http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, Early Online: 1–5 ! 2014 Informa UK Ltd. DOI: 10.3109/14767058.2014.968548
REVIEW ARTICLE
Use of lactoferrin in the newborn: where do we stand? J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Oregon Health Sciences University on 03/27/15 For personal use only.
Deepak Sharma1, Anuradha Murki2, Srinivas Murki1, and Oleti Tejo Pratap1 1
Department of Neonatology, Fernandez Hospital, Hyderguda, Hyderabad, Andhra Pradesh, India and 2Department of Obstetrics and Gynaecology, Kamineni Hospital, Hyderabad, Andhra Pradesh, India Abstract
Keywords
Sepsis and necrotizing enterocolitis (NEC) cause significant morbidity and mortality in the newborn. Their ill effects persist in spite of appropriate and effective antibiotic therapy. Lactoferrin as an adjunct to antibiotics in the treatment of sepsis or NEC in the newborn may improve the clinical outcomes by enhancing the host defense and modulating the inflammatory response. This review focuses on the various aspects of lactoferrin use in the newborn.
Anti-bacterial, anti-fungal, anti-viral, fungal sepsis, immunomodulation, lactoferrin, necrotizing enterocolitis, neonatal sepsis
Introduction The term ‘‘Lactoferrin’’ (LF) is derived from its past classification as a major iron-binding protein in milk. LF, also referred to as lacto-transferrin, was first identified in 1939 in bovine milk [1] and it was isolated from human milk in 1960 [2]. LF is produced mainly from the neutrophils [3] but other cells also contribute to its levels in milk. The complete amino acid sequences of human LF contain 703amino acid residues [4]. LF is present in human plasma in relatively low concentrations, but high levels are observed in colostrum, human breast milk, and seminal plasma. Markedly higher levels occur in cord blood, tears, and vaginal mucus. Masson et al, when studying ten different mammalian species, reported the highest levels of LF in human breast milk [5]. In human milk, its concentration peaks in the colostrum (7 mg/ mL) and then decrease (1 mg/mL) in mature milk, the rate of reduction being slower in the milk of mothers of premature neonates [6,7]. Bovine lactoferrin (BLF) and human lactoferrin (HLF) share a high degree (77%) of amino-acid homology, and also have the same N-terminal peptide, called lactoferricin. Both BLF and HLF resist proteolysis through the infant’s digestive tract, bind to specific receptors on enterocytes, and may be found intact in stools that are poorly absorbed in the gut [8,9]. LF receptors have been identified in the gastrointestinal tract, on leukocytes and macrophages, platelets, and on bacteria. BLF is transferred from the intestine into
Address for correspondence: Dr Srinivas Murki, Consultant Fernandez Hospital, Hyderguda, Hyderabad 500029, Andhra Pradesh, India. E-mail: [email protected]
History Received 27 January 2014 Revised 19 September 2014 Accepted 19 September 2014 Published online 8 October 2014
peripheral blood in a form with intact molecular weight (80 kDa] and localized within 10 to 20 min after oral administration in the liver, kidneys, gall bladder, spleen, and brain [10]. 54LF has been known to have antimicrobial properties, immunomodulatory and trophic activities on the developing gut. LF has broad spectrum antimicrobial activity with bacteriostatic activity against Escherichia coli, Klebsiella pneumoniae, Streptococcus mutans and Candida albicans [11]. LF antimicrobial activity acts by several mechanisms.
Antimicrobial and anti-fungal action LF has direct antimicrobial action against all types of bacteria, fungi, viruses, and parasites. LF acts by prevention of biofilm formation by Pseudomonas aeruginosa [12] and oral pathogens [13] and proteolysis of virulence factors of Haemophilus influenzae [14]. LF blocks bacterial adhesion to host cells by binding to glycosaminoglycans on bacteria and/or host cell membranes [15,16]. Prophylactic LF fed to neonatal rats before they are enterally infected with E coli initiates a mechanism called anoikis wherein cells containing viable bacteria develop apoptosis [17]. Human lactoferrin-derived peptide, HLF [1–11], exerts potent in-vitro candidacidal activity, in order to display antifungal activity against disseminated Candida albicans infections [18]. BLF has been proved as an effective adjuvant to 7-day triple therapy for eradication of Helicobacter pylori infection [19].
