rapamycin significantly extended lifespan (−12), and this extension was lost upon gut-specific RNAi against FKH (−15). Hence, FKH function in the gut is necessary for rapamycin-induced longevity.Rapamycin reduces age-related intestinal pathologies in flies . Given tDrosophila lifespan . Hence, the decrease of IIS activity in the gut alone results in long-lived flies, and this not only requires FKH function but also is deleterious without it.Downregulating IIS activity in progenitor cells of the gut extends lifespan . We examOur results indicate that FKH function in the gut is necessary for both rapamycin- and reduced IIS-induced longevity. Given the detrimental effect of FKH knockdown in guts with reduced IIS, in our subsequent analysis we focused on the identification of genes regulated by FKH in the gut upon downregulation of IIS. We isolated guts from flies with ubiquitous reduction of IIS with or without knockdown of FKH, and controls, and we carried out genome-wide transcript profiling using next-generation RNA sequencing.daGS;UAS-InRDN> UAS-GFP RNAi + RU486 versus daGS;UAS-InRDN> UAS-GFP RNAi − RU486) and the set of genes differentially expressed in reduced-IIS guts with or without RNAi against FKH . We reasoned that the transcriptional response regulated by FKH in response to reduced IIS should be indicated by genes whose expression levels are reverted back to wild-type levels upon FKH knockdown in reduced-IIS flies , glucose (CG1208), phospholipids (mdr50), and fatty acids (pmp70)  B, suggesDrosophila enterocytes (ECs) regulate nutrient transport and absorption in the midgut , but zip1 expression levels remained significantly increased . Linear model analysis showed a significant interaction between RU and genotype (p = 0.0004), showing that the expression of genes involved in transport of nutrients was increased in the IIS mutant in an FKH-dependent manner.To confirm our transcriptomic data, we assessed by qRT-PCR the expression levels of transmembrane transporters, including d CG1208 A, and wectr1B, zip1, and mdr50 expression in guts with the induction of fkh  and metallothioneinC (mtnC) in guts isolated from flies with fkh knockdown alone or with reduced IIS with or without fkh knockdown. Reduced IIS increased expression of both metallothionein genes, and this increase was dependent on FKH , TAG levels in whole flies (−5).Previous studies highlighted a key role for the fly gut in absorption and metabolism of dietary lipids . The mide midgut , with a −7). This finding further supports the transcriptomic and molecular analysis, and it pinpoints the pro-longevity effect of FKH to EEs and ECs.Our findings suggest a role for FKH in nutrient absorption. We next assessed whether increasing FKH activity specifically in differentiated gut cells, namely EEs and absorptive ECs, affected longevity. Upregulation of FKH in differentiated cells resulted in a significant increase in lifespan  and controls (Irs1lox/lox) at 3 months of age. We quantified mRNA levels of Glut8, Abcd3, Slc39a3, and Slc31a1 , and we found a significant increase in Abcd3, Glut8, and Slc39a3 expression  brewer’s yeast, 5% (w/v) sucrose, and 1.5% (w/v) agar, unless otherwise noted. Please see the https://cran.r-project.org/web/packages/survival/index.html). qPCR analysis was performed by using either Student’s t test or a linear model.Statistical analyses were performed in JMP (version 9) software (SAS Institute), R, or Excel (Microsoft). Survival data were analyzed with either log rank test or CPH using Excel (Microsoft) or R using the survival package  mouse are described in the All mice were maintained at 22°C under a 12-hr light/dark cycle (lights on from 6:00 a.m. to 6:00 p.m.). Mice were housed in groups of three to five same-sex littermates under specific pathogen-free conditions within individually ventilated cages . Mice had ad libitum access to normal chow  and water. Female mice were sacrificed at 3 months. Duodenums were dissected and tissues were snap-frozen in liquid nitrogen. Details of the generation of tissue-specific This study was performed in strict accordance with the recommendations and guidelines of the Federation of European Laboratory Animal Science Associations (FELASA). The protocol was approved by the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen.E.B. and L.P. designed the experiments. E.B., M.K., J.C.R., J.A., M.C.D., and T.N. performed the experiments. E.B., N.A., and D.K.I. analyzed data. J.M.T. and L.P. supervised the project. E.B. and L.P. wrote the manuscript. All authors approved the final submission.


During the last decade there is a growing scientific interest in nonsuicidal self-injury (NSSI). The aim of the current paper was to review systematically the literature with a special focus on the associations between self-injurious behaviours and externalizing psychopathology. An additional aim was to review terminology and measurements of self-injurious behaviour and the connection between self-injurious behaviours and suicide in the included publications.A systematic literature search was conducted on 31st December 2016 in five databases  with two categories of search terms .Finally 35 papers were included. Eleven different terms were found for describing self-injurious behaviours and 20 methods for measuring it. NSSI has the clearest definition. All the examined externalizing psychopathologies had strong associations with self-injurious behaviours according to: higher prevalence rates in externalizing groups than in control groups, higher externalizing scores on the externalizing scales of questionnaires, higher symptom severity in self-injurious groups. Eight studies investigated the relationship between suicide and self-injurious behaviours and found high overlap between the two phenomena and similar risk factors.Based on the current findings the association between externalizing psychopathology and self-injurious behaviours has been proven by the scientific literature. Similarly to other reviews on self-injurious behaviours the confusion in terminology and methodology was noticed. NSSI is suggested for use as a distinct term. Further studies should investigate the role of comorbid conditions in NSSI, especially when internalizing and externalizing pathologies are both presented. In the past decade self-injury became a hot topic in scientific literature. Nonsuicidal self-injury (NSSI) became an individual diagnosis in the 5th edition of the Diagnostic and Statistical Manual of Mental Disorders ; for theIn the 4th edition of DSM , deliberSeveral risk factors of NSSI are described, such as prior history of NSSI, cluster b personality, hopelessness, female gender, depression, prior suicidal thought/behaviour, exposure to peer NSSI, eating disorder, abuse, etc. .Many studies examined the association between NSSI and internalizing pathology; recently Bentley et al.  summarizThe association between externalizing pathology and NSSI are understudied. Although Fox et al.  found thTherefore, our aim is to systematically review the studies that examined the association between self-injurious behaviour and externalizing psychopathology. Not only ‘NSSI’ was chosen as a search word for self-injurious behaviour, because the scientific literature is very confused about the terms . MoreoveBefore investigating the main aim of our study – 1) prevalence rates, odds ratios or other associations between externalizing pathology and self-injury – it seemed to be important to review: 2) terminology and definitions of self-injurious behaviour, and 3) methods for measuring it. Finally, we examined: 4) associations between self-injury and suicide among the included publications.A systematic literature search was made on 31th December 2016 in five computerised literature databases: PubMed, OVID Medline, OVID PsycINFO, Scopus, Web of Science. Search terms were: ‘ AND ’. We found 902 papers in PubMed, 127 papers in OVID Medline, 120 papers in OVID PsycINFO, 116 papers in Web of Science and 16 papers in Scopus, making a total of 1281 studies including duplicates. We used EndNote X7 software for the systematization of the papers. After a duplicate search  we excluded 222 articles, so altogether we had 1059 individual papers ’.In 7 papers there was no clear distinction between the purpose of the investigated self-injurious act  –17, but There were 11 different terms for self-injurious behaviour in the investigated 35 papers.‘NSSI’ was the most common term: this expression was presented in 10 studies , 43, 45.