Annually over 520,000 newborns die from neonatal sepsis, and 60,000 more from tetanus. Estimates of the effect of clean birth and postnatal care practices are required for evidence-based program planning.To review the evidence for clean birth and postnatal care practices and estimate the effect on neonatal mortality from sepsis and tetanus for the Lives Saved Tool (LiST).We conducted a systematic review of multiple databases. Data were abstracted into standard tables and assessed by GRADE criteria. Where appropriate, meta-analyses were undertaken. For interventions with low quality evidence but a strong GRADE recommendation, a Delphi process was conducted.Low quality evidence supports a reduction in all-cause neonatal mortality (19% (95% c.i. 1–34%)), cord infection (30% (95% c.i. 20–39%)) and neonatal tetanus (49% (95% c.i. 35–62%)) with birth attendant handwashing. Very low quality evidence supports a reduction in neonatal tetanus mortality with a clean birth surface (93% (95% c.i. 77-100%)) and no relationship between a clean perineum and tetanus. Low quality evidence supports a reduction of neonatal tetanus with facility birth (68% (95% c.i. 47-88%). No relationship was found between birth place and cord infections or sepsis mortality. For postnatal clean practices, all-cause mortality is reduced with chlorhexidine cord applications in the first 24 hours of life  and antimicrobial cord applications . One study of postnatal maternal handwashing reported reductions in all-cause mortality (44% (95% c.i. 18–62%)) and cord infection ((24% (95% c.i. 5-40%)).Given the low quality of evidence, a Delphi expert opinion process was undertaken. Thirty experts reached consensus regarding reduction of neonatal sepsis deaths by clean birth practices at home (15% (IQR 10–20)) or in a facility (27% IQR 24–36)), and by clean postnatal care practices (40% (IQR 25–50)). The panel estimated that neonatal tetanus mortality was reduced by clean birth practices at home (30% (IQR(20–30)), or in a facility (38% (IQR 34–40)), and by clean postnatal care practices (40% (IQR 30–50)).According to expert opinion, clean birth and particularly postnatal care practices are effective in reducing neonatal mortality from sepsis and tetanus. Further research is required regarding optimal implementation strategies. More than half a million newborns are estimated to die each year from serious neonatal infections, accounting for about 15% of all neonatal deaths globally , which iIt is estimated that 30-40% of infections resulting in neonatal sepsis deaths are transmitted at the time of childbirth and have early onset of symptoms (developing during the first 72 hours after birth) ,7. In loIn addition many neonatal deaths due to tetanus and other infections are acquired postnatally ,10. The The global burden of neonatal tetanus has reduced over from over 600,000 neonatal deaths in 1990 to fewer than 60,000 in 2008 . IncreasHygienic behaviours during childbirth and during the early postnatal period are variably defined. In this paper, we define clean birth and postnatal care practices in accordance with World Health Organisation’s (WHO) “six cleans” - hand washing of birth attendant before birth, clean birth surface, clean perineum, cutting of the umbilical cord using a clean implement, clean cord tie, and a clean cloth for drying Figure . These pAlthough such hygienic practices at birth and during the postnatal period are accepted as a standard of care, there is very limited evidence regarding their effect to guide policymakers in the potential mortality effect size. Individually randomized trials of hygienic versus unhygienic practices would clearly be unethical, and thus the quality of evidence will always be limited. There is increasing evidence from cluster randomized trials in South Asia of the significant effect on neonatal mortality of postnatal care practices, including clean practices, ,30-35. HThis review does not include other interventions to reduce deaths from neonatal infections, such as tetanus toxoid immunization , antibioThe objective of this review is to estimate, for use in the Lives Saved Tool (LiST), the effect of clean practices at birth and during the postnatal period on all-cause neonatal mortality, cause-specific mortality from sepsis and tetanus, and infection-related morbidity .This review uses an adaptation of the GRADE approach and is designed to provide estimates for use in LiST which models cause-specific deaths averted by increases in coverage of effective interventions. Details of the general review methods, the adapted GRADE approach and the LiST model have been described elsewhere .“clean/safe birth/delivery +/- kit”, “tetanus,”, “sepsis,” “meningitis,”, “infection,” “omphalitis/oomphalitis”, “hygiene,” “hand washing,” “umbilical cord care,” “skin care,” “neonatal/perinatal mortality”, “newborn care”, ”chlorhexidine”, “home birth”, “Latin America/Africa/Asia or developing country” limited to newborn 0 – 28 days. as defined below. We considered both randomised trials and observational studies  as follows. The s Figure . We exclInterventions considered in this review include clean birth practices at home or in a health facility, including hand washing, clean cord cutting and tying, clean birth surface, clean perineum, topical antiseptic applications to the cord and skin, and clean birth kit use. In addition clean postnatal newborn care practices and applications to the birth canal, umbilical cord or skin were reviewed Table . The effFrom a programmatic viewpoint, how to change complex behaviours and cultural norms that govern practices around the time of birth is of great importance, but is not the focus of this paper Figure .We used cause of death definitions consistent with the Child Health Epidemiology Reference Group (CHERG) based on ICD 10 rules . Our out2 and the chi-squared test. When evidence of heterogeneity was present (p<0.10), a random effects model was used, otherwise a fixed effect was assumed. Summary risk ratios and corresponding 95% confidence intervals (CI) are reported.All studies meeting the inclusion criteria were abstracted onto a standardised abstraction form for each outcome of interest . Each stFor interventions with low or very low quality evidence, we sought expert consensus via the Delphi method . The queOur searches identified 778 records, and snowball searching identified a further three papers. Figure  After inThe remainder of this review focuses on studies carried out in low/middle income countries where the effect of the clean practices is likely to be larger, and the evidence more applicable to decision makers in the countries using the LiST tool. The order of the results section follows the list in table 1Most studies of clean birth practices are carried out in populations with high rates of home birth. Direct evidence for the effect of clean birth practices in a facility compared to clean birth practices at home on overall neonatal mortality or sepsis-related mortality is complex to assess given multiple confounders. Four studies reported a reduced risk of neonatal tetanus associated with facility delivery after adjusting for potential confounders .-63.60-63One large cohort study  reported lower neonatal mortality associated with reported birth attendant hand-washing with soap and water (aRR=0.81: 95% CI 0.66 – 0.99) . No caseTwo case-control studies reported no association after adjustment between cleaning the perineum, with soap and water, and reduction in the incidence of neonatal tetanus ,65. No eTwo case-control studies controlling for potential confounders examined the association between using a clean plastic sheet as a birth surface and incidence of neonatal tetanus and reported aOR=0.31 (95% CI 0.10 – 0.91) and aOR=Seven observational studies  were identified which examined the association of using a clean implement (a new/ boiled/ sterile blade or scissors) to cut the cord with neonatal sepsis or tetanus.Five of these studies from Asia and one from Senegal examined the effect on neonatal tetanus incidence or mortality. Two case-control studies and one cohort study which sought to adjust for confounders reported strong evidence of lower neonatal tetanus mortality associated with use of clean cord cutting tools . However, the level of new blade use was >95%, and those not using new blades may have used boiled blades .Four case-control studies examined the association between use of a ‘new clean thread’ to tie the umbilical cord at birth and neonatal tetanus incidence or mortality. A study from Uganda using hospital-based cases reported an aOR of 0.1 (95% CI 0.01 – 1.1) for clean cord tie use . The thrAn urban community case-control study compared use of a cord clamp versus ‘thread’ to tie the cord, and found no evidence of a difference in tetanus incidence between the two groups in multivariate analysis, where home birth and cord applications were the most important risk factors . A cohorVaginal washing with chlorhexidine has been shown to reduce colonization rates with Group B streptococcus in high income countries . The mosA large community-based cluster RCT of skin cleansing with chlorhexidine (0.25%) as soon as possible after birth without vaginal cleansing did not find evidence of a reduction in neonatal mortality among all treated infants . In a sub-analysis among low birth weight infants, those wiped with chlorhexidine experienced lower neonatal mortality than the placebo group  ) and sepsis-specific neonatal mortality (RR=0.69 (95% CI 0.4 – 1.18) if applied within the first 24 hours . ChlorheFour case-control studies reported that cord applications of topical antimicrobials were associated with reduced neonatal tetanus incidence compared with dry cord care. The reported associations found for topical antibiotics were [aOR=0.21(95% CI 0.05-0.97))] and aOR); for diEvidence from case-control studies supports an association between neonatal tetanus and applications of cow dung or ash to the cord [aOR=2.31 (95% CI 1.03 – 5.03)], traditiConsistent with the effect on neonatal tetanus, in one cohort study in Nepal, mustard oil application was associated with an increased risk of cord infection [aRR=1.29 95% CI 1.11 – 1.51)] [ – 1.51] Two RCTs of hospitalized preterm infants of less than 33 weeks gestational age demonstrated reductions in nosocomial bloodstream infections with topical application of emollients(sunflower oil)   and all-Three studies using clean birth kits have reported effects on overall neonatal mortality. One before-and-after study reported a 22% reduction in all-cause neonatal mortality, OR 0.78 (95% CI 0.50 – 1.21), after introduction of clean birth kit use into an existing lady health worker-delivered package of antenatal/ birth care . An 80% One further study, also reporting on neonatal sepsis and cord infection, was excluded as ‘newborn care kits’ were given, which included soap and spirits, but no blade or cord tie ,82. ThreOne before-and-after study reported an 89% reduction in neonatal sepsis after the