Immunological outcomes in infants with ROP after dexamethasone and aminophylline.

This research aims to improve anaesthesia services given to preterm infants by the use of dexamethasone and aminophylline administrated under sevoflurane, and to analyze its effect on the cell-mediated immunity (CD4+CD25+Foxp3+(T-reg) and CD4+CD25highFoxp3+CD127low). We have examined 74 premature babies with retinopathy of prematurity (ROP) at the 3-5 stages during the 25-32 week gestation period (1-6 months after birth). Both immunomodulators had no significant effect on clinical parameters after one dose (P > .05). Aminophylline (2.4% solution, 0.1 mL/kg or 0.132 mL per infant on average) and dexamethasone (0.4% solution, 0.1 mg/kg or 0.132 mL per infant on average) were intravenously injected 15 minutes before the end of the surgery. Required anaesthesia depth was maintained with inhalation anaesthetic (1.5-2.0 IAC), and the minimum fresh gas flow was not less than 2 L. Blood samples were taken from the vein (anaesthesia induction stage) into the tubes containing EDTA (the anticoagulant), stored at 20-25°C, and then, processed and stained within 24 hours after sampling. Both immunomodulators had no significant effect on clinical parameters after one dose (P > .05). Short-term shift in regulatory T-cell level affected by dexamethasone has a negative effect combined with further withdrawal effect that this hormonal drug has. Aminophylline has such clinical effects as improving pulmonary ventilation, decrease in apnoea frequency, and improving blood gas indices. Aminophylline has less expressed but more prolonged positive effect during the day when used for several days. It may lead to a persistent positive effect with progressive treatment outcomes.

An exclusive human milk diet for very low birth weight newborns-A cost-effectiveness and EVPI study for Germany.

OBJECTIVES: Human milk-based fortifiers have shown a protective effect on major complications for very low birth weight newborns. The current study aimed to estimate the cost-effectiveness of an exclusive human milk diet (EHMD) compared to the current approach using cow's milk-based fortifiers in very low birth weight newborns. METHODS: A decision tree model using the health states of necrotising enterocolitis (NEC), sepsis, NEC + sepsis and no complication was used to calculate the cost-effectiveness of an EHMD. For each health state, bronchopulmonary dysplasia (BPD), retinopathy of prematurity (RoP) and neurodevelopmental problems were included as possible complications; additionally, short-bowel syndrome (SBS) was included as a complication for surgical treatment of NEC. The model was stratified into birth weight categories. Costs for inpatient treatment and long-term consequences were considered from a third party payer perspective for the reference year 2017. Deterministic and probabilistic sensitivity analyses were performed, including a societal perspective, discounting rate and all input parameter-values. RESULTS: In the base case, the EHMD was estimated to be cost-effective compared to the current nutrition for very low birth weight newborns with an incremental cost-effectiveness ratio (ICER) of €28,325 per Life-Year-Gained (LYG). From a societal perspective, the ICER is €27,494/LYG using a friction cost approach and €16,112/LYG using a human capital approach. Deterministic sensitivity analyses demonstrated that the estimate was robust against changes in the input parameters and probabilistic sensitivity analysis suggested that the probability EHMD was cost-effective at a threshold of €45,790/LYG was 94.8 percent. CONCLUSION: Adopting EHMD as the standard approach to nutrition is a cost-effective intervention for very low birth weight newborns in Germany.

A Genome-Wide Analysis of Clinical Chorioamnionitis among Preterm Infants.