‘DSH’ was presented in 6 publications , 34, 44.‘Self-mutilation’ was found in 5 publications , 29, 32.‘SIB’ was a term in 4 papers , 40, 42,‘Self-injury’ was presented in 4 papers , 30, 45,There were some other phrases describing self-injurious behaviour: ‘parasuicide’ : there iAmong the studies included in our review, diagnostic interviews, self-reported questionnaires and institutional records were used to measure self-injurious behaviours: altogether we found 20 different instruments among the investigated 35 papers.In 9 cases, the authors designed a longer diagnostic interview, and some questions about self-injurious behaviours were included in it , 36, 41.An interview specially developed for measuring self-injurious behaviours was used in 10 studies , 42, 43.Self-report questionnaires were used for measuring self-injurious behaviours in 9 studies , 44, 45 In the remaining 7 publications, some kind of institutional  records or national databases were the sources of the data on self-injurious behaviours , 27, 37.Epidemiological data about self-injurious behaviours in different groups can be found in 20 publications (see details below).Six studies examined the normal population , 36, 45.Five studies investigated general psychiatric populations  , 37, 41.In the study of Carli et al.  there waIn the remaining 8 studies, the aim of the investigation was to compare a group having a specific psychopathology with a healthy control group or with a group having another psychopathology.Four publications examined attention deficit hyperactivity disorder (ADHD) as a group with specific psychopathology , 30, 31.Two studies examined intermittent explosive disorder (IED) as a specific psychopathology. McCloskey et al.  found thOnly in one publication could data be found on the prevalence of self-mutilation among participants with conduct disorder (CD) : they foFifteen publications investigated the associations between self-injurious behaviours and ADHD , 41–44. Twelve publications examined the possible connection between conduct disorder (CD) or CD symptoms or antisocial personality disorder in adulthood and self-injurious behaviours , 40, 42.Four papers studied the link between oppositional defiant disorder (ODD) and self-injurious behaviours or SIB. Cerutti et al.  found, tThere are 3 publications that strengthen the relationship between intermittent explosive disorder and self-injurious behaviours  and DSH  than those who did not have the disorder. Female gender and depression/anxiety were also associated with both suicidal ideation and DSH, while behavioural disorder, substance use and certain family factors  were associated with DSH but not with suicidal ideation and acts.In an adolescent community sample , those w1(N = 75) = 3.91, p < .05); major depressive disorder  = 13.74, p < .01;) and dysthymia (χ21 (N = 76) = 4.82, p < 0.05), and had higher scores of loneliness , anger , risk taking  and reckless behaviour   and self-mutilation 3.9-fold  among adolescent girls with DSM-IV-diagnosed CD.Ilomaki et al.  did not In the current systematic review, 35 papers examining the correlation between externalizing psychopathology and self-injurious behaviours are summarized.Consistent with the review on internalizing pathology and NSSI , we founThe heterogeneity that is present in the terminology and definitions of self-injurious behaviours is also reflected in the diversity of instruments used to assess the phenomenon. The most frequently used instruments  and in normal population based samples (2.9–41.9%). This variance can be explained partially by the definition and measurement heterogeneity, described above. However, using one term (e.g. NSSI) does not lead to more homogenous prevalence rates. In the specific psychopathology groups investigated, the diversity of prevalence rates could also be observed. Due to this heterogeneity, and with the aim of avoiding confusion, the original terms used by the authors of the papers are used in the ‘Results’ section, and the prevalence rates are mentioned in parallel. These various results underline the importance of standardized terminology and measurement for self-injurious behaviours, which would make the results of future studies comparable.There were significantly higher prevalence rates in ADHD groups than in control groups , 30, 31.In addition to the finding that ADHD and self-injurious behaviours are strongly associated, which is in agreement with a previous review on this topic , we founAlthough the aim of this review was not specifically to explore the relationship between NSSI and suicidality, 8 out of the 35 articles contained results regarding this topic. Although the two phenomena might overlap , 34 and The main limitation of our review is the heterogeneity of both the terminology and instruments, as we mentioned above, which makes it hard to compare the results of the included papers. In most of the cases, the distinction that the act was with or without suicidal intent was clear, however, among these 35 papers there were 7 papers, where this distinction was not clarified –17. The A further limitation is that we may be missing some relevant papers on this topic. Firstly, because we included only articles that are written in English. Secondly, we have chosen the standard method and searched for publications in large indexed literature databases. This led to 14 papers on the topic of ADHD and self-injury, however, Allely  used a lAnother methodological limitation could be that our work is a narrative review. A meta-analytic review, or a full meta-analysis would be more useful to compare the results of these very heterogenous papers. To handle this limitation and make the comparisons easier, sample sizes, design of the original studies and statistical results are presented in Table Another limitation could be that since our focus in this review was to investigate the association between NSSI and externalizing pathology, the role of externalizing problems in the relationship between NSSI and suicidality might be overrepresented, while other important factors might remain hidden. A review focusing directly on the NSSI-suicidality relationship, including two of the articles discussed in this paper , 34 was In conclusion, reviewing these 35 papers on self-injury and externalizing psychopathology underlines the confusion in terminology on self-injurious behaviours, which could lead to difficulty in comparing studies on the topic. Based on our findings, we suggest the use of the term NSSI in future studies, as NSSI has the most precise definition. Furthermore, it became an individual diagnostic category in DSM-5 .Summarizing the results of these papers – although the connection between internalizing pathology and self-injurious behaviours are well-studied  – there


Gaussia princeps luciferase (GpL) fusion protein of TNF to TNFR1 and TNFR2 and blocked binding of soluble GpL fusion proteins of TNFR1 and TNFR2 to membrane TNF expressing cells to >95%, too. Purified PGRN, however, showed in both assays no effect on TNF–TNFR1/2 interaction even when applied in huge excess. To rule out that tags and purification- or storage-related effects compromise the potential ability of PGRN to bind TNF receptors, we directly co-expressed PGRN, and as control TNF, in TNFR1- and TNFR2-expressing cells and looked for binding of GpL-TNF. While expression of TNF strongly inhibited binding of GpL-TNF to TNFR1/2, co-expression of PGRN had not effect on the ability of the TNFR1/2-expressing cells to bind TNF.Progranulin (PGRN) is a secreted anti-inflammatory protein which can be processed by neutrophil proteases to various granulins. It has been reported that at least a significant portion of the anti-inflammatory effects of PGRN is due to direct high affinity binding to tumor necrosis factor receptor-1 (TNFR1) and TNFR2 and inhibition of tumor necrosis factor (TNF)-induced TNFR1/2 signaling. Two studies failed to reproduce the interaction of TNFR1 and TNFR2 with PGRN, but follow up reports speculated that this was due to varying experimental circumstances and/or the use of PGRN from different sources. However, even under consideration of these speculations, there is still a striking discrepancy in the literature between the concentrations of PGRN needed to inhibit TNF signaling and the concentrations required to block TNF binding to TNFR1 and TNFR2. While signaling events induced by 0.2–2 nM of TNF have been efficiently inhibited by low, near to equimolar concentrations (0.5–2.5 nM) of PGRN in various studies, the reported inhibitory effects of PGRN on TNF-binding to TNFR1/2 required a huge excess of PGRN . Therefore, we investigated the effect of PGRN on TNF binding to TNFR1 and TNFR2 in highly sensitive cellular binding studies. Unlabeled TNF inhibited >95% of the specific binding of a  Tumor necrosis factor-alpha (TNF) is a pleiotropic cytokine which has not only crucially implicated in a variety of immunoregulatory processes in innate and adaptive immunity, but has also manifold roles in the control of tissue homeostasis . TNF is Progranulin (PGRN) or granulin precursor (Gene ID GRN) is a phylogenetically conserved unique protein without stringent homology to other proteins , 6. PGRNin vitro and animal model evidence that the immune modulatory effects of PGRN are based, at least in part, on the modulation of TNF signaling  measured in the same study with the same methodology (Gaussia princeps luciferase (GpL) fusion protein of soluble TNF (GpL-TNF) to TNFR1 and TNFR2. The luciferase from the mesopelagic copepod Gp is a secreted, small (19 kDa), monomeric luciferase with superior brightness. GpL fusion proteins offer, therefore, an exquisite sensitivity and a for several orders of magnitude linear signal strength (Adi) and R&D Systems (PGRNRD) which have been cited in the studies reporting direct TNFR1/2–PGRN interaction  reached up to approximately 10,000 ng/ml and lysates of PGRN expressing cells (PGRNlys) contained approximately 30,000 ng/ml of the protein  compared to the PGRNAdi standard  which was derived of HEK293 cells, too. Thus, PGRN of different sources appears to be differentially modified . The sizes of PGRNlys and PGRNSN are in accordance with the literature typically indicating a size of 88 kDa for PGRN. Next, we subjected PGRNlys and PGRNSN along with corresponding samples of empty vector (EV)-transfected cells to competitive binding studies with plate-bound TNFR2-Fc and GpL-TNF. There was again no evidence for an interference of PGRN with the interaction of GpL-TNF and TNFR2. Neither pretreatment with PGRNlys nor with PGRNSN showed a significant inhibitory effect on GpL-TNF binding to TNFR2-Fc  supplemented with 2,000 ng/ml soluble TNF showed efficient inhibition of binding of GpL-TNF  were not possible due to the cell lytic effects of the lysis buffer. It should also be stressed that the lysis buffer used was prepared according to Tang et al. reporting PGRN-TNFR2 co-immunoprecipitation in this buffer  had no effect on GpL-binding. In contrast, pretreatment with sTNF or Flag-LTα (F-LTα) inhibited GpL-binding for >99 and >98% indicating efficient blockade of TNFR1 and TNFR2 .To minimize possible unknown negative effects of the purification process and storage conditions of the commercially obtained PGRN samples on their ability to bind to TNFR1 and TNFR2, we used next cultures supernatants (SNs) and lysates of HEK293 cells transiently transfected with an expression plasmid encoding human PGRN. The human cell line HEK293 has been used here because HEK293 cells not only ensure high transfection efficiency, but has also used for PGRN production by groups reporting PGRN–TNFR interaction , 10, 23.s buffer . DespiteD-value of the interaction of GpL-TNF and TNFR2 , soluble TNF (sTNF), or membrane TNF (memTNF), and analyzed the transfected cells finally again for GpL-TNF binding. As expected transfection of plasmids encoding sTNF or memTNF resulted in strong reduction of GpL-TNF binding to TNFR2 Figures A,B. Once2 Figure B. In thilys and GpL-PGRNSN) and PGRN-GpL (PGRN-GPLlys and PGRN-GpLSN) expressing HEK293 cells despite using concentration of up to 5,000–30,000 ng/ml  and investigated their binding to TNFR1 and TNFR2. While 4–250 ng/ml GpL-TNF showed significant binding to plastic-bound TNFR1-Fc and TNFR2-Fc, there was no significant binding with lysates and SNs of GpL-PGRN .In sum, we found no evidence for a direct and TNF binding-competing interaction of PGRN and TNF receptors even not in experimental settings, where PGRN and TNF receptors were expressed directly by the cells in the assay and where their potential interaction can thus not be affected by unknown factors related to experimental processing  and R&D Systems, Wiesbaden, Germany . The expression vector (pCMV6-XL5) encoding untagged human PGRN (Ac. No.: NM_002087) was from Origene, Rockville, MD, USA (#SC118822). The anti-PGRN mouse monoclonal antibody C-11 and the TNF-specific goat IgG N-19 were from Santa Cruz Biotechnology, Dallas, USA . The pEF-BOS-based expression vector encoding membrane TNF have been described elsewhere . TNFR1-FThe expression vector encoding soluble Flag-TNF was generated by replacement of the TRAILR2 encoding part in PS435 , a pCR3.1-based expression vector  encoding the human Ig leader followed by a Flag-tag and TRAILR2, with a DNA amplicon encoding aa 85–223 of human TNF (ac. no.: NP000585). The TNFR1ed-GpL and TNFR2ed-GpL encoding expression plasmids are also based on pCR3.1 and encode expression cassettes comprising the ectodomain of TNFR1 (aa 1–211 of ac. no.: M58286.1) or TNFR2 (aa 1–257 of ac. no.: M55994.1) followed by the Flag epitope and aa 18–185 of ac. no.: GM037681 encoding mature, thus leader free GpL. Two aa insertions (GSAGEF and LE) resulting from molecular cloning furthermore separate the Flag tag from the receptor and GpL parts, respectively. The GpL-PGRN encoding expression plasmid is also a pCR3.1 derivative and encodes GpL including its leader sequence followed by a Flag tag and aa 21–593 of human PGRN whereby the Flag epitope is connected with the GpL and the PGRN domain by a five aa (SGAGS) and a two aa (EF) insertion.g). The resulting PGRN containing SN (PGRNSN) was directly used for experiments or after dilution in cell culture medium. To obtain cell-associated PGRN (PGRNlys), correspondingly transfected cells were harvested 48 or 72 h post transfection and lysed in RIPA buffer . Finally, concentration of the protein of interest was evaluated by Western blotting and an appropriate protein mass standard and/or by measuring the activity of the GpL domain. The complete Western blots of the cuttings shown in Figures Recombinant proteins were produced in HEK293 cells by transient transfection of the expression plasmids described above. For this purpose, the medium of tissue culture dishes with confluent HEK293 cells was replaced by 15 ml of serum-free RPMI 1640 medium containing penicillin–streptomycin. For each culture, 2 ml of RPMI 1640 medium containing 12 µg of the expression plasmid of interest were prepared and supplemented dropwise and under vortexing with 36 µl of a 1 mg/ml polyethylenimine . After 15 min at room temperature, the plasmid/PEI solution was added and transferred to the HEK293 cells. Next day, the serum-free plasmid/PEI-containing medium was replaced by fresh RPMI 1640 medium containing 2% FCS and penicillin–streptomycin. After 4–6 days, SNs were collected and cellular debris was removed by centrifugation  of the purified protein of interest  were subjected to black high bind ELISA plates . After overnight incubation at 4°C and three washes with PBS Tween, remaining free binding sites were saturated by incubation  with blocking buffer (10% FCS in PBS). After three washing cycles with PBS Tween, the actual binding studies were performed. In the case of equilibrium binding studies wells were incubated for 2 h with increasing concentrations of the GpL fusion protein of interest at room temperature. Unbound protein was then removed by five wash cycles with PBS Tween and finally well-associated luciferase activity was determined (see below). Values for non-specific binding were derived from wells coated with control protein or with coating buffer only and were subtracted from the corresponding total binding values obtained from the wells coated with the protein of interest to obtain specific binding values. In the case of competition binding studies, wells were treated for 0.5–1 h with increasing concentrations of the potential inhibitor  or remained untreated before the GpL fusion protein was added for an additional hour. Finally, wells were again washed five times and used for quantification of bound luciferase activity.6 cells in 150 µl RPMI 1640 medium with 10% FCS. Where indicated, cells were then pretreated at 37°C for 1 h with potential antagonistic proteins , otherwise cells remained untreated. Cells were then supplemented with the GpL fusion protein of interest and after an additional incubation period of 1 h, unbound proteins were removed (five washes with PBS). Finally, cells were collected in 50 µl of RPMI 1640 medium with 0.5% FCS to quantify the remaining cell-bound GpL fusion protein molecules. Binding values derived of EV-transfected cells were considered as non-specific binding and binding values obtained from the transfectants expressing the protein of interest were considered as total binding. Please note, the expression levels observed after transfection of expression plasmids encoding TNFR1 and TNFR2 were regularly >100-fold higher than those of endogenously expressed TNFR1. There was no evidence for endogenous expression of TNF in the HEK293 cells.For cellular binding studies, HEK293 cells were transiently transfected with expression plasmids encoding the protein of interest and EV. Next day, cells were divided into the required number of aliquots of 0.5–1 × 10Gaussia princeps luciferase activity was measured with the Gaussia luciferase Assay Kit  essentially as described by the supplier. After starting the reaction by adding substrate-buffer solution, light emission was immediately (<10 s) quantified  to minimize errors due to the decay of GpL activity. Please note, the LUmo Luminometer has a much higher sensitivity compared to the Lucy 2 luminometer. Data are reported as mean ± SEM and were analyzed by Bonferroni’s test or were analyzed with the “nonlinear regression to a one-site specific binding curve” or the “nonlinear regression to a one-site competitive binding curve” function of the GraphPad Prism5 software.HW designed the study and prepared the manuscript. IL and SF performed the experiments and contributed to study design. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila and vertebrates. Nociceptive sensitization in Drosophila larvae following UV-induced tissue damage is accompanied by epidermal apoptosis and requires epidermal-derived TNF/Eiger and the initiator caspase, Dronc. Major gaps remain regarding TNF function in sensitization, including the relationship between apoptosis/tissue damage and TNF production, the downstream signaling in this context, and the target genes that modulate nociceptive behaviors. Here, apoptotic cell death and thermal nociceptive sensitization are genetically and procedurally separable in a Drosophila model of UV-induced nociceptive sensitization. Activation of epidermal Dronc induces TNF-dependent but effector caspase-independent nociceptive sensitization in the absence of UV. In addition, knockdown of Dronc attenuated nociceptive sensitization induced by full-length TNF/Eiger but not by a constitutively soluble form. UV irradiation induced TNF production in both in vitro and in vivo, but TNF secretion into hemolymph was not sufficient to induce thermal nociceptive sensitization. Downstream mediators of TNF-induced sensitization included two TNF receptor-associated factors, a p38 kinase, and the transcription factor nuclear factor kappa B. Finally, sensory neuron-specific microarray analysis revealed downstream TNF target genes induced during thermal nociceptive sensitization. One of these, enhancer of zeste (E(z)), functions downstream of TNF during thermal nociceptive sensitization. Our findings suggest that an initiator caspase is involved in TNF processing/secretion during nociceptive sensitization, and that TNF activation leads to a specific downstream signaling cascade and gene transcription required for sensitization. These findings have implications for both the evolution of inflammatory caspase function following tissue damage signals and the action of TNF during sensitization in vertebrates.Tumor necrosis factor (TNF) signaling is required for inflammatory nociceptive (pain) sensitization in  It acts through its receptor, TNFR/Wengen, in nociceptive sensory neurons to mediate thermal allodynia – aversive withdrawal to previously innocuous temperatures. This role of TNF/TNFR is phylogenetically conserved, as they mediate various types of sensitization in vertebrates.12 TNF/Eiger is not required for larval epidermal apoptosis,13 as it is in Drosophila photoreceptors and in some vertebrate tissues.15 As TNF/Eiger originates from dying epidermal cells yet acts through its receptor in nociceptive sensory neurons, substantial questions remain regarding how this transmembrane ligand is activated to mediate sensitization.During 13 Dronc is the initiator caspase for apoptosis in most fly tissues.16 In the absence of an apoptosis-inducing signal, the death-associated inhibitor of apoptosis protein 1 (DIAP1) prevents the activation of Dronc.17 This inhibition is released by the expression of pro-apoptotic genes such as reaper, hid, and grim.18 Dronc interacts with death-associated APAF-1-related killer (Dark) to form an apoptosome that activates effector caspases such as Drice and Dcp-1.19 These cleave the cellular substrates that lead to apoptosis.21 In addition to their roles in apoptosis, Drosophila caspases also function non-apoptotically in sperm individualization,22 compensatory proliferation,23 and innate immunity.24Although TNF/Eiger is dispensable for UV-induced epidermal apoptosis, the initiator caspase Dronc is required within the epidermis for both epidermal apoptosis and thermal allodynia development.κB).25 Although the mechanism of NF-κB activation varies by cell type, assembly of downstream mediators at TNFR generally recruits the IκB kinase, resulting in the phosphorylation and degradation of NF-κB inhibitor (IκB).26 Free NF-κB then translocates to the nucleus and activates target gene expression. Caspases and mitogen-activated protein kinases (MAPKs) can also be targeted by TNF signaling and can lead to proliferation, cell death, and inflammation.28 TNF signaling and NF-κB are associated with both acute and chronic pain.35 The transcriptional consequences of TNFR signaling and NF-κB activation in damage-sensitized nociceptive sensory neurons, however, remain unclear.The binding of TNF to TNFR recruits adapters such as TNF receptor-associated factors (TRAFs) to transduce signals. This binding ultimately activates nuclear factor kappa B (NF-enhancer of zeste (E(z)), which is upregulated by TNF signaling in nociceptive sensory neurons, is also required for sensitization. Together, these data reveal the full TNF signaling architecture, from ligand activation/production to signal transduction to downstream target gene activation, that is necessary for nociceptive sensitization following tissue injury.Here we show that the initiator caspase, Dronc, is required for both apoptosis and generation of an active TNF signal in irradiated epidermal cells. These activities are genetically and procedurally separable. The allodynia-promoting function of Dronc requires TNF but not downstream caspases. Indeed, when TNF is expressed ectopically in nociceptive sensory neurons, an unexpected requirement for Dronc is revealed. We have identified multiple conserved downstream signaling factors and a novel target gene that are required for nociceptive sensitization following TNF-mediated activation of sensory neurons. The epigenetic factor versus unirradiated controls  or effector caspase (UAS-p35) activity41 using the epidermal-specific e22C-Gal4 driver.42 Overexpression of either transgene blocked cell death , and the NF-κB-like transcription factor dorsal for UV-induced thermal allodynia. Expression of UAS-RNAi transgenes targeting each of these factors decreased thermal allodynia relative to the Gal4 driver and UAS-alone controls (UAS-RNAi transgene that is effective in the epidermis)13 did not interfere with thermal allodynia, demonstrating that Dronc functions specifically in the epidermis during development of UV-induced thermal allodynia ,50 we tested mutants of all of them. Null mutants for dorsal and Relish failed to develop thermal allodynia, but null mutants for Dif exhibited normal thermal allodynia after UV  of UAS-controls . Importallodynia . To ruleaf6 ref.  and p38a38a ref. . These a38a ref. . As therafter UV . Nocicepafter UV .13 Thus, we examined whether the expression of UAS-RNAi transgenes targeting Traf3, Traf6, p38a, and dorsal can attenuate the ectopic allodynia caused by TNF/Eiger overexpression. As expected, expression of UAS-TNFR/WengenRNAi attenuated TNF-induced thermal allodynia  of potential transcriptional, translational, processing/secretion, or other regulatory mechanisms in regulating TNF activity during UV-induced thermal allodynia remain unclear. To understand how TNF/Eiger is regulated in the epidermis, we monitored changes in a transcriptional reporteradiation .Drosophila S2 cells. Active caspase-3 staining was performed 24 h after UV irradiation and 5 mJ/cm2 was selected as a sub-apoptotic dose  in TNF signaling-activated sensory neurons compared to controls. Twenty-eight of these have clear human orthologs (E(z).61To find target genes whose levels might be regulated by TNF signaling activation in sensory neurons, we performed cell-type-specific microarray analysis. Nociceptive sensory neurons were isolated from control larvae and from genetically- sensitized larvae by overexpressing TNF/Eiger in nociceptive sensory neurons  and 6a. rthologs . These lrthologs . Among tE(z) encodes a histone lysine methyltransferase and is induced 2.6-fold compared to controls. We hypothesized that increased expression of E(z) is functionally important for nociceptive sensitization. Expression of two UAS-E(z)RNAi transgenes in nociceptive sensory neurons was tested during both UV-induced and TNF/Eiger overexpression-induced thermal allodynia. Attenuated responses were observed in both types of thermal allodynia, suggesting that E(z) functions as an important target gene of TNF signaling in the development of thermal allodynia (E(z), off-target effects are highly unlikely (UAS-E(z)RNAi caused a mild defect in baseline thermal nociception (62 expression of UAS-E(z)RNAi transgenes did not alter the dendritic morphology of nociceptive sensory neurons  UV-induced thermal allodynia is completely genetically separable from UV-induced apoptosis. (2) A low dose of UV that does not cause apoptosis still results in Dronc-dependent thermal allodynia. (3) UV irradiation can trigger cellular production of soluble TNF/Eiger. (4) Soluble TNF/Eiger-induced thermal alloydnia does not require Dronc function. (5) Ectopic expression of TNF/Eiger in sensory neurons reveals a new requirement for Dronc in the genetically-induced thermal allodynia.64 Dronc could act on some component of the exocytic pathway to alter subcellular localization of TNF/Eiger. Alternatively, the irradiated epidermis might release Dronc itself and this extracellular Dronc could activate TNF/Eiger. There is precedence for extracellular caspase-6 release from axonal terminals that regulates TNFα secretion from microglial cells in the spinal cord.65How does Dronc function in producing active TNF/Eiger during sensitization? TNF/Eiger does not have a consensus caspase recognition site and any cleavage that would liberate extracellular TNF should necessarily occur outside of the cell. Thus, it is unlikely that Dronc cleaves TNF/Eiger directly. Alternatively, given the evidence that subcellular trafficking is important for TNF production,β, forming inflammasome complexes that cleave the cytokine prodomain.68 Inflammasome complexes have been identified in mice, fish, and humans but not in invertebrates.69 How then do invertebrates orchestrate cytokine-dependent inflammatory responses? Our work might shed new light on this evolutionary problem. The Drosophila apoptosome may be functionally equivalent to an inflammasome.68 Indeed, Dronc is most similar to human caspase-2, which, although it functions as an apoptotic initiator, is also closely related to the inflammatory caspase subgroup.69 Our data reveal that activation of the Drosophila apoptosome (Dark/Apaf-1 and Dronc) results in both apoptosis and production of active TNF/Eiger. This suggests that the Drosophila apoptosome may moonlight as an ancient version of the inflammasome.Inflammatory caspases mediate production of active cytokines such as interleukin-129 Interestingly, however, UV irradiation causes both sensitization in rats9 and production of TNF by epidermal keratinocytes.70Although the specific mechanism is as yet unclear, both full-length and soluble forms of TNF/Eiger were increased following UV treatment – transiently in S2 cells and in a more sustained manner in whole larvae. This suggests that UV increases both full-length expression and processing to a soluble form. Interestingly, local TNF/Eiger production in the epidermis appears more important for thermal allodynia, as overexpression of soluble TNF/Eiger in the epidermis was sufficient to induce ectopic allodynia, whereas hemolymph overexpression of soluble TNF/Eiger was not. Epidermal production of TNF/Eiger following UV irradiation may differ from what occurs during CFA-induced inflammatory pain in vertebrates. Here local TNF is thought to mainly derive from inflammatory cells.κB-like transcription factor. In vertebrates, TRAF6 and p38 have been implicated in TNFα-induced mechanical hypersensitivity in astrocytes and DRG neurons, respectively.72 In mouse sensory neurons, TRPV1 is required downstream of p38 for an electrophysiological correlate of sensitization,72 suggesting that phosphorylation of painless, the TRP channel required for thermal allodynia,51 is a plausible mechanism of sensitization in vivo in Drosophila. NF-κB p50 subunit knockout mice are defective in both acute nociception and inflammatory sensitization,73 but the specific cell(s) in which this activity is required are not yet clear.Our third main finding regards how TNFR/Wengen transduces TNF signaling to mediate thermal nociceptive sensitization. Thermal allodynia requires a number of canonical TNF signaling factors, including 2 TRAFs, a p38a MAP kinase and an NF-κB factor may mediate transcriptional regulation of neuronal target genes. We have here developed a methodology for identifying and validating TNF-activated target genes in this context. E(z) is induced 2.6-fold by TNF expression in nociceptive sensory neurons and is required for nociceptive sensitization. TNF has been found to activate EZH2, the human ortholog of E(z), in skeletal myogenesis.74 Functional roles for polycomb group genes have not been reported in nociceptive biology although histone acetylation has been implicated in injury-induced alterations in nociceptive sensory neurons.75 Conditional knockout mouse lacking histone deacetylase (HDAC) 4 exhibit a specific decrease in thermal sensitization and TRPV1 expression.76 Increased global histone acetylation and epigenetic suppression through HDAC are also involved in inflammatory pain.77 The mechanism by which E(z) contributes to nociceptive sensitization is not yet clear but presumably involves its histone lysine methylation activity. Our data highlight the utility of the Drosophila model for identifying new conserved regulators of nociceptive sensitization and suggest that polycomb group gene members and histone methylation may be evolutionarily conserved regulators of nociceptive sensitization.Because Dorsal functions downstream of TNF/Eiger signaling in sensory neurons during nociceptive sensitization, this NF-13 Briefly, a UV crosslinker  was pre-warmed for 100 s in a time-mode and right after warming up was over, larvae were irradiated in an energy-mode with 20 mJ/cm2 setting. A UV photometer (Spectronics Corporation) was used to measure actual amount of UV that the UV crosslinker emitted at each setting. Comparison between the UV crosslinker setting and the actual reading with a photometer is shown in UV treatment and thermal allodynia analysis were performed as previously described.13 Washed larvae were placed under a Leica  MZ6 light microscope and stimulated on their mid-dorsal side (third or fourth segment from the head) for a maximum of 20 s or until initiation of withdrawal behavior. Aversive withdrawal was defined as a 360° rolling along the anterior posterior body axis within 20 s of physical contact with the probe. Thermal allodynia was tested at 38 °C, 24 h after UV irradiation, whereas baseline nociception was tested at both 45 and 48 °C in the absence of injury. Behavioral responses were categorized as follows: fast (up to 5 s), slow (between 6 and 20 s), and no response (no response within 20 s). χ2-test was used to measure statistical significance in categorical data.For nociceptive behavior analyses, a custom-designed heat probe was used as described previously.52 Primary antibodies: anti-Fasciclin III , anti-activated Caspase-3 , and anti-GFP . Secondary antibodies: alexa488-conjugated anti-mouse IgG , Cy3-conjugated goat anti-rabbit IgG . TUNEL labeling kit  was used to label apoptotic cells.Dissection and immunostaining of larval epidermis were performed as previously described.56 Twnety-four hours after transfection, 50 μM of CuSO4 was added to induce expression. Twenty-four hours later, medium was removed, UV irradiation was performed, and fresh medium added. Cells and media were separated by centrifugation and cells were lysed in RIPA buffer (25 mM Tris/HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing protease inhibitor cocktail (Roche). Proteins in medium were concentrated using Vivaspin15R  in the presence of protease inhibitor. Lysate and concentrated medium were mixed with anti-Flag M2 affinity gel  and incubated at 4 °C overnight. Flag gel beads were collected by centrifugation, washed with RIPA buffer, and boiled with 2 × sample buffer for 5 min. Supernatants were run on SDS-PAGE gels. Immunoblotting was performed using anti-Flag antibody (1:1000) conjugated with HRP .S2 cells were transfected with Nextfect  or lipofectin  transfection reagent to overexpress TNF/Eiger via pMT vector.58 Anti-cv-d (1:2000),78 Anti-actin , HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch), and HRP-conjugate anti-guinea pig IgG (Jackson ImmunoResearch) antibodies. Detection was with ECL reagents .Whole larval extract was prepared by homogenizing PBS-washed larvae in RIPA buffer containing protease inhibitor cocktail. Protein concentration was measured (Bradford assay), and samples were boiled with 3 × sample buffer for 10 min and run on SDS-PAGE gels. Immunoblotting was performed using Anti-Egr (1:100),