introduction of clean birth kits for home births [unadjusted OR=0.11 (95% CI 0.01 – 0.84) [Four cross-sectional studies examined the association between clean birth kits and incidence of cord infection. They used different definitions for cord infection and showed marked heterogeneity. All studies compared clean birth kits users (adopters) with non-users (non-adopters). In three studies the kits were distributed free of charge by health workers ,57,59. IA study from Pemba, Tanzania found an increased risk of infection with use of a plastic cord cutting coin surface . This stFour studies report a protective association of neonatal tetanus with facility birth. No strong evidence of a relationship between place of birth and cord infections was found.Published data from four low or very low quality studies -63 are cAll studies are consistent with a beneficial effect of clean birth practices; however the data currently available are inadequate to evaluate the size of the effect of a clean cord cutting implement, clean perineum, clean cord tie or clean birth surface on mortality from neonatal sepsis. Limited data on the effects on cord infection were found, however, these were single studies providing low quality evidence and the outcome was distal to the outcome of interest .Low quality evidence of no association of a clean perineum with the incidence of neonatal tetanus, and a reduced incidence with a clean birth surface was found. Much heterogeneity was found in the six very low quality studies reporting the effect of a clean cutting implement and four very low quality studies reporting the effect of a clean cord tie on neonatal tetanus incidence. The overall of the evidence was low. Table Evidence from three very low quality studies suggests a 63% 95% CI 41 – 86%) reduction in neonatal tetanus mortality with antimicrobial applications to the cord. A community-based cluster RCT and two facility-based RCT have not found evidence of a benefit in mortality reduction of chlorhexidine skin applications with or without chlorhexidine vaginal cleansing -72. A si1 – 86% rTwo high quality studies found evidence that topical emollients reduce nosocomial infections and one study reported a reduction in neonatal mortality in hospitalized preterm infants -79.One study provided supportive evidence of an effect of maternal hand-washing in the postnatal period on all-cause neonatal mortality of 44% (95% CI 18 – 62%) .One very low quality study reported a reduction in neonatal sepsis of 89% (95% CI 16 – 99%) . Four stDue to concurrent interventions  and contextual factors, the generalisability of the remaining study findings reviewed is unclear. Hence, whilst the available data supports that clean birth kit use as part of a ‘package’ has an effect on neonatal mortality from sepsis and tetanus we were unable to estimate the individual contribution of clean birth kit use on this mortality reduction.Low quality evidence was found for the effect of hand washing and for antimicrobial applications to the cord on mortality from neonatal sepsis and tetanus. Very low quality or no evidence was found for the other clean practices reviewed. This is counter balanced with the ethical complexity of randomised trials of what is a standard of care throughout the world. The GRADE recommendation for clean practices is strong. Given this recommendation, and as the objective of this review is to establish mortality effect estimates for clean practices with transparent methodology, a Delphi expert consensus was undertaken. The panel invited to participate included experts in obstetrics, gynaecology and newborn health representing five WHO regions , and including multiple disciplines - programme management, research, obstetrics, and paediatrics. Thirty experts participated.Consensus was reached in the first round for all interventions to reduce neonatal mortality. Experts judged that clean birth practices at home with no skilled attendant could avert 15% (IQR 10 – 20) of sepsis-related and 30% (IQR 20 – 30) of tetanus-related neonatal deaths. Skilled attendance at home was judged to avert 23% (IQR 19 – 30) of sepsis-related and 35% (IQR 30 – 40) of tetanus-related neonatal deaths. Skilled attendance in a facility was judged to increase this to 27% (IQR 24 – 36) of sepsis-related and 38% (IQR 34 – 40) of tetanus-related deaths. Clean newborn care practices in the postnatal period were judged to avert 40% IQR 25 – 50) of sepsis-related and 40% (IQR 30 – 50) of tetanus-related deaths. , health worker/birth attendant training and specific vehicles such as clean birth kits. Possible unintended adverse effects of these strategies may exist e.g. dis-incentivising facility birth. Table  There isOne potential vehicle for promoting clean birth and postnatal care practices is the clean birth kit. In some settings, e.g. in conflict or humanitarian emergencies, or in settings where there is currently low coverage of facility birth, a working group of over 35 experts from multiple disciplines concluded that clean birth kits are to be recommended as long as they do not act as a disincentive for facility birth . The conWhile clean birth and postnatal care is widely accepted, there is understandably low-quality evidence for the effect of these interventions especially in low income settings. However, since there is strong biological plausibility and given that clean practices are an accepted standard of care, the GRADE recommendation is strong. Our Delphi expert consensus process judged that clean birth practices at home with no skilled attendant could reduce neonatal sepsis deaths by 15% and tetanus deaths by 30%. The panel judged that clean birth practices in a facility would reduce neonatal deaths from sepsis by 27% and tetanus by 38%. Postnatal newborn care practices were considered to have a higher effect on neonatal mortality with 40% reduction in both sepsis and tetanus deaths.More research is needed particularly on the content and quality of care during the early postnatal period. Given that most evidence to date is from South Asia, the results of ongoing studies in Africa are of especial importance. Use of standard definitions and outcome case definitions would improve future attempts at evidence synthesis. In addition population-based coverage data are lacking for clean birth practices or for use of clean birth kits.Clean practices at birth and in the postnatal period could prevent many needless deaths, especially in settings with high baseline neonatal mortality and where the majority of births still take place at home, although in many facilities in low income settings, hygienic practices may also be sub optimal. The benefits of a clean birth have been recognised for centuries and if this basic and feasible action was achieved for every mother and baby of the 135 million births each year, over 100,000 lives could be saved each year .JL planned the review with HB who undertook the searches and abstraction. HB and JL drafted the manuscript. ACL contributed to the design of the Delphi process. HB produced the meta-analysis. SC provided statistical support. All authors contributed to the data review and to the manuscript.This work was supported in part by a grant to the US Fund for UNICEF from the Bill & Melinda Gates Foundation (grant 43386) to “Promote evidence-based decision making in designing maternal, neonatal and child health interventions in low- and middle-income countries”, and by a grant to Save the Children USA from the Bill & Melinda Gates Foundation (Grant 50124) for "Saving Newborn Lives".The authors all declare no conflict of interest.Delphi formClick here for fileDetailed GRADE tableClick here for file


Previous research has documented heterogeneity in the effects of maternal education on adverse birth outcomes by nativity and Hispanic subgroup in the United States. In this article, we considered the risk of preterm birth (PTB) using 9 years of vital statistics birth data from New York City. We employed finer categorizations of exposure than used previously and estimated the risk dose-response across the range of education by nativity and ethnicity.Using Bayesian random effects logistic regression models with restricted quadratic spline terms for years of completed maternal education, we calculated and plotted the estimated posterior probabilities of PTB  for each year of education by ethnic and nativity subgroups adjusted for only maternal age, as well as with more extensive covariate adjustments. We then estimated the posterior risk difference between native and foreign born mothers by ethnicity over the continuous range of education exposures.The risk of PTB varied substantially by education, nativity and ethnicity. Native born groups showed higher absolute risk of PTB and declining risk associated with higher levels of education beyond about 10 years, as did foreign-born Puerto Ricans. For most other foreign born groups, however, risk of PTB was flatter across the education range. For Mexicans, Central Americans, Dominicans, South Americans and "Others", the protective effect of foreign birth diminished progressively across the educational range. Only for Puerto Ricans was there no nativity advantage for the foreign born, although small numbers of foreign born Cubans limited precision of estimates for that group.Using flexible Bayesian regression models with random effects allowed us to estimate absolute risks without strong modeling assumptions. Risk comparisons for any sub-groups at any exposure level were simple to calculate. Shrinkage of posterior estimates through the use of random effects allowed for finer categorization of exposures without restricting joint effects to follow a fixed parametric scale. Although foreign born Hispanic women with the least education appeared to generally have low risk, this seems likely to be a marker for unmeasured environmental and behavioral factors, rather than a causally protective effect of low education itself. A great deal of research in reproductive and social epidemiology has focused on the "Hispanic Paradox", in which Hispanic women of low socioeconomic status (SES) in the United States (US) have better than expected birth outcomes, compared to other similarly disadvantaged groups, such as African-Americans . Some auWe extend the previous research in several ways using a dataset from New York City. Although our sample size is smaller than that used by Acevedo-Garcia et al, by using Bayesian random effects regression models we are able to employ finer categorizations of both education and Hispanic subgroups to depict additional heterogeneity in risk of adverse birth outcomes and variations in the degree to which advancing education is associated with reduced risk across Hispanic subgroups, all on the absolute scale. We propose that the use of a single geographic region is advantageous in contrast with national data in order to avoid confounding by many unmeasured regional differences, such as the historically unique context of Cuban-Americans in South