OBJECTIVE: To identify single nucleotide polymorphisms (SNPs) associated with clinical chorioamnionitis among preterm infants. STUDY DESIGN: We reanalyzed a genome-wide association study (GWAS) from preterm newborns at less than 30 weeks' gestation. Cases and control definitions were determined using administrative records. There were 213 clinical chorioamnionitis cases and 707 clinically uninfected controls. We compared demographic and clinical outcomes of cases and controls. We performed a GWAS and compared the distribution of SNPs from the background genes and from the immunome genes. We used a Wilcoxon's rank-sum test to compare the SNPs normalized odds ratio and used odds ratios and p-values to determine candidate genes. RESULTS: Infants affected by clinical chorioamnionitis were more likely to have periventricular leukomalacia, high-grade retinopathy, and high-grade intraventricular hemorrhage (IVH). Although a GWAS did not identify SNPs associated with clinical chorioamnionitis at the genome-wide significance level, a direct test on the exonic variants in the human immunome revealed their significant increase of risk in clinical chorioamnionitis. CONCLUSION: Among very preterm infants, clinical chorioamnionitis was associated with periventricular leukomalacia, high-grade retinopathy, and IVH. Our analysis of variants in the human immunome indicates an association with clinical chorioamnionitis in very preterm pregnancies.

The role of the maternal and fetal inflammatory response in retinopathy of prematurity.

PROBLEM: There is a paucity of research on the contribution of placental inflammation to severe retinopathy of prematurity (ROP). METHOD OF STUDY: A retrospective cohort study (n = 1217) was conducted of infants screened for ROP (2006-2016). The outcomes of the study were severe ROP (type 1 or type 2 ROP) and low grade ROP. We categorized the placental pathology as the presence of (i) maternal plus fetal inflammatory response, (ii) maternal inflammatory response only, (iii) fetal inflammatory response only and, (iv) no evidence of a maternal or fetal inflammatory response. The data were analyzed using univariate and multivariate logistic regression analyses (P < .05). RESULTS: In this cohort, the number of infants with the maternal plus fetal inflammatory response, the maternal inflammatory response only, the fetal inflammatory response only, and no maternal or fetal inflammatory response was 305 (25%), 82 (7%), 8 (1%), and 822 (67%), respectively. Adjusted for covariates, the maternal plus fetal inflammatory response was a significant risk factor for severe ROP (AOR = 2.6, 95% CI 1.1-5.9, P = .03). None of the categories of placental inflammation were significantly associated with low grade ROP. CONCLUSION: Placental pathology distinguished by the maternal plus fetal inflammatory response was a significant risk factor for severe ROP. Our study supports a link between intrauterine inflammatory events and the subsequent development of severe ROP.

Endogenous developmental endothelial locus-1 limits ischaemia-related angiogenesis by blocking inflammation.

We have recently identified endothelial cell-secreted developmental endothelial locus-1 (Del-1) as an endogenous inhibitor of ß2-integrin-dependent leukocyte infiltration. Del-1 was previously also implicated in angiogenesis. Here, we addressed the role of endogenously produced Del-1 in ischaemia-related angiogenesis. Intriguingly, Del-1-deficient mice displayed increased neovascularisation in two independent ischaemic models (retinopathy of prematurity and hind-limb ischaemia), as compared to Del-1-proficient mice. On the contrary, angiogenic sprouting in vitro or ex vivo (aortic ring assay) and physiological developmental retina angiogenesis were not affected by Del-1 deficiency. Mechanistically, the enhanced ischaemic neovascularisation in Del-1-deficiency was linked to higher infiltration of the ischaemic tissue by CD45+ haematopoietic and immune cells. Moreover, Del-1-deficiency promoted ß2-integrin-dependent adhesion of haematopoietic cells to endothelial cells in vitro, and the homing of hematopoietic progenitor cells and of immune cell populations to ischaemic muscles in vivo. Consistently, the increased hind limb ischaemia-related angiogenesis in Del-1 deficiency was completely reversed in mice lacking both Del-1 and the ß2-integrin LFA-1. Additionally, enhanced retinopathy-associated neovascularisation in Del-1-deficient mice was reversed by LFA-1 blockade. Our data reveal a hitherto unrecognised function of endogenous Del-1 as a local inhibitor of ischaemia-induced angiogenesis by restraining LFA-1-dependent homing of pro-angiogenic haematopoietic cells to ischaemic tissues. Our findings are relevant for the optimisation of therapeutic approaches in the context of ischaemic diseases.

Inhibition of the Nuclear Receptor RORγ and Interleukin-17A Suppresses Neovascular Retinopathy: Involvement of Immunocompetent Microglia.

OBJECTIVE: Although inhibitors of vascular endothelial growth factor (VEGF) provide benefit for the management of neovascular retinopathies, their use is limited to end-stage disease and some eyes are resistant. We hypothesized that retinoic acid-related orphan nuclear receptor γ (RORγ) and its downstream effector, interleukin (IL)-17A, upregulate VEGF and hence are important treatment targets for neovascular retinopathies. APPROACH AND RESULTS: Utilizing a model of oxygen-induced retinopathy, confocal microscopy and flow cytometry, we identified that retinal immunocompetent cells, microglia, express IL-17A. This was confirmed in primary cultures of rat retinal microglia, where hypoxia increased IL-17A protein as well as IL-17A, RORγ, and tumor necrosis factor-α mRNA, which were reduced by the RORγ inhibitor, digoxin, and the RORα/RORγ inverse agonist, SR1001. By contrast, retinal macroglial Müller cells and ganglion cells, key sources of VEGF in oxygen-induced retinopathy, did not produce IL-17A when exposed to hypoxia and IL-1ß. However, they expressed IL-17 receptors, and in response to IL-17A, secreted VEGF. This suggested that RORγ and IL-17A inhibition might attenuate neovascular retinopathy. Indeed, digoxin and SR1001 reduced retinal vaso-obliteration, neovascularization, and vascular leakage as well as VEGF and VEGF-related placental growth factor. Digoxin and SR1001 reduced microglial-derived IL-17A and Müller cell and ganglion cell damage. The importance of IL-17A in oxygen-induced retinopathy was confirmed by IL-17A neutralization reducing vasculopathy, VEGF, placental growth factor, tumor necrosis factor-α, microglial density and Müller cell, and ganglion cell injury. CONCLUSIONS: Our findings indicate that an RORγ/IL-17A axis influences VEGF production and neovascular retinopathy by mechanisms involving neuroglia. Inhibition of RORγ and IL-17A may have potential for the improved treatment of neovascular retinopathies.

The peptidomimetic Vasotide targets two retinal VEGF receptors and reduces pathological angiogenesis in murine and nonhuman primate models of retinal disease.

Blood vessel growth from preexisting vessels (angiogenesis) underlies many severe diseases including major blinding retinal diseases such as retinopathy of prematurity (ROP) and aged macular degeneration (AMD). This observation has driven development of antibody inhibitors that block a central factor in AMD, vascular endothelial growth factor (VEGF), from binding to its receptors VEGFR-1 and mainly VEGFR-2. However, some patients are insensitive to current anti-VEGF drugs or develop resistance, and the required repeated intravitreal injection of these large molecules is costly and clinically problematic. We have evaluated a small cyclic retro-inverted peptidomimetic, D(Cys-Leu-Pro-Arg-Cys) [D(CLPRC)], and hereafter named Vasotide, that inhibits retinal angiogenesis by binding selectively to the VEGF receptors VEGFR-1 and neuropilin-1 (NRP-1). Delivery of Vasotide via either eye drops or intraperitoneal injection in a laser-induced monkey model of human wet AMD, a mouse genetic knockout model of the AMD subtype called retinal angiomatous proliferation (RAP), and a mouse oxygen-induced model of ROP decreased retinal angiogenesis in all three animal models. This prototype drug candidate is a promising new dual receptor inhibitor of the VEGF ligand with potential for translation into safer, less-invasive applications to combat pathological angiogenesis in retinal disorders.

What is the basis for a genetic approach in neonatal disorders?

Gene-environment interactions likely account for some degree of the variance in response rates that are clinically observed with antenatal corticosteroids, breast milk prophylaxis, surfactant administration, early recognition and treatment of sepsis, utility of non-invasive ventilation, and judicious exposure to supplemental oxygen. While these therapies and practice guidelines have significantly decreased overall neonatal mortality in the NICU, they have not made a marked impact on the frequency and severity of conditions such as bronchopulmonary dysplasia (BPD), necrotizing enterocolitis, and periventricular leukomalacia. One possible explanation is that genetic factors in the neonate modulate response to external intervention or preventative agents, culminating in variable levels of injury and different degrees of resolution and repair. Gene-environment explanations are supported by the observed heritability of BPD in twin studies, but they do not differentiate the interactions between neonate and offending toxin or pathogen, from interactions between neonate and intervention or therapeutic agent. Likely, both kinds of interactions are important in determining outcome.

The role of the human leukocyte antigen system in retinopathy of prematurity: a pilot study.

AIM: To assess the association between the human leukocyte antigen system and retinopathy of prematurity. METHODS: Neonates of <32 weeks of gestational age, born at two level III neonatal intensive care units from January 2000 to December 2001 and from January 2006 to June 2009, were included in the study. Demographic and clinical data were recorded, and retinopathy was classified according to the International Classification. Epithelial cells were collected from the oral cavity and the HLA were studied using the PCR/SSO method. Univariate and multivariate analyses were performed using SPSS® v.18. RESULTS: We evaluated 156 neonates, including 82 (52.6%) males. Median gestational age was 29 (23-31) weeks, and median birth weight was 1030 (525-1935) grams. Seventy (44.9%) of the neonates developed retinopathy. Alleles HLA-B*38, HLA-Cw*12, HLA-DRB1*09, HLA-DRB1*14 (univariate analysis) and HLA-A*68 and HLA-Cw*12 were associated to retinopathy (multivariate analysis). CONCLUSION: The results suggest that the HLA system may be associated with the development of retinopathy of prematurity. A large-scale population-based study should be performed to clarify this association.

Human milk feeding prevents retinopathy of prematurity (ROP) in preterm VLBW neonates.

BACKGROUND: Retinopathy of prematurity (ROP) is a multifactorial disease, but little is known about its relationships with neonatal nutritional policies. Human, maternal milk is the best possible nutritional option for all premature infants, including those at high risk for severe complications of prematurity, such as ROP. OBJECTIVE: This is a secondary analysis of data collected during two multicenter RCTs performed consecutively (years 2004 through 2008) by a network of eleven tertiary NICUs in Italy. The two trials aimed at assessing effectiveness of fluconazole prophylaxis (Manzoni et al., N Engl J Med 2007 Jun 14;356(24):2483-95), and of bovine lactoferrin supplementation (Manzoni et al., JAMA 2009 Oct 7;302(13):1421-8), in prevention of invasive fungal infection, and of late-onset sepsis in VLBW infants, respectively. We tested the hypothesis that exclusive feeding with fresh maternal milk may prevent ROP of any stage - as defined by the ETROP study - in VLBW neonates, compared to formula feeding. METHODS: We analyzed the database from both trials. Systematic screening for detection of ROP was part of the protocol of both studies. The definition of threshold ROP was as defined by the ETROP study. Univariate analysis was performed to look for significant associations between ROP and several possible associated factors, and among them, the type of milk feeding (maternal milk or formula for preterms). When an association was indicated by p < 0.05, multiple logistic regression was used to determine the factors significantly associated with ROP. RESULTS: In both trials combined, 314 infants received exclusively human maternal milk (group A), and 184 a preterm formula because their mothers were not expected to breastfeed. The clinical, demographical and management characteristics of the neonates did not differ between the two groups, particularly related to the presence of the known risk factors for ROP. Overall, ROP incidence (any stage) was significantly lower in infants fed maternal milk (11 of 314; 3.5%) as compared to formula-fed neonates (29 of 184; 15.8%) (RR 0.14; 95% CI 0.12-0.62; p = 0.004). The same occurred for threshold ROP (1.3% vs. 12.3%, respectively; RR 0.19; 95% CI 0.05-0.69; p = 0.009). At multivariate logistic regression controlling for potentially confounding factors that were significantly associated to ROP (any stage) at univariate analysis (birth weight, gestational age, days on supplemental oxygen, systemic fungal infection, outborn, hyperglycaemia), type of milk feeding retained significance, human maternal milk being protective with p = 0.01. CONCLUSIONS: Exclusive human, maternal milk feeding since birth may prevent ROP of any stage in VLBW infants in the NICU.

Complement involvement in neovascular ocular diseases.

Pathological neovascularization (NV) is a hallmark of late stage neovascular age-related macular degeneration (AMD), diabetic retinopathy (DR), and retinopathy of prematurity (ROP). There is accumulating evidence that alterations in inflammatory and immune system pathways that arise from genetic differences, injury, and disease can predispose individuals to retinal neovascular eye diseases. Yet the mechanism of disease progression with respect to the complement system in these maladies is not fully understood. Recent studies have implicated the complement system as an emerging player in the etiology of several retinal diseases. We will summarize herein several of the complement system pathways known to be involved in ocular neovascular pathologies. Current treatment for many neovascular eye diseases focuses on suppression of NV with laser ablation, photodynamic therapy, or anti-VEGF angiogenic inhibitors. However, these treatments do not address the underlying cause of many of these diseases. A clear understanding of the cellular and molecular mechanisms could bring a major shift in our approach to disease treatment and prevention.

Infection, oxygen, and immaturity: interacting risk factors for retinopathy of prematurity.

BACKGROUND: Interactions among known risk factors for retinopathy of prematurity (ROP) remain to be clarified. OBJECTIVES: The aim of this study was to identify risk factors associated with ROP and to explore the interrelationships between prominent risk factors for ROP. METHODS: From an institutional cohort of 1,646 very preterm newborns with gestational age <30 weeks or birth weight <1,501 g, we selected infants with a gestational age <30 weeks who met the criteria for ROP screening (n = 622) for a nested case-control analysis. RESULTS: Of the 622 eligible newborns, 293 (47%) were diagnosed with ROP. From multivariable analyses, gestational age <26 weeks (OR 2.9, CI 1.7-4.9), oxygen exposure at 28 days (OR 1.7, CI 1.0-2.7), and neonatal sepsis (OR 2.1, CI 1.4-3.2) emerged as prominent risk factors for ROP. Oxygen- associated ROP risk was more prominent among infants of 23-25 weeks' gestational age, while infection-associated ROP risk was higher among infants born at 28-29 weeks. The OR for the joint effect of all 3 risk factors (23.5) was higher than would have been expected under the additive (8.6) and the multiplicative (16.5) patterns of interaction. CONCLUSIONS: Our study suggests that neonatal sepsis, oxygen exposure, and low gestational age are not only independently associated with a significantly increased risk of ROP, but also interact beyond additive and even multiplicative patterns.

Complement-mediated inhibition of neovascularization reveals a point of convergence between innate immunity and angiogenesis.

Beyond its role in immunity, complement mediates a wide range of functions in the context of morphogenetic or tissue remodeling processes. Angiogenesis is crucial during tissue remodeling in multiple pathologies; however, the knowledge about the regulation of neovascularization by the complement components is scarce. Here we studied the involvement of complement in pathological angiogenesis. Strikingly, we found that mice deficient in the central complement component C3 displayed increased neovascularization in the model of retinopathy of prematurity (ROP) and in the in vivo Matrigel plug assay. In addition, antibody-mediated blockade of C5, treatment with C5aR antagonist, or C5aR deficiency in mice resulted in enhanced pathological retina angiogenesis. While complement did not directly affect angiogenesis-related endothelial cell functions, we found that macrophages mediated the antiangiogenic activity of complement. In particular, C5a-stimulated macrophages were polarized toward an angiogenesis-inhibitory phenotype, including the up-regulated secretion of the antiangiogenic soluble vascular endothelial growth factor receptor-1. Consistently, macrophage depletion in vivo reversed the increased neovascularization associated with C3- or C5aR deficiency. Taken together, complement and in particular the C5a-C5aR axes are potent inhibitors of angiogenesis.

Perinatal systemic inflammatory response syndrome and retinopathy of prematurity.

Fetal and neonatal inflammation is associated with several morbidities of prematurity. Its relationship to retinopathy of prematurity (ROP) has not been investigated. Our objective was to determine the relationship between cytokine levels and ROP in the first 3 postnatal wks. Data for this study were derived from the NICHD Cytokine Study. Dried blood spots (DBS) were obtained from infants <1000 g on days 0-1, 3 +/- 1, 7 +/- 2, 14 +/- 3, and 21 +/- 3. Infants were classified into three groups-no, mild, and severe ROP. Multiplex Luminex assay was used to quantify 20 cytokines. Temporal profiles of cytokines were evaluated using mixed-effects models after controlling for covariates. Of 1074 infants enrolled, 890 were examined for ROP and 877 included in the analysis. ROP was associated with several clinical characteristics on unadjusted analyses. Eight cytokines remained significantly different across ROP groups in adjusted analyses. IL-6 and IL-17 showed significant effects in early time periods (D0-3); TGF-beta, brain-derived neurotrophic factor (BDNF), and regulated on activation, normal T cell expressed and secreted (RANTES) in later time periods (D7-21) and IL-18, C-reactive protein (CRP), and neurotrophin-4 (NT-4) in both early and later time periods. We conclude that perinatal inflammation may be involved in the pathogenesis of ROP.

[Humoral immune response to the retinal S-antigen in premature infants and its role in the development and course of retinopathy of prematurity]. / Gumoral'nyi immunnyi otvet na S-antigen setchatki u nedonoshennykh detei i ego rol' v razvitii i techenii retinopatii nedonoshennykh.

The humoral immune response to the retinal S-antigen was studied in premature babies with active and cicatricle stages of retinopathy of the premature (RP) and in the controls (102 serum test). The presence of antibodies to the S-antigen of classes IgG and IgM was established by the enzyme multiplied analysis and by an authors' developed test-system. A definite specific humoral response to the S-antigen correlated with the nature of the course and prognosis of the disease was found in 71% of cases. The first peak of IgM-antibodies was found in babies of the risk-group (preretinopathy), it correlated with a poor prognosis (progression of RP). The second peak was highly pronounced in babies with the terminal cicatricle stages of RP. The wave-like dynamics of IgG-antibodies suggests that specific circulating immune complexes shape up. The role of immune responses, induced by the retinal S-antigen, in the RP pathogenesis is discussed.

[Granulocyte function in premature infants before the 34th week of pregnancy and in mature newborn infants]. / Granulozytenfunktion bei Frühgeborenen ab der 34. Schwangerschaftswoche und bei reifen Neugeborenen.

BACKGROUND: Preterm and term neonates have an increased risk to develop severe bacterial infections. Impairment of neutrophil function may be responsible for this increased risk. Other diseases related to prematurity like retinopathia of prematurity (ROP) or broncho-pulmonary dysplasia (BPD) on the other hand may be due to poorly controlled O2-radical production. PATIENTS AND METHODS: Blood samples of 112 premature (34 weeks of gestation and older) and term neonates were analysed. Blood samples of 23 healthy adults (18 to 50 years old) served as controls. O2-radical production and phagocytosis of neutrophils were determined by flow cytometry, using a commercial test system. RESULTS: Under the experimental conditions applied, the capacity to produce O2-radicals following vigorous stimulation (E. coli) is comparable between neutrophils of preterm/term neonates and healthy adults. However, unstimulated or weakly stimulated (fMLP) neutrophils of preterm and term neonates show a statistically higher O2-radical production as neutrophils of the control group. The production of oxygen radicals increases during the first 10 days of the life. The capability of neutrophils to phagocytose E. coli is significantly lower in newborns (preterm and term) compared to the adult controls. CONCLUSIONS: The values reported here for phagocytosis and O2-radical production utilizing a commercially available test system may serve as "preliminary normal values" for neonates. No differences were found between the groups of neonates with and without infection. Impaired neutrophil-phagocytosis possibly contributes to the increased risk of preterm and term neonates to acquire bacterial infections. The increased spontaneous O2-radical production, on the other hand, may play a role for the development of so called "free radical diseases" such as ROP or BPD. However, our results cannot add further proof to this hypothesis.