Proper monitoring of labor and childbirth prevents many pregnancy-related complications. However, monitoring is still poor in many places partly due to the usability concerns of support tools such as the partograph. In 2011, the World Health Organization (WHO) called for the development and evaluation of context-adaptable electronic health solutions to health challenges. Computerized tools have penetrated many areas of health care, but their influence in supporting health staff with childbirth seems limited.The objective of this scoping review was to determine the scope and trends of research on computerized labor monitoring tools that could be used by health care providers in childbirth management.We used key terms to search the Web for eligible peer-reviewed and gray literature. Eligibility criteria were a computerized labor monitoring tool for maternity service providers and dated 2006 to mid-2016. Retrieved papers were screened to eliminate ineligible papers, and consensus was reached on the papers included in the final analysis.We started with about 380,000 papers, of which 14 papers qualified for the final analysis. Most tools were at the design and implementation stages of development. Three papers addressed post-implementation evaluations of two tools. No documentation on clinical outcome studies was retrieved. The parameters targeted with the tools varied, but they included fetal heart (10 of 11 tools), labor progress (8 of 11), and maternal status (7 of 11). Most tools were designed for use in personal computers in low-resource settings and could be customized for different user needs.Research on computerized labor monitoring tools is inadequate. Compared with other labor parameters, there was preponderance to fetal heart monitoring and hardly any summative evaluation of the available tools. More research, including clinical outcomes evaluation of computerized childbirth monitoring tools, is needed. In 2015, an estimated 303,000 women died from pregnancy-related complications such as excessive bleeding, obstructed labor, and infections .Proper monitoring of the labor and delivery process with appropriate action based on findings is one of the keys to the prevention of pregnancy-related diseases and deaths ,6. LaborSince 1994, the paper partograph has been promoted by the World Health Organization (WHO) as the standard labor monitoring tool , but to Noteworthy, mobile health  was embraced in many settings, especially chronic conditions with concomitant improvement in medical care ,16. It wWe had a notion that the responses to various calls for better labor monitoring tools are still poor. Therefore, we set out to determine the volume and scope of research on computerized monitoring tools that can be used by health care providers in childbirth management.We undertook a scoping review, as defined elsewhere ,20, to aThe inclusion criteria were a paper written in English language, addressing an aspect of a computerized or mobile labor monitoring tool, for use by maternity service providers, and written between January 1, 2006 to May 31, 2016. We excluded literature on tools for use primarily by expectant parents and the numerous apps for nonprofessional use such as contraction monitoring at home or fetal growth monitoring.Our key terms in the search included “labor,” “monitoring,” “computer,” “mobile,” “tool,” “provider,” “delivery,” and “birth.” We combined them in various ways to get search strings. An example of such a string is “With all ‘labor monitoring’ + plus at least one of ‘computer$ tool$ mobile provider$ delivery birth - consumer’ (anywhere in article).” At the title review stage, we combined the term “labor” with one or more of the other terms to get relevant papers.Data collection started with the individual researcher or research assistant identifying and screening papers for allocated years. We then entered the search terms and filtered the results according to the desired years of publication. We sorted the results in ascending years to ease tracking of the viewed Web pages. Each collector exported the identified papers into Mendeley-1.16.1 reference manager (by Elsevier) and used it to remove duplicates.The data collector imported the Mendeley (by Elsevier) output into Google Scholar or PubMed and applied more specific filters to the titles. The filters helped eliminate the nonhuman birth-related and non-computer tools. The subsequent titles were saved to an online library for further screening. Abstracts to the saved titles were downloaded and qualitatively analyzed to determine the target users. Uncertainty about the eligibility of an article was consensually resolved based on the selection criteria., to mimic the preferred reporting items for systematic reviews and meta-analyses (PRISMA). Each paper was read to decipher the focus, developmental stage, and computing platform of the labor monitoring tool.Full papers to abstracts with all eligibility criteria were retrieved. Manually identified abstracts or papers that were not part of the controlled search were added to the pool. All papers were scrutinized against all selection criteria. The remaining papers were included in the quantitative final analysis. These steps are summarized, as shown in In the preliminary literature search, we retrieved 389,000 titles from Google Scholar and 156 from PubMed . These wAs shown in Five tools that were in the clinical trial or field testing -25 phaseThe main parameters of focus in the analyzed papers were monitoring all labor parameters (7 papers) and fetal heart (3 papers). Four tools were designed to monitor one of three labor monitoring sections, whereas the rest could monitor multiple sections, as shown in All tools were intended for clinical diagnosis support based on algorithms. The least basic was the capability to take measurements or give alerts and reminders to maternity care providers ,29,34. H, the tools were mostly usable on existing personal computer hardware, especially mobile gadgets such as phones, laptops, and desktop computers. Majority communicated through client-server networks, and 4 of 11 (36%) used stand-alone computers. Microsoft Windows was the most commonly used operating system in desktops, and Android was used in most mobile systems. One tool (PartoPen) uses custom-made software, whereas the application-based tools were developed in a Java environment. For half of the tools, the authors did not specify the software framework used in development. Of 11 tools, only 4 were reported as customizable to suit a user’s context of work.As shown in The authors of the analyzed papers planned for adaptable tools. The intended context of tool use was stated as low-resource settings apart from the ANGEL shield. This computer program was made for and operated in a university hospital, but there were plans of rolling it out to nearby rural health centers. For 5 of 11 tools, it was reported that they could be customized to different contexts of use and even more functions added, where necessary.In this review, out of over 380,000 papers, 14 qualified for the final analysis. They represented studies of 11 computerized tools capable of aiding health workers in maternity care. All labor parameters could be monitored by 7 of the 11 tools upon implementation. Only one tool had summative evaluations, which included cost and indicator studies. We did not find evidence of morbidity or mortality evaluation for any tool. Most tools used open source software that was also adaptable to common computers.In this study, we chose to conduct a scoping review due to an ostensibly low volume of systematic evaluations and publications but with potentially more works on the subject. To this effect, many papers on pregnancy care applications were identified, although they were excluded from analysis for failure to meet other inclusion criteria. PubMed and Google Scholar formed the basis for the main search results due to their popularity among authors and a wide coverage of subjects. They were augmented with a manual search that indeed yielded more papers ,23,32,33Many papers were identified, which echo the findings of Kortteisto et al 2014), that is, mHealth is widespread . However, that isThis state of affairs may be due to the human-intensive nature and high litigation potential of labor monitoring events that designing and testing a reliable computerized labor tool calls for more effort than is needed for an average medical condition. Another factor that could deter innovators is the difficulty in definitive summative evaluation of the tools in light of many confounders of labor outcomes. A similar situation in 2011 could have led the WHO to call for better research and evaluation of mHealth  such as After the data collection for this review, another call to action on improving quality of maternity and newborn care was sounded through the Lancet maternal health series . FurtherRegarding the focus of the tools, it was obvious that the three main sections—fetal, labor progress, and maternal states—of labor monitoring received unequal attention. Monitoring the fetal condition, especially the fetal heart, was most researched, perhaps due to the discovery of cardiotocogram (CTG) that is efficacious in detecting fetal distress. CTG is sonoelectric and improving it through computerization was easier than inventing tools as was necessary for the labor progress. On the other hand, cervical dilation and maternal conditions are subjectively dependent on provider skills, and sociocultural or religious norms. Hence, the diversity of these conditions could hamper the design, testing, and development of widely acceptable tools to monitor labor progress and maternal conditions. With the recognition of the diversity of contexts of use but limited resources, it is prudent that generic tools are designed and developed or adapted for specific contexts .The operating system platforms were generic in about half of the tools perhaps to cut development costs. This is also good for the diverse hardware that is increasingly portable. On the other hand, the programming software base was mostly undisclosed. This could be due to the early stages of development. Moreover, the authors were not yet committed to a specific program. However, the generic Java development environment was used in most specified cases. This would suggest that even the rest are more likely to use it in tool development.The sources of data for this review were not exhaustive of all literature , and as such, we could have omitted some papers on the computerized labor monitoring tools. This is a drawback, but the main sources are wide enough for an adequate sample and similar reviewers ,20,40 inIn conclusion, the scope and volume of research on computerized maternal labor monitoring tools is likely to be narrow and small. Fetal heart monitoring seems to dominate over other labor management parameters. Most tools are designed to use affordable computing platforms, but there is hardly any summative evaluation of the available tools. This may imply a slow response to the call for developing and evaluating computerized labor monitoring tools that could reduce labor-related disease. Further research, including clinical outcomes studies and publication of results, is needed on computerized tools for use in comprehensive childbirth monitoring.


The collection, management and visualization of clinical pedigree (family history) data is a core activity in clinical genetics centres. However, clinical pedigree datasets can be difficult to manage, as they are time consuming to capture, and can be difficult to build, manipulate and visualize graphically. Several standalone graphical pedigree editors and drawing applications exist but there are no freely available lightweight graphical pedigree editors that can be easily configured and incorporated into web applications.We developed ‘pedigreejs’, an interactive graphical pedigree editor written in JavaScript, which uses standard pedigree nomenclature. Pedigreejs provides an easily configurable, extensible and lightweight pedigree editor. It makes use of an open-source Javascript library to define a hierarchical layout and to produce images in scalable vector graphics (SVG) format that can be viewed and edited in web browsers.https://ccge-boadicea.github.io/pedigreejs/).The software is freely available under GPL licence (Bioinformatics online. BRCA1 and BRCA2 mutation carrier probabilities and breast cancer risks in the BOADICEA https://www.nice.org.uk/guidance/cg164).The collection, management and visualization of pedigree (family history) data is a core activity in clinical settings, such as genetic centres and primary care, where healthcare professionals manage patients at risk of disease. Pedigree data are used to identify individuals at risk of familial disease and to inform their clinical management, including referral of individuals at elevated risk for mutation screening and/or recommending enhanced screening. For example, generating a pedigree is a requirement for calculating Clinical pedigree datasets can be difficult to manage for several reasons: (i) they are supplied by humans not machines, and are therefore time consuming to capture in digital form; (ii) they include both family structure  and separate information for each family member (which must be linked); (iii) they often require updating over time; (iv) they can include data errors (e.g. broken family structures) and internal inconsistencies; and (v) they must be stored securely in order to fulfill data privacy requirements.http://www.progenygenetics.com, http://pedigreedraw.com/, http://www.pedigreexp.com). However, after evaluating available tools  to assist the layout of individuals’ nodes in the pedigree representation. Pedigrees are rendered as SVG images in the browser window which results in a fast and responsive user experience to facilitate clinical data entry. The standard nomenclature for representing pedigrees Pedigreejs has been implemented as a configurable JavaScript library to provide a graphical pedigree editor in a web-based environment. The pedigreejs algorithm uses the d3-hierarchy JavaScript module . This means that confidential patient data can be stored on the client computer and not transmitted over the Internet. For reasons of data privacy the default is to hold these data for the session so that they do not persist after the browser or tab is closed. Previous versions of the pedigree dataset are also stored so that when a change is made it can be undone. Basic validation of the dataset is provided by default  and this can be overridden to define specific validations.https://www.w3schools.com/js/js_json_intro.asp) to facilitate data interchange with other applications. JSON is easily extendable, so that the individual level data that are gathered are not limited. Once constructed, the pedigree dataset can be saved to a JSON format data file and loaded back into pedigreejs at a later date, so that pedigrees can be easily updated. In addition, pedigree data files in BOADICEA Web Application (v4) pedigree format and PED format  and uses D3.js to assist in the rendering and does not send requests to a backend server. Creating and deleting individuals is made easy by clicking on widgets that appear on mouseover of a family member. ‘Undo’, ‘redo’ and ‘reset’ options and the ‘fullscreen’ option add to this being a user friendly editing tool.Future developments will include further support for other commonly used pedigree data file formats . Pedigreejs will also be updated to support more complex family structures including consanguineous partners beyond those at the same level in the tree such as cousins. Pedigreejs will be used in future releases of the BOADICEA Web Application.Supplementary DataClick here for additional data file.


During Eugene Garfield’s (EG’s) lengthy career as information scientist, he published about 1500 papers. In this study, we use the impressive oeuvre of EG to introduce a new type of bibliometric networks: keyword co-occurrences networks based on the context of citations, which are referenced in a certain paper set (here: the papers published by EG). The citation context is defined by the words which are located around a specific citation. We retrieved the citation context from Microsoft Academic. To interpret and compare the results of the new network type, we generated two further networks: co-occurrence networks which are based on title and abstract keywords from (1) EG’s papers and (2) the papers citing EG’s publications. The comparison of the three networks suggests that papers of EG and citation contexts of papers citing EG are semantically more closely related to each other than to titles and abstracts of papers citing EG. This result accords with the use of citations in research evaluation that is based on the premise that citations reflect the cognitive influence of the cited on the citing publication. Science (Garfield clarivate.com) which provided later the Science Citation Index, the Social Sciences Citation Index, and the Arts and Humanities Citation Index—the predecessors of the Web of Science (WoS). With algorithmic historiography, Garfield et al. . In this study, we use the impressive oeuvre of EG to introduce a new type of bibliometric networks: keyword co-occurrence networks based on the context of citations, which are referenced in a certain paper set (here: the papers published by EG). The citation context is defined by the words which are located around a specific citation. Most of the networks published hitherto are citation, co-citation, bibliographic coupling, co-authorship networks, or keyword co-occurrence . Jha et al. . First, we retrieved all publications by EG and their MA ID. Only 327 of EG’s 1558 publications were found in MA. Second, we retrieved all publications citing EG’s papers. Citations contexts were available for 59 papers. Third, for the 59 papers and their citing papers (n = 343 papers), we retrieved the title and abstract information from the WoS by searching for the Digital Object Identifier (DOI). These two sets of publications show an overlap of two papers. Finally, we extracted the citation context (428 citation contexts due to multiple citations in some citing papers) of the citing papers.All MA data were collected via the AK API using the www.vosviewer.com; van Eck and Waltman We produced three co-occurrences networks using MA data and the VOSviewer software  and names of cited authors in citation contexts  were removed.n = 59) to get an impression of his papers (included in this study). From the titles and abstracts of these 59 papers published by EG, 607 keywords were extracted of which 27 occurred four or more times. Based on the criteria mentioned in “In the first step of the analysis, we generated a network based on EG’s papers for which a citation context was available ;SNIP ;h-index ;PageRank ;Journal Citation Report ;Science Citation Index ;Publication output of regions or nations ;Rise of output of nations ;Self-citation ;Coverage of databases ;Mapping and visualization ;Citation networks and prediction or recommendation ;Diffusion of ideas ;Scholarly communication ;Lancet, New England Journal of Medicine, PubMed);Publication and citation analysis in medicine ;Digital libraries ;Peer review and research evaluation .The 18 clusters identified by VOSviewer can be characterized with the following extracted keywords:The 18 clusters cover a broad range of bibliometric topics, which refer—among other things—to products of providers of bibliometric data (e.g. JCR), indicators (e.g. h-index), units of bibliometric analysis (e.g. nations), types of citations (e.g. self-citations), bibliometric methods (e.g. citation networks), and the use of citations in peer review and research evaluation. The broad range of topics show that EG’s papers are cited by publications with a very heterogeneous thematic spectrum.In the third step of the analysis, we contrast the results on the cited and citing papers with those of the citation context in the citing papers. From the 428 citation contexts that enclose references to EG’s publications, a total of 2347 keywords were extracted of which 184 occurred four or more times. Based on the inclusion criteria, 97 keywords were retained in the map. The following three clusters were identified: ‘Journal Impact Factor’, ‘historiographs’, and ‘Eugene Garfield as founder of the ISI’  the ‘normalization’ is among the most frequent keywords. It refers to the problem of the JIF that it cannot be used for cross-field comparisons. Citation data have field-specific patterns which can be normalized by different methods  are associated with different keywords in the citation context network than in the other two networks. In particular, the JIF is discussed in relation to other indicators such as SNIP, percentiles, and the h-index, JCR in relation to JIF scores and the Eigenfactor, and the SCI in relation to patents.Taken as a whole, the comparison of the three networks suggests that papers of EG and citation contexts of paper citing EG are semantically more closely related to each other than to titles and abstracts of papers citing EG. This result accords to our expectation that the citation context network is related to title and abstract keywords from the cited but not citing publications. This is in line with the use of citation data in research evaluation purposes see “” section


In 2016, 55 jurisdictions in 49 states and six cities in the United StatesIISs, also known as immunization registries, are confidential, computerized, population-based systems that collect and consolidate vaccination data from providers in a jurisdiction  programHealthy People 2020 objective of ≥95% child IIS participation. Among the 55 jurisdictions, 33 (60%) reported that ≥95% of children aged 4 months through 5 years in their geographic area participated in their IIS in 2016, compared with 24 (44%) in 2013. In 2016, provider participation was 85% among VFC provider sites enrolled in an IIS. The number of VFC provider sites enrolled in an IIS decreased from 41,710 in 2014 to 41,393 in 2016. Among these enrolled sites, the number of VFC provider sites participating in an IIS increased slightly from 33,266 in 2013 to 34,662 in 2016  to IISs, and to request and receive immunization information from IISs into EHRs for the patients they serve. HL7 messaging is a nationally recognized platform-independent standard that supports the bidirectional exchange of health-related information, including immunization-related messaging. In 2016, 91% of jurisdictions had an IIS that used HL7 version 2.5.1 to receive vaccination histories from providers and returned acknowledgment messages, compared with 87% in 2013. Furthermore, in 2016, 67% of jurisdictions had an IIS that received requests for vaccination histories and returned responses to those requests, compared with 45% in 2013 . FinallyClinical Decision Support (CDS) functionalities enable providers to evaluate the validity of vaccine doses administered to patients and forecast future vaccines that will be needed, based on recommendations developed by the Advisory Committee on Immunization Practices. From 2013 to 2016, all jurisdictions’ IISs had CDS capabilities that were available to providers through the IIS’s user interface. In 2016, 58% (32 of 55) of jurisdictions reported sending a vaccine forecast to another system via HL7 messaging. This is an 87% increase from 2013, when 31% (17 of 55) of jurisdictions reported performing this task.IISs can be used to generate coverage estimates for childhood vaccinations at the jurisdictional level  and at the provider level to identify vulnerable subpopulations. In 2016, 89% of jurisdictions (49 of 55) provided a predefined, automatic report on immunization coverage by geography. This is 11% higher than in 2013, when 80% of jurisdictions provided these reports. In 2016, 95% of jurisdictions (52 of 55) provided a predefined, automatic report on immunization coverage by provider site. This is 7% higher than in 2013, when 89% of jurisdictions reported providing these reports.Since 2013, incremental progress was noted in each of the four priority areas for immunization programs that were assessed. Notably, the increased number of jurisdictions that had IIS estimates that were within 10 percentage points of the corresponding NIS-Child coverage estimate suggests that more jurisdictions have IISs with more complete data, or at least that the IIS and NIS are similar in their ability to estimate vaccination coverage for that jurisdiction’s population. Jurisdictions with IIS coverage estimates that were at least 10 percentage points lower than the corresponding NIS-Child estimate might have less complete IIS data, particularly at sites with the largest IIS–NIS discrepancies.By prioritizing resources to the identified priority areas, jurisdictions can make substantial progress in this important subset of activities rather than incremental progress across all Functional Standards. Improvements in priority areas can also support a broader range of immunization services; for example, improved data completeness for children aged <6 years would strengthen immunization delivery for this population  and increase VFC program accountability (2.1–2.6). In addition, as IISs identify more children and record all doses administered within their jurisdiction, IIS-based vaccination coverage estimates will be able to supplement estimates from surveys like the NIS-Child  had ≥2 doses recorded in immunization information systems (IISs).Healthy People 2020 objective of ≥95%. However, variability in IIS pediatric data quality persists: 30 of 55 IISs produced 7-vaccine series coverage rates that were at least 10 percentage points lower than the corresponding National Immunization Survey-Child coverage rate in 2016, suggesting incompleteness of IIS data. Across all IISs, there was progress in achieving bidirectional information exchange with electronic health record systems, pediatric clinical decision support for immunizations, and the ability to generate jurisdictional and provider-level childhood vaccination coverage estimates.From 2013 to 2016, the percentage of children with ≥2 immunizations recorded in IISs increased from 90% to 94%, approaching the To realize the full benefits of IISs, immunization programs need to implement strategies that prioritize and align resources to achieve functionality and high data quality in four focus areas: 1) pediatric data completeness, 2) bidirectional data exchange with electronic health record systems, 3) clinical decision support for immunizations, and 4) ability to generate childhood vaccination coverage estimates. Strategies such as implementing best practices, adhering to national standards, and incorporating independent third-party assessments can reduce variability across IISs, and support IIS’ full potential to facilitate complete vaccination of U.S. children against vaccine-preventable diseases.