Florida or Mexican-Americans in the Southwest.The absolute scale is a more useful contrast to make in a public health context because it directly represents the number of attributable cases, and therefore the actual public health burden . The useLow birthweight, defined as births of less than <2500 g, has been widely used as a convenient measure of an adverse birth outcome. However, LBW encompasses infants with a mix of underlying pathologies: those who are growing normally but are born too early (i.e. preterm), and those that are full term but small from stunted fetal growth (i.e. intra-uterine growth retardation). Therefore, we focus in the current report on preterm birth (PTB) as a more etiologically distinct outcome , especiaWe used publicly available vital statistics birth data for 1995 to 2003 from the NYC Department of Health and Mental Hygiene. To remain consistent with the previous analysis by Acevedo-Garcia et al [Preterm birth was defined as delivery for any reason prior to 37 completed weeks of gestation using the clinical estimate. Maternal education was based on self-reported years of completed schooling. Although previous authors categorized years of education broadly, we included the exact number of years reported, up to a maximum of 17 years, using a flexible regression model. Nativity was dichotomized as foreign-born or native-born, where for Puerto Rican women this corresponded to the distinction between being born on the island of Puerto Rico ("foreign born") and being born in the mainland United States ("native born"). Hispanic subgroups were based on maternal self-reported ancestry and ethnicity and categorized as: Mexican, Puerto Rican, Cuban, Dominican, Central American , South American  and Other/Unknown.Models were adjusted either for age alone, or for covariates selected to replicate the previously published analysis. These included: prenatal care, defined by the Kessner Index as adequate, intermediate or inadequate care , sex of We estimate the risk of PTB by years of maternal education, nativity and Hispanic subgroup using a Bayesian random-effects logistic regression model with restricted quadratic splines and knot locations placed at 8, 11, and 13 years. The form of the model is:ijk is defined as the binary PTB outcome for the ith woman (for i = 1...njk) in the jth ethnic group (for j = 1...7) and the kth nativity group , with adjustment for m baseline covariates, x, by means of estimated coefficients βm. The terms q1ijk to q3ijk are restricted quadratic spline terms, where the qualifier "restricted" implies a linear dose response relationship in the region less than 8 years and the region greater than 13 years of education. The α1jk ... α3jk coefficients determine what the education dose-response curve looks like for the infants of mothers in ethnic group j and nativity class k. The α coefficients are specified as random effects so that the education functions may borrow information between ethnic groups of the same nativity class (but not across different nativity classes). In particular, we specify:where Ygjk; g = 1...3, j = 1...7, k = 0,1 is shrunk toward the group mean for that particular nativity class [3, for example, indicates that the spline coefficients for foreign born mothers will borrow a larger amount of information from one another.Each of the αty class . That is0 ...τ3 were assumed to be independently gamma distributed with shape and rate parameters 0.1 and 0.1. Finally, the prior distribution of the remaining coefficients in expression [m, were assumed independent and identically normally distributed with mean 0 and variance 10.The Bayesian approach requires priors to be specified for all unknown parameters. The specifications for these priors were selected to be relatively uninformative since little data exists to specify informative priors on the spline coefficients in our model. Further, given the large number of observations prior information is unlikely to have any substantial impact on the results. The prior mean parameters, th iteration to reduce autocorrelation between samples as well as for analytic tractability. Convergence was assessed by visual examination of traceplots . We excluded from the analysis observations missing birthweight (n = 81), nativity status , ethnic ancestry  or education , for a cumulative exclusion of n = 26,550 (2.7%). We also excluded all observations that were missing any covariate when fitting the fully adjusted model, although no covariate was missing more than 1.2% with the exception of prenatal care (11.4%), which left a final sample size of 258,680 for the fully adjusted analyses.Similar to findings in previous reports, demographics and maternal education varied by nativity and Hispanic subgroup Table . The risa and b) and foreign born women in the two lower panels (c and d) of Figure Figure Figure Using vital statistics birth records from New York City, we have extended previous investigations of the "Hispanic Paradox", but with a novel statistical methodology that allowed for numerous improvements. The use of Bayesian hierarchical modeling allowed for the estimation and graphing of all effects on the absolute scale, which has more direct public health relevance and allows for direct comparison between groups without having to specify a referent group for the contrast parameter. The methodology also permits easy calculation of posterior intervals, whereas the calculation of variances for posterior probability estimates from multilevel logistic models in a frequentist setting would have been enormously difficult. Moreover, the shrinkage accomplished with random coefficient terms allowed for flexible modeling over relatively fine categorizations of education and ethnicity in order to produce more specific patterns than reported previously, and with the extent of shrinkage determined by the data. Finally, we reported effects for a more pathologically specific outcome, preterm birth, which represents a more homogeneous etiology  than the composite outcome of low birthweight (LBW) that has often been reported ,5.We also highlight the age-adjusted rather than the fully-adjusted estimates because we argue that these are particularly important for understanding disease burden in populations, since the real situation of these women and their pregnancies is more readily revealed in the age-adjusted values. Moreover, we would argue that for etiologic inference, the covariate-adjusted estimates may be less helpful because the covariates are, except for age, arguably consequences of the primary exposures: nativity and ethnicity . FurtherRandom-effects regression models have the advantage of shrinking group-specific estimates toward the adjusted education-category mean risks, which guarantees a reduced mean square error for the ensemble of results . This imPrevious analyses defined low education categorically as 0-11 years, and then reported a monotonic decrease in risk of adverse outcomes with increasing education ,5. By usAdditionally, plotting predicted probabilities rather than graphing relative measures of effect allowed us to look directly at risk dose-response across groups. For example, the plotting of predicted absolute risks not only reveals the substantially higher risk for Puerto Ricans, but also shows that for this group in particular, there is no apparent advantage for the "foreign born"  as there is for other ethnicities. This makes sense substantively, since all Puerto Ricans are US citizens, whether born in the mainland or on the island. Greater mobility is therefore possible between populations, with less migrant selectivity among those who relocate. The other group with little apparent effect of nativity was Cubans, although the very small number of foreign born Cuban mothers in New York City limited the precision of these estimates severely. Evidence that this is not a distinctively Caribbean phenomenon can be seen by contrasting Dominicans, whose risk profile looks very much like the other Central and South American groups.It should be noted that because we display absolute risk estimates, the adjusted values shown must depend on the choice of level at which covariates are fixed in the analysis. We chose to set all covariates to values with lowest risk, meaning that the graphs displayed are the "best case" scenarios for each stratum defined by ethnicity and nativity; when additional risk factor are "switched on", the absolute risks will be greater than those shown.This paper uses novel statistical methods to extend previous findings concerning risk of adverse birth outcomes for Hispanic women as a function of ethnicity, nativity and years of completed education. We confirmed the previously published findings that the education gradient is much flatter for foreign-born women, with the exception of island-born Puerto Ricans. We went beyond previous research, however, to demonstrate that more substantively interpretable analyses are possible through the use of flexible hierarchical models. Finer categorizations help reveal evidence that the benefit associated with additional years of schooling may not be monotonic, but rather that women with the lowest levels of education show reduced risk compared to those with 8-11 years. Furthermore, by displaying Bayesian posterior risk estimates and their differences, rather than ratio measures of effect, we show heterogeneity between groups that is obscured when each group is used as its own referent. These results show a consistent disadvantage for Puerto Rican women at all education levels and for all outcomes. Furthermore, results that are adjusted for measured risk factors will also be more moderate than those that occur in the real world. If Puerto Ricans also have a more disadvantaged profile of these factors, their true risks will be even more disparate.The authors declare that they have no competing interests.JSK and EAT conceived the study, carried out initial analyses and wrote the manuscript. RFM developed the current analytic strategy and fit the models. DAS provided the data and subject matter expertise. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/11/51/prepubAnnotated Winbugs Software Code. Annotated Winbugs Software CodeClick here for fileAppendix Figures. Appendix Figure A1. Age-adjusted risks of preterm birth (PTB) by nativity, education and Hispanic subgroup, women ≥20 years old, New York City, 1995-2003. Appendix Figure A2. Fully-adjusted risks of preterm birth (PTB) by nativity, education and Hispanic subgroup, women ≥20 years old, New York City, 1995-2003. Appendix Figure A3. Age-adjusted risk differences for the effect of nativity on preterm birth (PTB) by education and Hispanic subgroup, women ≥20 years old, New York City, 1995-2003. Appendix Figure A4. Fully-adjusted risk differences for the effect of nativity on preterm birth (PTB) by education and Hispanic subgroup, women ≥20 years old, New York City, 1995-2003. Appendix Figure A5. Comparison of age-adjusted to fully-adjusted risk differences for the effect of nativity on preterm birth (PTB) by education and Hispanic subgroup, women ≥20 years old, New York City, 1995-2003. Appendix Figure A6. Trace plots for several selected terms in the fully adjusted model  for native born Mexican-American women.Click here for file


Two of these, LIF and IL-11, induced EGR2 expression. Although these data would need to be extended to a larger number of cytokines and the biological validation should be done using more robust in vivo models, rather then just one cell line, this study shows the feasibility of this approach. This type of functional cluster analysis could be extended to other fields of cytokine research and help design biological experiments.The discovery of the tissue-protective activities of erythropoietin (EPO) has underlined the importance of some cytokines in tissue-protection, repair, and remodeling. As such activities have been reported for other cytokines, we asked whether we could define a class of tissue-protective cytokines. We therefore explored a novel approach based on functional clustering. In this pilot study, we started by analyzing a small number of cytokines (30). We functionally classified the 30 cytokines according to their interactions by using the bioinformatics tool STRING , followed by hierarchical cluster analysis. The results of this functional clustering were different from those obtained by clustering cytokines simply according to their sequence. We previously reported that the protective activity of EPO in a model of cerebral ischemia was paralleled by an upregulation of synaptic plasticity genes, particularly early growth response 2 (EGR2). To assess the predictivity of functional clustering, we tested some of the cytokines clustering close to EPO  in an  The cytokine field was initially focused on the role of cytokines as humoral factors in host defense against infection and cancer, and the discoveries of interferon (IFN), interleukins (IL)-1 and -2 , as thisThe current focus on stem cell therapy and regenerative medicine has put an emphasis on the protective functions of some cytokines. Our earlier finding that erythropoietin (EPO) has neuroprotective activities in models of brain and spinal cord injury  led to tIt should be noted, however, that protective/regenerative functions of some cytokines have long been known. For instance transforming growth factor beta (TGFb) has been extensively studied in the context of wound repair , and IL-We recently performed a microarray study to investigate the effect of EPO on the gene expression profile in rat ischemic brain and identified several genes associated with neuronal plasticity . These iAnother major action of some cytokines that can be regarded as protective/reparative is the stem cells-mobilizing action of EPO, Kit ligand/stem cell factor (KITLG), G-CSF, and GM-CSF , 15.Despite studies in the literature reporting protective or reparative actions of various cytokines, no attempt has been made to define a functional family of tissue protective cytokines [see Ref.  for a reIn the present study, we identify a cluster of tissue-protective cytokines using a bioinformatics approach. In particular, we have used the STRING  database . The STRTo assess the effectiveness of this strategy, four cytokines were chosen and their ability to protect tissue from ischemic damage was assessed experimentally by determining the degree to which they induce EGR2 expression in neuronal cells, an experimental model in which EPO shows activity .We find that this strategy is far more effective at identifying cytokines with common biological functions than clustering simply based on amino acid sequence similarity.A list of functional interactors for each cytokine was obtained using the STRING database, available onlineFor each cytokine, search criteria were: a maximum of 500 interactors, combined confidence score >0.2 and use of all active prediction methods, including text mining. No “white nodes” were requested in the search criteria. This refers to secondary interactors, proteins that are predicted functional partners of primary interactors with the search cytokine, but which do not interact directly with the search cytokine.Data for each cytokine was saved as a text file and imported to Microsoft Excel for processing. Primary interactors with each cytokine and the combined confidence score for each respective interaction were extracted from all 30 datasets and pooled onto a single spreadsheet. The lowest scoring interactions that straddled the 500 interactors search limit were deleted as it would not be clear how many other predicted functional interactors with this score existed without increasing the upper search limit past 500. A Perl program was used to integrate all the interaction data into a single data matrix for further analysis.The interactors were functionally annotated using the DAVID database, accessible onlineClustalwThe rat neuroblastoma B104 cell line, genetically modified to express EPOR constitutively, as reported , was cult-test.Total RNA was extracted from cultured cells using TRIzol (Invitrogen/Life Technologies). RNA quality and concentration were determined using a NanoDrop ND-1000 . Reverse transcription and real time qPCR were carried out as reported , using TThe main steps involved in data processing are summarized in Figure To compare the patterns of interaction of the cytokines, a matrix was constructed that combined all the interactions identified. The matrix was populated by the interaction confidence for each cytokine, scored against all 4250 identified interactors. Where a functional interaction had been identified by STRING, the interaction was defined by the combined confidence score. If no interaction was identified, a score of −1 was recorded.All the interactors were annotated using the DAVID functional annotation resource (see text footnote 2) and GO terms were assigned to each. Interactors with no associated GO terms were excluded, thus obtaining a dataset of 4338 interactors out of 4520.Since our aim was to identify cytokines functionally similar in terms of protective/reparative functions, therefore sharing interactors involved in “repair or protection,” we chose to select only the interactors identified by GO terms containing the text listed in Table The respective interactors were then extracted from the complete list. Therefore, only 1035 interactors involved in repair or protection were used for the subsequent cluster analysis .The final dataset, consisting of the 30 cytokines scored against the 1035 genes extracted with DAVID , was imported into the GENESIS analysis suite (Genesis version 1.7.6 for Mac OSX). Hierarchical clustering was then performed and the results are shown in Figure It can be seen that the distribution of cytokines within primary, secondary, and tertiary clusters to EPO are markedly different in the “functional” cluster and in the sequence alignment cluster.Cytokines selected from primary, secondary, and tertiary clusters with respect to EPO  were chosen as experimental candidates, to test their effect on Egr2 mRNA expression in serum-starved EPOR-B104 neuroblastoma cells exactly as we described previously .As shown Figure As the experimental results confirmed that EPO, LIF, and IL-11 exhibit similar tissue protective functions, we decided to explore the similarities on their protein interaction networks. We identified the genes predicted to have a functional association with EPO, LIF, and IL-11. Venn diagrams were used to display the similarities among EPO, LIF, and IL-11 in terms of shared interactors Figure . Thirty-To further explore the potential of this strategy, we also performed an analysis where cytokines were functionally clustered in terms of inflammatory functions. To this aim, we selected only the interactors having “inflammatory” in their GO terms, and obtained 299 interactors . We then clustered the cytokines based on these interactors Figure , and obtin vitro.This study shows a pragmatic approach to identify a functional class of cytokines. In particular, we have tested the possibility of defining a functional family of neuro- and tissue-protective cytokines. We have also validated the results of the prediction by testing some of the cytokines in vivo models  this was neither feasible nor within the scopes of this study and we decided to use an in vitro test for this pilot study. In fact we could demonstrate that IL-11 and LIF can induce EGR2 expression in a system where EPO is effective. Of note, the induction of EGR2 expression by LIF or IL-11, in neurons as well as in other cells, was not previously known, indicating that functional clustering can actually be helpful in predicting an unknown biological activity.While, ideally, the way to validate the predictions would have been to test all 30 cytokines in multiple in vitro experiments are not necessarily confirmative because the cell line used may not express sufficient levels of the receptor for the specific cytokine, and this could be the reason for the lack of activity of KITLG and THPO in our system. Another limitation is that the in vitro model used in this study (a human neuroblastoma cell line) is obviously tailored toward the nervous system and is not appropriate for studying other tissue-protective activities. For instance, KITLG administration via a lentiviral vector was reported to protect against myocardial infarctions, and the assay we used is clearly unsuitable to study a cardioprotective activity. Furthermore, in vivo testing using the appropriate models depending on the functional family explored would be desirable to validate the results obtained with the cluster analysis.However, one limitation of this study is that the The observed induction of EGR2 by LIF and IL-11 might be important in the common pro-myelinating effects of these cytokines , 20, as The identification of common interactors for EPO, LIF, and IL-11 (listed in Table In conclusion, this study reports for the first time a strategy to cluster cytokines by their function. The results, although performed on a limited number of cytokines, support the concept of a functional family of tissue-protective cytokines that share the tissue-protective activities of EPO.Finally, although this study was aimed at defining a class or “tissue-protective cytokines,” we also tried to use the same strategy to identify other functional families by choosing to cluster only for interactors containing other specific GO terms, in particular only those related to inflammation. Indeed, EPO clustered with different cytokines depending on the interactors selected for the cluster analysis.We therefore propose that this approach, if performed on a large number of cytokines, will be helpful in identifying other functional families.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.www.frontiersin.org/journal/10.3389/fimmu.2014.00115/abstractThe Supplementary Material for this article can be found online at: Click here for additional data file.Click here for additional data file.