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BLF has also been seen useful in antibiotic associated diarrhea caused by Clostridium difficile [20] and enteritis caused by Shigella Flexnieria [21] in adults. LF has bifidogenic activity, enhancing the growth of the normal commensal bifidogenic microflora in the gut.
Anti-viral properties
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LF is found to decrease serum alanine transaminase and Hepatitis C Virus (HCV) RNA concentrations in patients with chronic hepatitis C [22]. LF has anti-viral activity against Human Immunodeficiency Virus (HIV] virus [23] and is shown to increase the percentage of CD4+ cells count [24].
Immunomodulation properties Talactoferrin (TLF) which is human recombinant LF acts on alarmin receptors and promotes activation and recruitment of antigen presenting cells [25]. TLF increases the ability of dendritic cells to trigger proliferation and release of interferon-gamma. It is proposed that activated dendritic cells promote secretion of interleukin (IL)-12 [26] which in turn inhibits production of IL-4. This protects the T helper cell Th1 cells from apoptosis mediated by IL-4 and helps to overcome the neonatal bias for Th2 cells over Th1 cells [27].
Immature gut growth LF promotes growth and differentiation of immature gut epithelium. The higher the LF levels the better the effects. LF is found in high concentrations in preterm human milk.
Anticancer properties LF can inhibit the growth of head and neck squamous cell carcinoma via direct cellular inhibition as well as systemically via immune modulation [28].
Bone growth properties LF has also been reported as a novel bone growth factor. At physiological concentrations, LF potently stimulates the proliferation and differentiation of primary osteoblasts and also acts as a survival factor inhibiting apoptosis induced by serum withdrawal [29].
Clinical uses of Lactoferrin Prevention of necrotizing enterocolitis (NEC). In the preclinical trial, feeding human recombinant LF to neonatal rats before an intestinal infection with Escherichia coli (E. coli) significantly reduced translocation, bacteremia, and death [30]. In another preclinical trial feeding recombinant human LF and Lactobacillus rhamnosus GG (LGG) had more effect than feeding LGG alone in reducing gut-related translocation after an enteral infection with E. coli; recombinant HLF enhanced intestinal colonization with LGG [31]. This research was the basis for the first clinical trial of LF in preterm infants. Manzoni et al. in their prospective, multicenter, double-blind, placebo controlled randomized trial found out that oral LF alone did not reduce the incidence of NEC (1.9% versus 6%, p ¼ 0.09, but a significant reduction in
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NEC was noted with the combination of LF with LGG (n ¼ 0/151 versus n ¼ 10/168, p ¼ 0.002). This study was not powered for detection of a difference in NEC in the intervention and the placebo groups [32]. In a Peruvian study that reported its results at the 2012 Pediatric Academic Society meeting, 190 infants weighing less than 2500 g at birth were randomized to BLF or maltodextrin, 200 mg/d in 3 divided doses over the first 4 weeks of age. Although the incidence of sepsis and NEC was lower in the BLF group the study was powered to detect the intended results of decrease in sepsis and NEC [33]. One clinical trial is underway in Ankara University, Turkey ‘‘Oral Lactoferrin Prophylaxis to Prevent Sepsis and Necrotizing Enterocolitis of Very Low Birth Weight Neonates in Neonatal Intensive Care Unit and Effect on T-regulatory Cells’’ (grant NCT01287507). The primary outcome is late onset sepsis (LOS), i.e. sepsis occurring ‘‘more than 72 hours after birth’’ with isolation of any pathogen from blood or from peritoneal or cerebrospinal fluid and NEC. This study is in the phase of recruiting the patients. Multi-center studies of LF to prevent NEC are needed and those investigations should be sufficiently powered to demonstrate effectiveness in preterm infants weighing less than 1000 g and 1000–1500 g at birth. Lactoferrin and sepsis LOS is an increasing concern in neonatal intensive care units (NICUs) worldwide, given the associated high morbidity and related mortality. Infections are frequently related to severe late neuro-developmental impairment and are the primary cause of death in pre-term infants and a major risk of poor outcomes [34–36]. Manzoni et al. conducted the study ‘‘Bovine Lactoferrin Supplementation for Prevention of Late-Onset Sepsis in Very Low-Birth-Weight Neonates’’. The primary outcome measured was first episode of LOS. In their study, infants were randomly assigned to receive orally administered BLF (100 mg/d) alone (n ¼ 153), BLF plus Lactobacillus rhamnosus GG (LGG) (6 109 colony-forming units/d) (n ¼ 151), or placebo (n ¼ 168) from birth until day 30 of life (day 45 for neonates 51000 g at birth). Overall LOS occurred less frequently in the BLF and BLF plus LGG group than in the control group (RR 0.34, 95% CI: 0.17–0.70, p ¼ 0.002 for BLF versus control; RR 0.27, 95% CI: 0.12– 0.60, p50.001 for BLF plus LGG versus control). When stratification for birth weight was done, the decrease in LOS was significant in extremely low birth-weight (ELBW) neonates (RR 0.31, 95% CI: 0.14–0.70; p ¼ 0.002 for BLF versus control and RR 0.30, 95% CI: 0.13–0.69; p ¼ 0.002 for BLF plus LGG versus control), whereas no significant difference was seen in neonates weighing 1001 to 1500 g [32]. Death from sepsis was significantly lower in very low birth-weight (VLBW) infants when supplemented with BLF. In a recent study by Kaur et al. 121 low birth weight infants with birth weight 52000 grams were randomized in the first 12 hours of life to either BLF or placebo. BLF was supplemented until day 28 of life. The primary outcome was LOS. In their study there was a significant reduction in the incidence of LOS in the BLF group than in the placebo group [n ¼ 2/59 versus n ¼ 9/62, p ¼ 0.033). There was a trend towards lower sepsis attributable mortality in the BLF
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DOI: 10.3109/14767058.2014.968548
supplemented group (n ¼ 0/59 versus 5/62, p ¼ 0.058) [38]. The Peruvian study also reported reduction in the cumulative incidence of sepsis in the BLF group as compared with placebo group [33]. Gunutupalli et al. studied the role of Talactoferrin alpha, a recombinant form of HLF, in 194 adult patients within the first 24 hours of onset of severe sepsis. They reported a significant reduction in the all cause mortality at day 28 of hospital admission; 26.9% in the placebo group and 14.4% in the talactoferrin group (p ¼ 0.052) representing 12.5% absolute and 46.5% relative reduction in mortality. Reduction in all-cause mortality was sustained at 6 months (p ¼ 0.039). No adverse effect was noted of drug intake [37]. Ongoing clinical trials of LF in children include (1) LF for prevention of neonatal sepsis (grant NCT01264536), (2) supplementation with LF in preterm newborns (grant NCT01172236), (3) study of TLF oral solution for nosocomial infection in preterm infants (grant NCT00854633), (4) study of biotene oral Balance gel for oral care in critically-ill mechanically ventilated neonates (grant NCT01314742). Oral LF prophylaxis reduces the incidence of LOS in infants weighing less than 1500 g and most effective in infants weighing less than 1000 g. Further studies confirming these findings are warranted in order to establish the role of BLF in the armamentarium of preventative strategies for sepsis. Lactoferrin and fungal sepsis Preterm infants in NICUs are at great risk for fungal sepsis due to a number of specific risk factors, such as immature immunity, prolonged need for intensive care, and disorders in gut micro ecology with proliferation of saprophytes and pathogens including Candida spp. Invasive fungal infections (IFIs) in preterm infants are caused mainly by the various Candida spp, and are responsible for considerable short and long-term morbidity and high attributable mortality [39]. In the study by Manzoni et al. fungal colonization rates were similar in the treatment and control groups. However, IFI occurred less frequently in the treatment groups (p ¼ 0.004 in BLF and p ¼ 0.07 in BLF plus LGG). The rate of progression from colonization to IFI was significantly reduced in BLF group (p ¼ 0.005) in comparison with controls [32]. Kaur et al. in their study reported decreasing trend in incidence of fungal sepsis in intervention group in comparison to control group [38]. In an in-vitro experiment, Venketesh et al. evaluated the synergic effect of talactoferrin with vancomycin (VAN) and naficillin (NAF) on coagulase negative Staphylococcus aureus (CoNS) and with amphotericin (AMB) or fluconazole (FLU) on Candida albicans. It was found that TLF acted synergistically with NAF and VAN against CoNS, and with AMB and FLC against C. albicans, at multiple dose effects and drug-dose ratios. They concluded that it is a promising agent and should be further evaluated [40]. The role of lactoferrin in prevention of fungal sepsis has to be evaluated further by more randomized controlled trials (RCTs). Lactoferrin and threshold Retinopathy of prematurity Manzoni et al. found decreasing trends in incidence of threshold Retinopathy of prematurity (ROP) requiring surgery
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in BLF group and in BLF plus LGG group as compared to control group. Further trials are needed to prove the beneficial effect of LF on this issue [32]. Lactoferrin and Bronchopulmonary dysplasia Manzoni et al. found no statistical difference in Bronchopulmonary dysplasia (BPD) in both BLF and BLF with LGG group after oral lactoferrin supplementation though there was decrease in incidence in intervention group [32]. Further research is needed to support this hypothesis. Lactoferrin and prevention of preterm delivery LF decreases the level of mediators which are involved in preterm delivery like IL-6 in both serum and cervico-vaginal fluids, prostaglandin F2a in cervico-vaginal and suppresses uterine contractility preventing preterm delivery. Paesano et al. in a cohort of 161 pregnant women with iron deficiency and iron deficient anemia evaluated the role of oral BLF in improving the hematological parameters and the role of oral with intravaginal BLF in a sub-cohort at threat of preterm delivery (PTD). In their study, BLF significantly improved the hematological parameters of anemia, iron deficiency and in the sub-cohort BLF resulted in better cervical length, lower values of IL-6 and PGF2a; both markers of preterm delivery. None of the women in the sub-cohort delivered before 37 weeks of gestation. No side effects were noted in the study. All the newborns exposed to BLF in the antenatal period had normal birth weights and good Apgar scores. These results provide strong evidence for a role of BLF in PTD treatment, thus extending the therapeutic potential of this multifunctional natural protein [41]. There are currently no studies showing effect of LF on neurological outcome at two years of age or more, periventricular leucomalacia, and duration of assisted ventilation through an endotracheal tube or length of hospital stay.
Future and conclusion A review published by Cochrane advised for further studies for evaluating the effect of LF on NEC and sepsis [42,43]. Future studies should answer the questions which are unanswered until now. As the primary outcome of most neonatal clinical trials is long-term neurodevelopmental outcomes, the impact of BLF on subsequent neurodevelopment and growth need to be studied. Infants in resource poor areas of the developing world have a higher frequency of infections and higher case fatality rate, role of LF need to be studied, as well as, the impact of LF use in the larger late preterm infant population. While the overall neonatal mortality rate is low for late preterm infants, infections among these infants increase the risk of complications, prolonged hospital stay, and increased mortality. Lastly, there is a need to understand the optimal dose and duration of LF supplementation and include infants in resource poor areas of the developing world which have higher frequencies of infections and higher case fatality rates. Ongoing clinical trials will hopefully be able to give answers to many of these unanswered questions Table 1.
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Table 1. Showing various lactoferrin studies done. Study Population
Intervention group
Control group
[32]
472 very low birth weight (VLBW) neonates
[33]
190 low birth weight (LBW) neonates
[37]
194 adults within 24 h of the onset of severe sepsis
[38]
121 low birth weight (52000 grams) neonates
First episode of late onset Control group received Infants were randomly sepsis was taken as placebo (2 mL of a assigned to receive outcome. There was 5% glucose solution) orally administered significant reduction of Bovine Lactoferrin incidence of Late onset (BLF) (100 mg/d) sepsis (LOS) in both alone (n ¼ 153), BLF the intervention group plus Lactobacillus in compare to control rhamnosus GG (LGG) group. Both fungal and (6 109 colony-forming bacterial sepsis was units/d) (n ¼ 151), or decreased. placebo (n ¼ 168) from birth until day 30 of life (day 45 for neonates 51000 g at birth). There was reduction in BLF was given enterally at Control group received incidence of neonatal placebo (maltodextrin) 200 mg/d in 3 divided sepsis and NEC in interenterally at 200 mg/d in doses over the first 4 vention group though the 3 divided doses over the weeks of life. difference was not first 4 weeks of life significant. Enterally administered pla- Intention-to-treat analysis Enterally administered was used to assess 28-day cebo every 8 h for up to talactoferrin 1.5 g every all-cause mortality. 28 days or until discharge 8 h for up to 28 days or Enteral administration from the ICU until discharge from the of talactoferrin reduced Intensive care unit (ICU). 28-day all-cause mortality in patients with severe sepsis. Primary outcome measure Bovine Lactoferrin [BLF] Control group (n ¼ 62) were incidence of culture received placebo daily (n ¼ 59) was suppleproven gram positive, from first to 28th day mented daily from first to gram negative and of life. 28th day of life. Candida sepsis at any day, after 72 hours of life. Secondary outcome measure were sepsis attributable mortality after 72 hours of life. They found that incidence of first episode of culture proven LOS was significantly lower in the BLF group than in the placebo group [2/59 (3.4%) versus 9/62 (14.5%); p ¼ 0.033]. The sepsis attributable mortality after 72 hours of life was comparable among the BLF supplemented and placebo group [0/59 (0) versus 5/62 (8.1%); p ¼ 0.058]
Declaration of interest There is no conflict of interest and there was no funding involved.
References 1. Soerensen M, Soerensen SPL. The protein in whey. C R Trav Lab Carlsberg 1939;23:55–99. 2. Johansson BG. Isolation of an iron containing red protein from human milk. Acta Chem Scand 1960;14:510–12. 3. Iyer S, Lo¨nnerdal B. Lactoferrin, lactoferrin receptors and iron metabolism. Eur J Clin Nutr 1993;47:232–41.
Outcome
Limitations Generalizability was a potential limitation. Trial was underpowered to detect adverse events or to compare the 2 treatment groups.
The study number was small
Adult study
Small sample size. Study can only be extrapolated to similar set up.
4. Metz Boutigue MH, Jolles J, Mazurier J, et al. Human lactotransferrin: amino acid sequence and structural comparisons with other transferrins. Eur J Biochem 1984;145:659–76. 5. Masson PL, Heremans JF. Lactoferrin in milk from different species. Comp Biochem Physiol 1971;39B:119–29. 6. Lonnerdal B. Nutritional and physiologic significance of human milk proteins. Am J Clin Nutr 2003;77:1537–43. 7. Hirai Y, Kawakata N, Satoh K, et al. Concentrations of lactoferrin and iron in human milk at different stages of lactation. J Nutr Sci Vitaminol 1990;36:531–44. 8. Orsi N. The antimicrobial activity of lactoferrin: current status and perspectives. Biometals 2004;17:189–96. 9. Farnaud S, Evans RW. Lactoferrin – a multifunctional protein with antimicrobial properties. Mol Immunol 2003;40:395–405.
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DOI: 10.3109/14767058.2014.968548
10. Fischer R, Debbabi H, Blais A, et al. Uptake of ingested bovine lactoferrin and its accumulation in adult mouse tissues. Int Immunopharmacol 2007;7:1387–93. 11. Bhimani RS, Vendrov Y, Furmanski P. Influence of lactoferrin feeding and injection against systemic staphylococcal infections in mice. J Appl Microbiol 1999;86:135–44. 12. Singh PK, Parsek MR, Greenberg EP, Welsh MJ. A component of innate immunity prevents bacterial biofilm development. Nature 2002;417:552–5. 13. Wakabayashi H, Yamauchi K, Kobayashi T, et al. Inhibitory effects of lactoferrin on growth and biofilm formation of Porphyromonas gingivalis and Prevotella intermedia. Antimicrob Agents Chemother 2009;53:3308–16. 14. Qiu J, Hendrixson DR, Baker EN, et al. Human milk lactoferrin inactivates two putative colonization factors expressed by Haemophilus influenzae. Proc Natl Acad Sci USA 1998;95: 12641–6. 15. Nascimento de Arau´jo A, Giugliano LG. Human milk fractions inhibit the adherence of diffusely adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) to HeLa cells. FEMS Microbiol Lett 2000;184:91–4. 16. