The impact of HIV exposure status and maternal feeding practice on the concentration of SLPI and E/tr-2 protein in infant saliva and maternal breast milk

Background Among infants, postnatal HIV transmission occurs via the oral route when ingesting breastmilk containing HI-virions (Kuhn et al., 2007). HIV acquisition via this route is however very low, possibly due to the presence of innate proteins in the oral secretions. Such innate proteins with anti-HIV activity include secretory leukocyte protease inhibitors (SLPI) and Elafin/trappin-2 (E/tr-2). At physiological concentrations SLPI and E/tr-2 have been shown to inhibit in vitro HIV replication in human monocytes and epithelial cells, respectively. Study Aims 1) To determine the impact of HIV infection/ exposure on the concentration of SLPI and E/tr-2 in maternal breast milk and infant saliva; 2) To investigate the impact of of feeding modes on the concentration of innate proteins in infant saliva. Methods This study compared the concentrations of SLPI and E-tr2 among three groups of maternal-infant pairs to address the study aims. Saliva was collected from HIV unexposed breast fed (UBF, n=135), HIV exposed breastfed (EBF, n=151) and HIV exposed formula fed (EFF, n=141) infants together with breast milk from HIV negative (HIV-ve, n=144) and HIV positive (HIV+ve, n=165) mothers. SLPI and E/tr-2 concentrations were measured in samples collected at birth, 15 and 36 weeks of infant age using ELISA. Results Breast milk concentrations of SLPI and E/tr-2 significantly decreased over time in both HIV+ve and HIV-ve women. The salivary SLPI concentration in HEU infants significantly increased over time. In the HU infants, significantly high SLPI concentrations were observed at birth, however the concentration of the analyte was significantly decreased at week 15 and then increased again significantly at week 36. The concentration of E/tr-2 in infant saliva remained the same at all-time points. No significant differences were observed in breast milk SLPI and E/tr-2 concentrations between HIV+ve and HIV-ve mothers at birth and 15 weeks of infant age. However, breast milk SLPI concentrations were significantly higher in the HIV-ve mothers compared to the HIV+ve mothers at 36 week of infant age. Salivary SLPI concentrations were significantly higher in the HU infants at birth, however, at 15 weeks of age an inverse was observed where HEU infants had significantly high SLPI concentrations compared to the HU infants. Additionally, salivary SLPI concentration was significantly higher in HIV exposed formula fed infants compared to HIV exposed breast fed infants at birth. However these differences dissipated with advancing age. No differences were observed for E/tr-2 concentration for both breast fed and formula fed infants. Lastly, no correlation was observed between maternal breast milk and infant salivary SLPI and E/tr-2 concentration at all-time points. Discussion The production of SLPI and E/tr-2 in infants is possibly due to inflammation as a results of bacterial LPS or other factors and not breastfeeding as originally hypothesized. In our cohort, we observed that elevated or low concentrations of SLPI in maternal breast milk is independent of HIV infection. However, in infant saliva HIV exposure played a significant role. An interesting finding in our study was that infants who received breast milk at birth had significantly low SLPI concentrations compared to those who were exclusively formula fed. However, at 15 and 36 weeks of infant age the concentration of SLPI increased probably due to ongoing exposure. It was also notable that we did not see any changes in E/tr-2 concentrations over time nor differences at all time points. Conclusion The data suggest that HEU infants produce sufficient SLPI concentrations capable of blocking HIV infection at 15 weeks of life, as previously shown (Mcneely et al., 1995, Bacqui et al., 2002). Further studies are required to investigate the ability of this protein to block HIV infection in vitro using human saliva

Early provision of maternal colostrum by the oropharyngeal route to preterm infants

Preterm infants are those born before 37 completed weeks of gestation. Worldwide, about one million children die each year due to complications of prematurity and survivors may face lifelong disabilities. Approximately 50% of neonatal deaths and 17% of deaths among children under five are affected by prematurity. Colostrum is the first milk produced by the mother within the early few days after birth. Colostrum is very rich in immunological and growth factors that indicates its primary functions are protective and trophic. Oropharyngeal administration of colostrum (OPC) is a novel route that involves coating the infant’s oropharynx with a small amount of colostrum (0.1 to 0.5 ml) during the early neonatal period. Immune and growth factors in colostrum might interact with the oropharyngeal mucosal-associated lymphoid tissues to modulate the infant’s immune system and promote intestinal growth, potentially reducing infection and necrotising enterocolitis (NEC), improving survival and health outcomes. Ultimately OPC could provide a potential target to prevent mortality and morbidities of preterm and sick infants. This thesis aimed to investigate whether OPC administration during the early neonatal period prevents deaths, improves health outcomes and promotes the growth of preterm infants. To achieve the aim of this thesis; initially, an online survey targeted neonatal professionals was performed to evaluate the current practice and perception of OPC administration in the UK neonatal units. Oropharyngeal colostrum has been introduced into UK neonatal practice despite a lack of high-quality evidence regarding its efficacy and safety. OPC practice was variable, frequently without written guidelines. A Cochrane systematic review was conducted to synthesis and appraise the currently available randomised controlled trials (RCTs), which evaluated if early OPC given within the first 48 hours has a positive impact in preterm infants (< 37 weeks gestation) compared with control. Six RCTs were eligible for inclusion in this systematic review. Meta-analysis showed that early OPC could shorten the time to reach full enteral feeds but did not reduce the incidence of late-onset infection (LOI), NEC and death nor the length of hospital stay. Available evidence is insufficient due to lack of participants and very low quality to demonstrate the benefits effects of OPC for preterm infants. The third study, a matched case-control study evaluated the effects of OPC administration on the short-term health outcomes in preterm (≤ 32 weeks) infants. Eligible infants who were admitted to the Nottingham neonatal units after the implementation of OPC in the care of preterm infants, and received OPC, compared with those who were admitted before the use of OPC in the units. Preterm infants who received OPC within the first 96 hours of life achieved full enteral feeding (150 ml/Kg/day for consecutive 72 hours) earlier than those infants who did not receive OPC. A higher rate of receiving breast milk at discharge to home was also observed. However, the two groups had a similar length of hospital stay, weight Z-score at hospital discharge, and incidences of NEC, LOI and deaths. Finally, a non-randomised observational study evaluated the response of gut hormones to OPC administration in preterm (< 37 weeks of gestation) and ill infants requiring neonatal intensive care (NIC). Preliminary results demonstrated a rising trend in plasma gut hormone concentrations in response to OPC administration in the participant preterm and full-term infants. This study is ongoing, and more infants are required before final conclusions can be elicited. In conclusion, OPC administration is a potentially feasible intervention that shortens time to attain full enteral feeds in preterm infants. Given the high risk for preterm infants and the benefits of maternal colostrum, OPC may have preventive implications for improving the health outcomes of this vulnerable population. This work expands the current knowledge about the use of OPC in the care of preterm and sick newborn infants and could benefit efforts to improve preterm birth outcomes by informing guidelines, clinical decision and future research. Larger, well-designed, high-quality research with sufficient power are needed to assess the efficacy and safety of this intervention

Lactoferrin supplementation during pregnancy — a review of the literature and current recommendations

Pregnancy is a period which requires special care and attention. Maintaining health during pregnancy helps to avoid birth related complications and is the best way of promoting a healthy birth. Besides a daily intake of folic acid, iron, iodine, vitamin D3 and A, calcium and polyunsaturated fatty-acids, as recommended by health agencies, supplementation of lactoferrin — a protein of multidirectional biological activity and proven safety of use — seems to be beneficial. A wide range of lactoferrin biological roles (including regulation of iron balance, modulation of immune responses, antimicrobial, antiviral, antioxidant, and anti-inflammatory activity) may contribute to better pregnancy and birth related outcomes

Early provision of maternal colostrum by the oropharyngeal route to preterm infants

Preterm infants are those born before 37 completed weeks of gestation. Worldwide, about one million children die each year due to complications of prematurity and survivors may face lifelong disabilities. Approximately 50% of neonatal deaths and 17% of deaths among children under five are affected by prematurity. Colostrum is the first milk produced by the mother within the early few days after birth. Colostrum is very rich in immunological and growth factors that indicates its primary functions are protective and trophic. Oropharyngeal administration of colostrum (OPC) is a novel route that involves coating the infant’s oropharynx with a small amount of colostrum (0.1 to 0.5 ml) during the early neonatal period. Immune and growth factors in colostrum might interact with the oropharyngeal mucosal-associated lymphoid tissues to modulate the infant’s immune system and promote intestinal growth, potentially reducing infection and necrotising enterocolitis (NEC), improving survival and health outcomes. Ultimately OPC could provide a potential target to prevent mortality and morbidities of preterm and sick infants. This thesis aimed to investigate whether OPC administration during the early neonatal period prevents deaths, improves health outcomes and promotes the growth of preterm infants. To achieve the aim of this thesis; initially, an online survey targeted neonatal professionals was performed to evaluate the current practice and perception of OPC administration in the UK neonatal units. Oropharyngeal colostrum has been introduced into UK neonatal practice despite a lack of high-quality evidence regarding its efficacy and safety. OPC practice was variable, frequently without written guidelines. A Cochrane systematic review was conducted to synthesis and appraise the currently available randomised controlled trials (RCTs), which evaluated if early OPC given within the first 48 hours has a positive impact in preterm infants (< 37 weeks gestation) compared with control. Six RCTs were eligible for inclusion in this systematic review. Meta-analysis showed that early OPC could shorten the time to reach full enteral feeds but did not reduce the incidence of late-onset infection (LOI), NEC and death nor the length of hospital stay. Available evidence is insufficient due to lack of participants and very low quality to demonstrate the benefits effects of OPC for preterm infants. The third study, a matched case-control study evaluated the effects of OPC administration on the short-term health outcomes in preterm (≤ 32 weeks) infants. Eligible infants who were admitted to the Nottingham neonatal units after the implementation of OPC in the care of preterm infants, and received OPC, compared with those who were admitted before the use of OPC in the units. Preterm infants who received OPC within the first 96 hours of life achieved full enteral feeding (150 ml/Kg/day for consecutive 72 hours) earlier than those infants who did not receive OPC. A higher rate of receiving breast milk at discharge to home was also observed. However, the two groups had a similar length of hospital stay, weight Z-score at hospital discharge, and incidences of NEC, LOI and deaths. Finally, a non-randomised observational study evaluated the response of gut hormones to OPC administration in preterm (< 37 weeks of gestation) and ill infants requiring neonatal intensive care (NIC). Preliminary results demonstrated a rising trend in plasma gut hormone concentrations in response to OPC administration in the participant preterm and full-term infants. This study is ongoing, and more infants are required before final conclusions can be elicited. In conclusion, OPC administration is a potentially feasible intervention that shortens time to attain full enteral feeds in preterm infants. Given the high risk for preterm infants and the benefits of maternal colostrum, OPC may have preventive implications for improving the health outcomes of this vulnerable population. This work expands the current knowledge about the use of OPC in the care of preterm and sick newborn infants and could benefit efforts to improve preterm birth outcomes by informing guidelines, clinical decision and future research. Larger, well-designed, high-quality research with sufficient power are needed to assess the efficacy and safety of this intervention

Antibiotic Therapy for Neonatal Sepsis - Studies on epidemiology, gentamicin safety, and early adverse effects of antibiotics

Paper 1, 2, 3 & 4 are not available in Munin. Paper 1: Fjalstad, J.W., Stensvold, H.J., Bergseng, H., Simonsen, G.S., Salvesen, B., Rønnestad, A.E. & Klingenberg, C. (2016). Early-onset Sepsis and Antibiotic Exposure in Term Infants: A Nationwide Population-based Study in Norway. Available in The Pediatric Infectious Disease Journal, 35(1), 1-6. Paper 2: Fjalstad, J.W., Laukli, W., van den Anker, J.N. & Klingenberg, C.(2013). High-dose gentamicin in newborn infants: is it safe? Available in European journal of pediatrics (2014),173, 489-495. Paper 3: Esaiassen, E., Fjalstad, J.W., Juvet, L.K., van den Anker, J.N. & Klingenberg, C. (2017). Antibiotic exposure in neonates and early adverse outcomes: a systematic review and meta-analysis. Available in Journal of Antimicrobial Chemotherapy, 72(7), 1858-70. Paper 4: Fjalstad, J.W., Esaiassen, E., Juvet, L.K., van den Anker, J.N. & Klingenberg, C. (2017). Antibiotic therapy in neonates and impact on gut microbiota and antibiotic resistance development: a systematic review. Available in Journal of Antimicrobial Chemoterapy (2018), 73(3), 569-580. Objectives: The overall aim of this thesis was to investigate different aspects of antibiotic therapy for neonatal sepsis. Material and Methods: The epidemiology of early onset sepsis (EOS) and systemic antibiotic exposure in the first week of life was studied in a nationwide population-based study from the Norwegian Neonatal Network between 2009-2011. A high-dose extended-interval gentamicin regimen was studied in the neonatal unit in Tromsø from 2004-2012. Early adverse effects of antibiotic therapy were studied in a systematic review. We included observational studies and randomized controlled trials that provided data on different categories of antibiotic therapy and either the risk of necrotizing enterocolitis, invasive fungal infection, death, antibiotic resistance development, or changes in the gut microbiota. Results: There were 0.54 cases of culture-confirmed EOS per 1000 live-born term infants with a mortality rate of 1%. Intravenous antibiotics were administered to 2.3% of all live-born term infants in Norway, and half of them were not diagnosed with an infection. In the neonatal unit in Tromsø, gentamicin trough concentrations were above the threshold of 2 mg/L in 6% of cases. Only 1% of these infants suffered from permanent hearing loss. In our systematic reviews, prolonged antibiotic exposure was significantly associated with necrotizing enterocolitis and/or death in preterm infants and reduced gut microbial diversity in all infants. Broad-spectrum antibiotic treatment increased the risk of invasive fungal infection. All categories of antibiotic exposure were associated with an increased risk of antibiotic resistance development. Main Conclusions: The incidence of culture-confirmed EOS in Norway was in line with previous international reports, and the mortality was very low. A large proportion of infants were treated with antibiotics without an infection. The extended-interval high-dose gentamicin regimen studied in this thesis seems safe. Neonatal antibiotic treatment was associated with several adverse effects

Proteomic Characterization of the Chorion

Fetal membranes were collected from term, vaginal deliveries with no known pregnancy-related complications. Samples were collected from spontaneously rupturing (SROM; n=5) and artificially rupturing (AROM; n=2) membranes. For each membrane, samples were taken proximal to and distal from the rupture initiation site. Mass spectrometry identified several proteins involved in assembly, wound response and immune response as differentially expressed between the placental and rupture regions. In particular, basement membrane proteins including fibrillin and laminin were observed exclusively in the artificially rupturing samples, and fibrinogen was found in the rupture region in AROM but not SROM samples. Talin and vinculin, proteins involved in focal adhesions, were observed exclusively in AROM rupture

Maternal milk feedings and cytomegalovirus infection in preterm infants in sweden

In Sweden, preterm infants are preferably fed human milk. Very preterm infants (< 32 weeks), who are unable to breastfeed, are fed with expressed maternal milk via a nasogastric tube. Mothers of these infants often experience difficulties in establishing and maintaining lactation. The majority of women excrete cytomegalovirus (CMV) in their breast milk. CMV transmitted through maternal milk can cause symptomatic infection in preterm infants presenting as a sepsis like syndrome, pneumonitis, hepatopathy or enterocolitis. Routine freezing of maternal milk decreases the CMV load in breast milk and is used in some neonatal centers to reduce CMV transmission to preterm infants. The aims of the studies in this thesis were to document existing routines pertaining to breast milk use for preterm infants in Sweden, to investigate predictors of maternal milk feedings in extremely preterm infants (EPIs, < 28 weeks), to evaluate the rate and clinical expression of postnatal CMV infection in EPIs, to evaluate the effect of routine freezing of maternal milk on CMV transmission rate, CMV associated disease and neonatal morbidity and mortality in EPIs and to evaluate the prevalence of CMV infection in intestinal specimens from infants with necrotizing enterocolitis (NEC), spontaneous intestinal perforation (SIP) and related surgical conditions. In a national cross sectional study in 2006 in Sweden, we found that 27 of 36 (75%) neonatal units had their own milk bank. Milk donors were screened for human immunodeficiency virus, human T-lymphotropic virus, and hepatitis B and C viruses by 27 (100%), 14 (52%), and 22 (81%) of the milk banks, respectively. Bacterial culture was performed on donor milk in 24 (89%) milk banks. Donor milk was pasteurized in 22 (81%) milk banks. In 11 of the 36 (31%) neonatal units maternal milk was frozen to reduce the risk ofCMV transmission. Nutritional analysis of donor and/or maternal milk was performed in 25 (69%) units. In a prospective cohort study at the neonatal units in Stockholm, including 97 mothers and their singleton EPIs, predictors of maternal milk feedings in EPIs during the first 6 weeks of life and at discharge were evaluated. Favorable predictors of maternal milk feedings the first 6 weeks of life were high maternal milk feedings (>90%) at second week of life, maternal university education and Nordic origin of the mother. The proportion of maternal milk feedings the first 6 weeks of life and maternal age were positively associated to the provision of maternal milk feedings at discharge while maternal overweight was an unfavorable predictor. High maternal milk feedings (>90%) at second week of life, assisted reproduction technology and maternal employment were predictive factors for exclusive maternal milk feedings at discharge. Ten EPIs and their 6 mothers were included in a pilot study at the neonatal unit, Astrid Lindgrens Children´s hospital to evaluate the rate and clinical expression of breast milk induced CMV infection. Five (83%) mothers were CMV-seropositive; of these, 4 (80%) excreted CMV-DNA in breast milk and 2 (40%) had a positive CMV culture. CMV was detected in the urine of 2/7 (29%) EPIs fed with CMV-positive milk; both were fed with breast milk positive for CMV culture. One EPI, later diagnosed with cystic fibrosis, developed hepatic affection concurrent with CMV urine excretion. In a randomized study at the neonatal units in Stockholm, evaluating the effect of routine freezing of maternal milk on postnatal CMV infection and neonatal outcome, 140 EPIs were randomized to be fed only freeze-thawed maternal milk (intervention group, IG) or both fresh maternal milk and freeze-thawed maternal milk (control group, CG). Outcome measures were CMV transmission rate and symptomatic infection in EPIs, neonatal mortality and morbidity during hospital stay. Fifty-six EPIs in the IG and 65 EPIs in the CG were included in the final per protocol analysis. We observed an overall low CMV transmission rate (8%) to EPIs from mothers with detectable CMV in breast milk. Routine freezing of maternal milk did not reduce the rate of CMV transmission (9% in IG vs 6% in CG). Congenital CMV infection was detected in 2% of screened infants. No infected EPI presented with clinical symptoms of CMV infection. Mortality rates were similar; 7% in the IG and 6% in the CG. Neonatal morbidity did not differ except for late onset Candida sepsis; the incidence was 12% in the CG while no case was observed in the IG. In a retrospective observational study, we investigated the occurrence of the CMV in 70 intestinal specimens from 61 infants with NEC, SIP and related surgical conditions at the Karolinska University Hospital Solna and Uppsala University Hospital. Ten intestinal specimens from autopsied infants without bowel disease were controls. By using immunohistochemistry (IHC), we detected the CMV specific proteins CMV-immediate early antigen (CMV-IEA) in 81% (57/70) and CMV-late antigen (CMV-LA) in 64% (45/70) of the intestinal specimens; 2/10 (20%) of the control specimens were positive for both antigens. Although CMV antigens were prevalent irrespective of pathologic diagnosis, they were most frequent in specimens with the pathologic diagnosis NEC and intestinal perforation; 95% and 89% ofthese tissue specimens were positive for CMV-IEA and CMV-LA, respectively. CMV infection was confirmed by CMV-DNA analysis in 4/10 (40%) CMV-IHC-positive intestinal samples using Taqman PCR after laser capture microdissection and in 13/13 (100%) CMV-IHC-positive intestinal samples by in situ hybridization. To conclude, human milk handling routines vary between neonatal units in Sweden and need to be standardized. Mothers of EPIs should aim for a high breast milk production immediately after delivery to optimize lactation success. Mothers who are young, overweight, ofnon-Nordic origin or without university education may need special lactation support. Postnatal CMV transmission from mothers excreting CMV in breast milk to EPIs was low (8%) and was not reduced by routine freezing of maternal milk. However, congenital CMV infection in EPIs was unexpectedly high (2%). No EPI infected by CMV presented with clinical symptoms. Routine freezing of maternal milk did not affect neonatal death in EPIs although it may have protected against fungal late onset sepsis. CMV infection was prevalent in intestinal specimens from infants with NEC, SIP and related surgical condition implicating a possible role of the virus in disease pathogenesis. More studies are needed to further evaluate the risk/benefit ratio ofmaternal milk feedings in EPIs with regard to the short-term and long-term effects of postnatal CMV infection

Evaluation of immune function in preterm infants using Immuknow® assay

Background. Neonatal immune system is not fully developed at birth; newborns have an adequate leukocytes and lymphocytes count at birth but these cells lack of function. Objective. To assess the functional activity of T-cells at birth and at 30 days of life and the influence of the main perinatal factors in a population of preterm infants. Design. A prospective longitudinal study was carried out in a population of 59 preterms. Fifteen healthy adults were included as a control group. Blood samples were collected at birth and at 30 days of life to evaluate CD4+ T cell activity using the Immuknow® assay. Results. CD4+ T cell activity at birth and at 30 days of life were significantly lower compared with adult controls (p < .001). Twins showed lower activity compared to singletons (p= .005). Infants born to vaginal delivery had higher CD4+ T cell activity compared to those born to c-section (p=0.031); infants born after pPROM showed a higher activity at birth (p= .002). Low levels of CD4+ T cells activation at birth were associated with necrotizing enterocolitis development in the first week of life (p=.049). Conclusions. Preterm infants show a lack in CD4+ T cells activation at birth. Perinatal factors such as intrauterine inflammation, mode of delivery, zygosity can influence the levels of adaptative immune activity at birth and can contribute to expose these infants to serious complications such as NEC