Short and long birth intervals have previously been linked to adverse neonatal outcomes. However, much of the existing literature uses cross-sectional studies, from which deriving causal inference is complex. We examine the association between short/long birth intervals and adverse neonatal outcomes by calculating and meta-analyzing associations using original data from cohort studies conducted in low-and middle-income countries (LMIC).We identified five cohort studies. Adjusted odds ratios (aOR) were calculated for each study, with birth interval as the exposure and small-for-gestational-age (SGA) and/or preterm birth, and neonatal and infant mortality as outcomes. The associations were controlled for potential confounders and meta-analyzed.Birth interval of shorter than 18 months had statistically significant increased odds of SGA , preterm  and infant mortality  after controlling for potential confounding factors (reference 36-<60 months). It was also significantly associated with term-SGA, preterm-appropriate-for-gestational-age, and preterm-SGA. Birth interval over 60 months had increased risk of SGA  and term-SGA , but was not associated with other outcomes.Birth intervals shorter than 18 months are significantly associated with SGA, preterm birth and death in the first year of life. Lack of access to family planning interventions thus contributes to the burden of adverse birth outcomes and infant mortality in LMICs. Programs and policies must assess ways to provide equitable access to reproductive health interventions to mothers before or soon after delivering a child, but also address underlying socioeconomic factors that may modify and worsen the effect of short intervals. Providing access to family planning in low- and middle-income countries (LMIC) has social and economic benefits, but is also a critical public health intervention that may increase survival and improve health of mothers and newborns. Short and long birth intervals, or the time between previous and index live births, have been linked to adverse neonatal outcomes, including child mortality, low birthweight, preterm birth, and intrauterine growth restriction (IUGR) -3. StudiSeveral mechanisms have been proposed linking short birth intervals with adverse pregnancy outcomes, which were recently systematically reviewed by Conde-Agudelo et al . The matLong birth intervals, whether intended or unintended, may also have negative outcomes, and thus important to understand the associations. A woman’s physiologic and anatomic capacity to accommodate fetal growth may revert to a nulliparous state if she has undergone a long period since her last birth, and that the infant subsequent to a long interval may experience the same risks as a first birth . The lonThere are limitations to the current literature on this topic. Much of the literature on this subject utilizes cross-sectional studies such as Demographic and Health Surveys (DHS), especially with long recall periods -3,8. CauLiST). LiST is a computer-based tool that estimates the impact of scaling up various health interventions, such as family planning, on maternal and child mortality [Thus, the aim of this work is to address some of these limitations by examining the association between birth intervals and poor neonatal outcomes , preterm, neonatal and infant mortality), using original data from prospective birth cohort studies conducted in LMIC, and conducting analyses using standardized categorizations and definitions of risk exposure and outcome variables. We controlled for available socioeconomic, nutritional, and reproductive health confounders in each dataset. The ultimate objective is to generate estimates to feed into the Lives Saved Tool . Medline, WHO regional database, and bibliographies of key articles were searched, and additionally, Child Health Epidemiology Reference Group investigators were also consulted to identify potential datasets by word-of-mouth. Investigators with birth cohorts containing the minimum required data were contacted to conduct a standard set of analyses or to contribute their data to the analyses. More details on the search strategy are available elsewhere . For incIn total, five birth cohort datasets from three countries  were included -15, totaThe independent variable was birth interval (the time between the previous and index live birth). Birth interval was categorized as <18 vs. 18-<24 vs. 24-<36 vs. 36-<60 (reference) vs. ≥60 months. The reference category was chosen in light of previous literature reporting a plateau in risk between roughly 36 and 60 months [th percentile of a gender-specific reference distribution at each completed gestational week, using births in the US in 1991 [SGA was defined as below the 10For each birth cohort, logistic regression was performed with the birth outcome as the dependent variable and birth interval, parity, age, and other potential confounders included as independent variables. In each dataset, the available socioeconomic and maternal nutrition variables were placed in the multivariate model to calculate adjusted odds ratios (aOR). The covariates are listed in Supplemental Table The percentage of births in each birth interval category are shown in Figure Table Supplemental Tables Birth interval <18 months was statistically significantly associated with almost all adverse neonatal outcomes. It had a pooled aOR of 1.51  for SGA and 1.58  for preterm. Examining the SGA and preterm combination outcomes, the short interval increased the odds of term-SGA  and preterm-AGA , and had a 3-fold increase in odds of preterm-SGA . See Figure Birth interval <18 months did not increase the risk of neonatal mortality to a level of statistical significance , however we observed an 83% increase in odds of infant mortality . See Figure Birth interval of ≥60 months was associated with a slight increase in odds of SGA  and term-SGA . The risks for other outcomes were small and non-significant.In our meta-analysis, short birth interval (<18 months) was significantly associated with SGA (aOR 1.51), preterm (aOR 1.58), and infant mortality (aOR 1.83). We observed a dose response relationship, with the magnitude of risk increasing as the birth intervals got shorter from the reference 36-<60 month category. Birth interval <18 months carried a substantially higher (three-fold) risk of delivering an infant who is both preterm and SGA compared to those who had a reference birth interval; preterm-SGA babies carry substantially higher risk of mortality than those born term-AGA .Our findings produced a similar magnitude of associations as previous literature for short intervals with SGA and preterm outcomes, although the results cannot be directly compared due to different birth interval cut-offs and definitions. Conde-Agudelo et al.’s meta-analysis found an adjusted odds ratio of 1.26 (95% CI: 1.18-1.33) for SGA and 1.40  for preterm, examining an interpregnancy interval (IPI) (period between birth and conception) of <6 months, against a reference of 18 to 23 months , pretermIn our meta-analysis, short birth interval was significantly associated with increased infant mortality risk, however had no significant association with neonatal mortality risk. This finding may be driven by the smaller number of neonatal deaths, compared to infant deaths; we noticed increased risk in all datasets, but confidence intervals were wide and crossed unity in the pooled association. Incomplete neonatal mortality information in the Zimbabwe dataset may also have affected the association. Another possible explanation may be the confounding effect of breastfeeding. Those who fail to breastfeed will regain their fecundity sooner than those who do, leading to shorter birth intervals. We also expect mothers to repeat negative breastfeeding patterns for the subsequent child , which iLong intervals do not appear to have a strong adverse association with neonatal outcomes; we only observed a statistically significant 12% increase in odds in SGA but no association with any other adverse outcomes. A meta-analysis of DHS data showed no adverse association for long birth intervals as well [Some researchers have hypothesized that maternal depletion drives the association between short birth intervals and adverse neonatal outcomes; a mother may not have nutritionally and physiologically recovered enough before conceiving the next child. Upon controlling for available maternal nutritional variables, we witnessed no significant change in the magnitude of the associations. This may imply that nutritional depletion either plays no or a small role. A systematic review also found weak evidence to support the maternal depletion hypothesis , examiniNumerous other hypotheses on mechanisms linking birth intervals to adverse health outcomes have been identified . MechaniThe strength of our analysis is the use of high quality exposure, outcome, and confounder data from prospective birth cohorts. Unlike some cross-sectional survey data, the outcome information is collected soon before or around the time of birth. Also, by standardizing the categorization of birth intervals and outcomes, we were able to meta-analyze five studies with the same exposure and outcome definitions. One of the largest methodological issues with other meta-analyses is the heterogeneity of birth interval categorization, definition of SGA, use of birth-to-birth intervals versus birth-to-conception intervals (or IPI), and other exposure and/or outcome definitions.The main weakness of this study and of almost all other studies reporting on birth interval is the inability to examine the associations taking into account the length of each component of a birth interval  Non-live birth outcomes  in between two live births may attenuate the association between short intervals and adverse outcomes by attributing more adverse outcomes to the reference or long birth interval. A study conducted in Bangladesh  noted diThe associations we see between short birth intervals and adverse outcomes emphasize the importance of family planning interventions and the timing of the interventions. As Conde-Agudelo et al. have also stated , unmet nOur results suggest that birth intervals under 18 months are associated with adverse neonatal outcomes, with as high as an 83% increase in odds of infant mortality. Policymakers have the responsibility to secure family planning access to all women, which would benefit the health and the economy of the population. Programs and policies also need to focus on vulnerable mothers, as they may have higher risks associated with short intervals and are least likely to have interventions reach them. Finally, more operations research needs to be conducted to determine the most effective ways to delay subsequent births in LMICs.LiST, we recommend the inclusion of the associations between short birth intervals and adverse outcomes, with the understanding that birth intervals may have differential impact on neonatal and infant outcomes, depending on the baseline condition of the mother.In We report no competing interests.NK was responsible for study design, data collection, literature review, analyses of primary datasets and the meta-analyses, interpretation of results, and writing of the manuscript. ACL and JK were responsible for study design, data collection, interpretation of results, and helped draft the manuscript. MFS analyzed the primary datasets and reviewed the manuscript. CV, LA, JH, RN contributed data and reviewed the manuscript. REB contributed to study design and interpretation of results, and reviewed the manuscript. All authors read and approved the final manuscript.Supplemental material.Click here for file


Angiogenesis plays a critical role in synovial inflammation and joint destruction in rheumatoid arthritis (RA). Vascular endothelial growth factor A (VEGF-A) and angiopoietins are two important mediators of synovial angiogenesis. We have previously developed a novel chimeric decoy receptor, namely, double-antiangiogenic protein (DAAP), which can both bind VEGF-A and angiopoietins and block their actions. This study was performed to evaluate the antiarthritic effect of DAAP and the combination effect with the tumor necrosis factor α (TNF-α) inhibitor in collagen-induced arthritis (CIA).Recombinant DAAP, VEGF-Trap, Tie2-Fc and dimeric Fc proteins were produced and purified from CHO cells in large-scale bioreactors. CIA was induced in DBA/1 mice with type II collagen. The preventive effect of DAAP was determined and compared with other decoy receptors such as VEGF-Trap or Tie2-Fc, which block VEGF-A or angiopoietins, respectively. The clinical, radiographic, pathologic and immunohistochemical analyses were performed in CIA mice. The levels of matrix metalloprotease 3 (MMP-3) and interleukin 1β (IL-1β) were quantified by enzyme-linked immunosorbent assay, and receptor activator of nuclear factor κB ligand (RANKL) mRNA levels were measured by polymerase chain reaction. Finally, we investigated the combination effects of DAAP with a low dose of TNF-α decoy receptor (etanercept 10 mg/kg).On the basis of clinical and radiographic evaluation, DAAP had a much greater inhibitory effect than VEGF-Trap or Tie2-Fc on arthritis severity and bone destruction. These inhibitory effects were accompanied by significantly diminishing pathologic abnormalities, CD31-positive vasculature and synovial infiltration by F4/80-positive macrophages. The levels of MMP-3, IL-1β and RANKL were much lower in the DAAP-injected group than those of the control. Furthermore, DAAP showed a therapeutic effect and a combination effect with etanercept when injected after arthritis onset in established CIA.DAAP has not only potent prophylactic effects on both inflammation and bone destruction but also therapeutic effects, alone and in combination with a TNF-α inhibitor in CIA mice. These results suggest that DAAP could be used as an effective new therapeutic agent for RA. Rheumatoid arthritis (RA) is the most common inflammatory arthritis and a major cause of disability due to joint destruction and permanent deformity . AngiogeMultiple mediators have been implicated in the process of angiogenesis ,6. AmongAngiopoietins, including angiopoietin 1 (Ang-1), Ang-2 and Ang-3/Ang-4, are other important angiogenic factors. They interact with tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2 (Tie2) receptors . NumerouWe have developed a novel chimeric decoy receptor, double-antiangiogenic protein (DAAP), which can bind the VEGF-A, placenta growth factor (PIGF) and the angiopoietins and thereby simultaneously block their actions . A previRecombinant proteins, including DAAP, VEGF-Trap, Tie2-Fc and dimeric Fc (Fc), were produced as previously described . BrieflyDBA/1 mice were purchased from The Jackson Laboratory , and male DBA/1 mice (seven to nine weeks old) were immunized with 150 μg of bovine collagen type II  emulsified with an equal volume of Complete Freund's Adjuvant (Chondrex). The day of the first immunization was defined as day 0. The mice were then boosted with an equal amount of bovine collagen type II emulsified in Incomplete Freund's Adjuvant on day 21. For the preventive experiment, CIA mice were given intraperitoneal injections of phosphate-buffered saline (PBS), Fc, VEGF-Trap, Tie2-Fc or DAAP  from day 21 to day 40. For the therapeutic experiment, following the development of clinical arthritis (average arthritis score of 2) on day 26, CIA mice were randomized and given intraperitoneal injections of Fc, DAAP (25 mg/kg every two days), etanercept (10 mg/kg every two days) or DAAP + etanercept. Clinical arthritis scores were evaluated using a scale of 0 to 3 for each paw with a total possible score of 12. Ankle thickness was measured with a caliper placed across the ankle joint at the widest point. All experimental animals used in this study were maintained under the protocol approved by Gyeongsang National University Institutional Animal Care and Use Committee (GLA-080822-M0060).Plain radiographs of the knees and feet were obtained using a mammographic imager as previously described . The deg+ cells and CD31+ blood vessels were counted in eight randomly selected high-power fields (×400) for each knee and ankle joint.Fixed joint tissues were decalcified and embedded in paraffin. Sections (5 µm) were stained with hematoxylin and eosin and Safranin O. The joint sections were scored for changes in synovial inflammation, pannus formation, cartilage damage and bone erosion, all on a 0- to 3-point scale. CD31 and F4/80 were immunohistochemically detected using a rabbit polyclonal antibody against CD31  or a rat monoclonal antibody directed against F4/80 . The number of F4/80Ankle joints and serum were harvested at day 40 from the CIA mice. The frozen joint tissues were pulverized in liquid nitrogen, and total protein extracts from the ankle joints were isolated from individual homogenized joints in lysis buffer  containing Protease Inhibitor Cocktail . IL-1β, MMP-3 and VEGF were measured using commercially available enzyme-linked immunosorbent assay kits according to the manufacturer's instructions .Total RNA was isolated and transcribed into cDNA using the iScript cDNA Synthesis Kit  according to the manufacturer's protocol. After denaturing at 85°C for five minutes and then cooling to 4°C, cDNA was amplified using PCR. Two microliters of denatured cDNA were amplified in a 50-μl final volume containing 1 U of ExTaq DNA polymerase , 0.4 μM concentrations of each primer, Taq polymerase buffer and 2 mM concentrations of each deoxyribonucleotide triphosphate. PCR was performed in a thermal cycler (Bio-Rad Laboratories) using a program of 35 cycles at 95°C for 30 s, 56°C for 40 s and 72°C for 50 s with a final seven-minute extension at 72°C. The sense primer for RANKL was 5'-CCAGCATCAAAATCCCAAGTT-3', and the antisense primer was 5'-TCAAGGTTCTCAGTGGCACAT-3'. The sense primer for glyceraldehyde 3-phosphate dehydrogenase was 5'-AATGCATCCTGCACCACCAA-3', and the antisense primer was 5'-GTAGCCATATTCATTGTCAT-3'. The amplified products were subjected to electrophoresis on 1.2% agarose gel, and the results were visualized with ethidium bromide staining.g for 20 min at 4°C to remove insoluble debris. Equal amounts of the protein lysate were resolved on reducing SDS polyacrylamide gels and then electrophoretically transferred onto a nitrocellulose membrane . After blocking for one hour with 5% skimmed milk in a TBST buffer solution , the membrane was incubated with primary antibodies against Ang-1 and Ang-2 , followed by incubation with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) (Santa Cruz Biotechnology). The membrane was developed by SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology) and exposed to an X-ray film.Lysates were generated from ankle tissue in a radioimmunoprecipitation assay lysis buffer  Nonidet P-40, Protease Inhibitor Cocktail). The homogenated tissue was centrifuged at 12,000 U test was used to analyze arthritic severity and the radiographic and histologic findings. An unpaired Student's t-test and one-way analysis of variance were used to assess the other results. P ≤ 0.05 was considered significant.Values are expressed as the mean ± SE. Fisher's exact test was used to analyze the differences in incidence among groups. The Mann-Whitney P < 0.01)   Figure . There wThe joints of Fc-injected CIA mice exhibited prominent synovial inflammation, cartilage damage, pannus formation and bone erosion. In contrast, VEGF-Trap- or Tie2-Fc-treated mice showed decreased pathological manifestations, and DAAP-treated mice exhibited dramatically reduced pathological abnormalities Figures  and 3B. + blood vessels and F4/80+ macrophages in the joint synovium compared with Fc  , active (day 38) and chronic (day 52) phases and analyzed for temporal patterns of expression of VEGF-A, Ang-1 and Ang-2. In the early phase, Ang-1 and Ang-2 were increased. In contrast, VEGF-A was not increased. In the active phase, VEGF-A rose abruptly, Ang-1 continued to increase and Ang-2 decreased. In the chronic phase, Ang-1 was persistently high despite decreased VEGF-A (Figure , active + vasculature and synovial infiltration by F4/80+ macrophages, suggesting that DAAP acts primarily by inhibiting angiogenesis. We further demonstrated the therapeutic effect of DAAP and the beneficial combinatory effect of DAAP with TNF-α inhibitor in the established CIA mice. These data provide evidence for the usefulness of DAAP, which simultaneously blocks VEGF-A and angiopoietins as an effective new therapeutic agent for RA.In this study, we found that DAAP produces better protective effects on both inflammation and bone destruction in CIA mice when compared with VEGF-Trap or Tie2-Fc. These effects of DAAP were correlated with significantly diminished pathologic abnormalities, CD31DAAP treatment showed more pronounced suppressive effects on arthritis severity than VEGF-Trap or Tie2-Fc alone in CIA mice. We propose the following explanations for why and how DAAP is more effective than VEGF-Trap or Tie2-Fc. First, arthritic synovium has strong expression and activation levels of VEGF-A, PIGF, Ang-1 and Ang-2, and their expression patterns were different according to disease stages of RA in our data and other reports ,24-27. OApart from its important function as a VEGF, angiopoietin/Tie-2 signaling induces the proliferation of RA synovial fibroblasts and promotes the proinflammatory activation of macrophages ,17. EspeNumerous efforts to create a promising strategy for treating cancer and inflammatory arthritis have focused on disrupting the VEGF-A pathway ,6. DespiEtanercept is a soluble TNF-α type II receptor linked to an IgG1-Fc moiety that binds to and inactivates TNF-α . AlthougThis study has some limitations. Our previous study showed that DAAP is superior to the combined therapy of VEGF-Trap plus Tie2-Fc in suppressing tumor growth, angiogenesis and metastasis . Those fWe found that DAAP induces better protective effects than VEGF-Trap or Tie2-Fc on both inflammation and bone destruction in CIA mice. Additionally, we proved that DAAP has therapeutic effects and a beneficial combination effect with the TNF-α inhibitor on CIA. Our results raise the possibility that the coordinated blocking of angiogenic activities by DAAP might represent a promising and powerful strategy for the treatment of RA.CIA: Collagen-induced arthritis; DAAP: Double-antiangiogenic protein; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; IL: Interleukin; MMP: Matrix metalloprotease; PCR: Polymerase chain reaction; RANKL: Receptor activator of nuclear factor-κB ligand; RA: Rheumatoid arthritis; TNF-α: Tumor necrosis factor α; Tie2: Tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2; VEGF: Vascular endothelial growth factor; VEGFR: Vascular endothelial growth factor receptorThe authors declare that they have no competing interests.YSH, YJK, GYK and SIL designed, organized and performed the experiments, analyzed the data, generated the figures, and wrote the manuscript. HSL, HOK and YHC performed the CIA experiments. YJK and GML generated the recombinant proteins. HSN, KYJ and SYL performed the radiological and histological analyses. All authors read and approved the final version of the manuscript.


Intelligence is a life-long trait that has strong influences on lifestyle, adult morbidity and life expectancy. Hence, lower cognitive abilities are therefore of public health interest. Our primary aim was to examine if prenatal bereavement measured as exposure to death of a close family member is associated with the intelligence quotient (IQ) scores at 18-years of age of adult Danish males completing a military cognitive screening examination.We extracted records for the Danish military screening test and found kinship links with biological parents, siblings, and maternal grandparents using the Danish Civil Registration System . The prenatal exposure period was defined as 12 months before conception until birth of the child. We categorized children as exposed in utero to severe stress (bereavement) during prenatal life if their mothers lost an elder child, husband, parent or sibling during the prenatal period; the remaining children were included in the unexposed cohort. Mean score estimates were adjusted for maternal and paternal age at birth, residence, income, maternal education, gestational age at birth and birth weight.When exposure was due to death of a father the offsprings' mean IQ scores were lower among men completing the military recruitment exam compared to their unexposed counterparts, adjusted difference of 6.5 standard IQ points . We did not observe a clinically significant association between exposure to prenatal maternal bereavement caused by death of a sibling, maternal uncle/aunt or maternal grandparent even after stratifying deaths only due to traumatic events.We found maternal bereavement to be adversely associated with IQ in male offspring, which could be related to prenatal stress exposure though more likely is due to changes in family conditions after death of the father. This finding supports other literature on maternal adversity during fetal life and cognitive development in the offspring. Several stressors during prenatal life have been shown to affect neurological development and cognitive outcomes in the offspring In general, the human placental enzyme 11β-HSD2 that converts active glucocorticoids to inactive metabolites Prenatal bereavement has been associated with several neurological outcomes, such as attention/deficit/hyperactivity disorder st 1976 and December 31st 1993. Among these recruits, we found kinship links for N = 205,393 records using the Danish Civil Registration System (CRS). We eliminated missing and invalid test scores as well as test scores of female conscripts (N = 6713), due to a lack of sufficient sample size. Since 1968, all live born children and new residents in Denmark are assigned a unique civil personal registration number, allowing for accurate linkage at the individual level between all national registries. Kinship links were made for N = 167,900 males with their biological parents (mother and father), siblings, and maternal grandparents. Information on birth outcomes, such as gestational age at birth and birth weight, were obtained from the Danish Medical Birth Registry which has been computerized since 1973. Annual information on maternal education, residence, and income was retrieved from the Fertility Database at Statistics Denmark, available since 1979. These variables were treated as time dependent for each pregnancy for women who contributed to the cohort more than once. The date of conception was estimated by subtracting gestational age from the date of birth. We defined the prenatal exposure period as 12 months before conception until the birth of the child. We categorized children as exposed to bereavement during prenatal life if their mothers lost an elder child, husband, parent or sibling during the prenatal period; the remaining children were included in the unexposed cohort.We extracted N = 241,222 individual records from the Danish Conscript Register. All test-takers were born between January 1IQ was estimated using the Danish military draft board's Børge Priens Prøve (BPP) test st, 2nd, 3rd, and 4th quartile); maternal education ; gestational age at birth ; and birth weight . Age, residence, maternal income and parental cohabitation were treated as time-dependent variables.We compared mean IQ scores among individuals exposed to bereavement and defined the unexposed group as those pregnancies unaffected by prenatal bereavement. F-tests provided by SAS version 9.1.3 PROC MIXED were used to evaluate differences between exposure groups. The models used minimum variance quadratic unbiased estimation method and a compound symmetry covariance structure. To control for the correlation between siblings, the models included a repeated subject statement with an identifying variable for the subjects' mother. All mean score estimates were adjusted for maternal and paternal age at birth ; residence ; income ; and death from other causes. This study was approved by the Danish Data Protection Agency (Ref no. 2008-41-2680) and the Ethics Committee for Central Jylland in Denmark (Ref no. M-20100252). Individual written consent was not obtained from study participants as we had no record linking the subjects to individual identifying information.(see ). Approximately 2.8 percent of our cohort was exposed to maternal bereavement prenatally (N = 4651) and among these exposed pregnancies 0.4 percent of deaths were due to traumatic events. We observed a lower mean IQ test score among all exposed groups, though only some groups reached both clinical and statistical significance. Compared to the standard deviation of the total sample (9.7), the 4.2 adjusted BPP IQ point different amounts to 0.43 standard deviation. A typical IQ scale has a mean of 100 and a standard deviation of 15, thus 0.43 BPP standard deviation corresponds to about 6.5 standard IQ points. The adjusted mean differences in BPP IQ scores transformed to a typical IQ scale are also presented in Almost all maternal demographic variables - maternal age, paternal age, household income, maternal education at birth, maternal residence, birth weight and gestational age were associated with exposure We found prenatal bereavement due to death of a father was adversely associated with IQ scores in early adulthood among Danish men completing the military screening exam. This association remained even after stratifying on deaths due to traumatic causes – which may partially control for background factors associated with reduced cognitive abilities. The lack of association detected when bereavement was due to a sibling death may however indicate that prenatal stress does not modify IQ in the offspring. It is possible that death of a father leads to loss in household income and overall socio-economic status, factors both associated with IQ, and that the observed association is due to post-natal changes rather than a prenatal gestational insult. Alternatively, the lack of association for bereavement due to a sibling death may be due to overcompensation in caring for a surviving child - although there is no current literature that supports this hypothesis. In either case, our findings support that IQ has more than genetic determinants.Several prenatal factors have been identified with offspring intelligence. A study on cognitive function found that infants born small for gestational age had lower IQ scores than their non-SGA counterparts Adverse fetal exposures during gestation and subsequent programming of the fetus has been established in animal models, and human studies utilizing data from natural occurrences/disasters are expanding research in this area. The hypothesis that stress increases maternal cortisol (corticosterone in rodents) which causes a range of short and long-term adverse health effects in fetal development is well documented in the literature The main strengths of our study are the large cohort size and high quality of exposure data. Information in the CRS has been made available for research purposes by the Danish Data Protection Agency However, it is unlikely that exposure contrast between the bereaved and non-bereaved exposure groups in our cohort is insubstantial. Furthermore, longitudinal sampling to estimate allostatic load would be impossible due to legal, logistic and economic reasons. Another possible limitation is the possibility of the study being over-powered. In a sub-analysis, we attempted to address this issue by using pregnancies exposed to a grandparents' death as controls and found no significant difference in interpretation of the results. The difference in standard IQ points using a control group bereaved to maternal grandparents was 5.7, only slightly lower than the 6.5 point difference when comparing to the unexposed group .A concern often raised with the validity of the Børge Priens Prøve is the motivation for optimal test performance in males reluctant to be drafted. Since some men try to avoid service in the military, it is expected that this may negatively influence test performance. However, it is difficult to avoid military service by deliberately performing poorly during IQ testing. A study of 2,236 men found that the small minority of men expressing negative attitudes of military participation had in fact higher educational status and that this sentiment was associated with a desire for uninterrupted pursuit of higher education. These individuals also performed better on the IQ test In our study we found maternal bereavement by death of the father to be adversely associated with intelligence in male offspring. This finding supports other literature on maternal adversity during fetal life and cognitive development in the offspring. However, it is also possible that the causes of the association are related to a change in social conditions rather than fetal programming of the HPA axis due to stress, since we found no clear association when bereavement was due to loss of a child, or other relationship status. Our results are limited to male offspring and a replication of this study is warranted among females.


Omphalomesenteric duct (OMD) remnants and omphalocele are not infrequently seen in paediatric patients. In most of the cases, OMD remnant in an omphalocele is a Meckel’s diverticulum; however rarely there may be other lesions. A one-day old male baby underwent surgery for omphalocele. At exploration a 10 x 12 cm cyst containing gut contents was found as the content of the omphalocele, with proximal and distal ileal loops running in continuity with it. Resection of the cyst with end to end primary gut anastomosis was done. Baby also had complex associated cardiac anomalies and died few days after surgery due to sepsis. Omphalomesenteric duct is a normal embryological structure that connects the fetal midgut with the yolk sac. It usually regresses by 9th week of gestation. However if that does not happen, a spectrum of anomalies may result.1 Remnants of OMD may be present in 2% of the population with Meckel’s diverticulum (MD) as the commonest of remnants. OMD remnants may form part of an omphalocele however, omphalocele containing an OMD cyst is extremely rare and very few cases have been reported.2,3 Some of these cysts are serous, probably occurring due to the sequestration of the OMD remnants during intrauterine life. Meconium or faecal cysts probably develop as a consequence of partial obstruction at the neck of the umbilical defect where gut contents can flow into the OMD and forms a large cyst. Herein we report a case of a newborn with an omphalocele in whom an omphalomesenteric cyst was found.A one-day old, full term male, weighing 2.6 kg, was referred with a swelling at umbilical region. Baby was delivered at a local hospital through spontaneous vaginal delivery. On arrival, he had normal morphological features. There was a large omphalocele about 10 x 12 cm, covered with a thin, intact, semi-transparent membrane with a narrow pedicle of around 3 cm diameter . Umbilical clamp was well away from the omphalocele. The small bowel loops and a thick sac like structure were visible through the thin membrane. Rest of the neonatal examination was unremarkable. iameter . UmbiliEchocardiography showed a large ventricular septal defect with overriding of aorta, hypertrophied right ventricle, infundibular pulmonary stenosis and bunching of atrial septum – correlated with Tetralogy of Fallot. After optimization of the general condition surgery was done. At surgery, a 10 x 12 cm meconium filled cyst located at the distal ileum was found as the major content of the sac, along with small intestinal loops. The proximal and distal ileal loops were communicating with the cyst. ( and ) Rest of the bowel had no gross anomaly. Excision of cyst with primary end-to-end ileo-ileal anastomosis was performed. e cyst.  and 3Post operative course was stormy. During his stay at PICU the baby developed uncontrolled septicaemia and died in spite of best of efforts. Histopathology of the excised specimen revealed wall of small intestine with thin walled blood vessels surrounded by fibromyxomatous tissue. Ectopic mucosa was not identified.Omphalomesenteric duct cyst is infrequently reported.2,4 Vane et al found only three cases of OMD cyst in 217 children with OMD remnants.5It may be found attached to the umbilicus, bowel or both.4 Males have higher incidence than females (4:1) as with other OM anomalies, and are not associated to maternal age, gravidity, race or prematurity.4,6 It has varied presentations.4,7-9In newborns, OMD remnants may present in an omphalocele as reported by Chattopadhyay A et al and Nicol JW et al.3,10 Ratan SK et al reported a case similar to ours in a newborn but had a cyst containing translucent fluid rather than meconium.2In the present case, a large cyst was seen in the omphalocele sac, outside peritoneal cavity. It was a localized dilatation in the area of Meckel’s diverticulum and was in communication at both the ends with the small intestine and contained meconium. The possibility of a variant of cystic duplication of the small bowel was ruled out as it was located on anti-mesenteric side of the bowel with no aberrant vascular supply and sharing of the common wall. So an impression of a variant of OMD remnant and possibly omphalomesenteric cyst was made. OMD cysts have a variable diameter and appear as a cystically dilated segment of small bowel. They are usually lined by columnar epithelium resembling that of the small bowel, colon or stomach. Ectopic pancreatic tissue may be present.6 The gross and histological characteristics of the specimen in our case were similar to that reported in the literature supporting the possibility of omphalomesenteric cyst. However, no ectopic tissue was identified in our case. In conclusion OMD cyst may be present in a large omphalocele and the prognosis depends upon the associated anomalies.


Children with poor nutrition are at increased risk of pneumonia. In many tropical settings seasonal pneumonia epidemics occur during the rainy season, which is often a period of poor nutrition. We have investigated whether seasonal hunger may be a driver of seasonal pneumonia epidemics in children in the tropical setting of the Philippines. In individual level cohort analysis, infant size and growth were both associated with increased pneumonia admissions, consistent with findings from previous studies. A low weight for age z-score in early infancy was associated with an increased risk of pneumonia admission over the following 12 months (RR for infants in the lowest quartile of weight for age z-scores 1.28 [95% CI 1.08 to 1.51]). Poor growth in smaller than average infants was also associated with an increased risk of pneumonia (RR for those in the lowest quartile of growth in early infancy 1.31 [95%CI 1.02 to 1.68]). At a population level, we found that seasonal undernutrition preceded the seasonal increase in pneumonia and respiratory syncytial virus admissions by approximately 10 weeks . This lag appears biologically plausible. These results suggest that in addition to being an individual level risk factor for pneumonia, poor nutrition may act as a population level driver of seasonal pneumonia epidemics in the tropics. Further investigation of the seasonal level association, in particular the estimation of the expected lag between seasonal undernutrition and increased pneumonia incidence, is recommended. Pneumonia is a major cause of child mortality globally, with most deaths occurring in the tropics We have investigated the association between undernutrition and pneumonia incidence in the tropical setting of the Philippines. In this paper we have three aims: A) to confirm that poor nutrition is a risk factor for pneumonia in individual children in this setting, B) to assess the seasonal pattern of nutritional status in the child population in this setting, and C) to assess whether seasonal patterns of nutritional status could be driving yearly pneumonia and RSV epidemics in children in this setting.The trial “Effectiveness of an 11-valent pneumococcal conjugate vaccine against pneumonia in Philippine children: A double-blind, placebo-controlled, randomised, multicentre, effectiveness study” was approved and monitored by the Ethical and Institutional Review Board of the Research Institute for Tropical Medicine (RITM), the Philippines. Written informed consent was obtained from parents/guardians prior to enrolling infants into the trial. In a small minority of cases both parents were illiterate. In these instances a witness independent of the research team (a relative or neighbour) attested to (1) that the information provided to the parent was from the information sheet, (2) that the parent was given the opportunity to ask questions, and (3) that the parent provided verbal consent for the child to participate in the study. The witness then signed the consent form on behalf of the parent. The Ethical and Institutional Review Board of the RITM approved the consent protocol and information sheets. This retrospective analysis used de-identified data collected during the trial. Ethical approval for the analysis in this paper was granted without the need to seek additional consent, by the University of Queensland School of Population Health Research Ethics Committee in June 2011 .The data used in this study were collected during a randomised controlled trial to investigate the efficacy of pneumococcal conjugate vaccine (PCV) in Bohol Province in the Philippines between July 2000 and December 2004 Previous individual level studies have used one of two methods to assess nutritional status: either a single point measure of nutritional status We constructed time series of two growth indices to assess seasonality. For the first time series we used weight for age z-score at first vaccination. Weight for age z-score at first vaccination appeared strongly influenced by birth weight: there was a strong individual level correlation between weight for age z-score at first vaccination and at third vaccination (r = 0.75). We therefore considered weight for age z-score at first vaccination to be a proxy measure of birth weight, and used this data to construct a time series of weekly mean birth weight z-scores (attributing each infant’s weight for age z-score at first vaccination to their birth date rather than the date of first vaccination). We used data from all children in the trial who were weighed at first vaccination . To assess the validity of this time series, we compared it to a monthly time series of the incidence of low birth weight in Bohol between 1979 and 1990, using data collected as part of a case control study examining risk factors for child mortality (n = 3663) We also assessed the seasonality of nutrition more directly using quarterly palay (unhusked rice) production in Bohol, available from the Philippines Bureau of Agricultural Statistics We compared weekly pneumonia (n = 1571) and RSV (n = 245) admissions to the seasonality of birth weight, infant growth, and palay production over the period from July 2000 to September 2003. For birth weight seasonality we used the three month moving average of weekly mean birth weight z-scores. We have used three month moving averages in order to smooth out short term fluctuations, in order to concentrate on seasonal scale fluctuations . For infant growth we used the three month moving average of weekly mean growth between first and third vaccination. We removed year on year trend from this infant growth time series by subtracting the 12 month moving average (giving a time series of relative infant growth according to season). For palay production seasonality we used the quarterly data. We compared these seasonal scale time series to weekly pneumonia and RSV admissions using cross-correlation analysis, and performed pairwise correlation at points of maximum cross-correlation to assess statistical significance Infants in the lowest quartile of weight for age z-score at third vaccination had a significantly increased rate of pneumonia admission over the following 12 months . The surWe have confirmed in our study setting the well known association between poor growth and increased pneumonia incidence in individual infants. In addition, we have shown that at a population level in our study setting, seasonal undernutrition precedes the seasonal peak of pneumonia and RSV incidence in infants. The seasonal level association between poorer nutritional status in the third calendar quarter and subsequent pneumonia and RSV admissions was maximal at a lag of approximately 10 weeks. This lag is plausible considering the non-linear dynamics of infectious disease transmission, where seasonal scale increases in transmission may take weeks to force seasonal increases in disease incidence Our time series of growth between first and third vaccination showed two troughs each year. One trough, occurring in the third calendar quarter, coincided with the yearly troughs in palay production and estimated birth weight z-scores. The other trough occurred in the first calendar quarter. This additional trough in growth may be due to diarrheal disease rather than poor nutrition. A recent study from the Philippines found that rotavirus prevalence was highest in infants aged three to five months, and most cases occurred between December and February Although our birth weight measure was estimated from weight for age z-scores at approximately two months of age, it is reassuring that the seasonal pattern of our estimated birth weight z-score time series is similar to that of actual birth weights from the earlier Bohol study. The correlation between birth weight and infant weight that we assumed when estimating our birth weight measure has been noted elsewhere: a study in Bangladesh found that weight at 12 months of age was still largely a function of birth weight In our individual level analysis we found that infant growth between first and third vaccination was associated with an increased risk of pneumonia only in the infants who were smaller than average at first vaccination. There are two possible explanations for this. Firstly, it is possible that larger infants have more energy reserves, and so can better withstand acute nutritional shocks. Secondly, it may be that our growth measure has poor validity in larger infants . Our results are consistent with two previous studies examining the association between poor growth and hospital admissions in children: both studies found this association was stronger in children who were smaller to begin with Household crowding is an independent risk factor for respiratory infection Several other environmental factors may play a role in driving seasonal patterns of respiratory infection in the tropics. There is good evidence to show that a larger number of children within the household is associated with an increased risk of RSV infection The results from our study suggest that in addition to being an individual level risk factor for pneumonia, poor nutrition may act as a population level driver of seasonal pneumonia epidemics in the tropics. Further investigation of the seasonal level association, in particular the estimation of the expected lag between seasonal undernutrition and increased pneumonia incidence, will be required to assess causality.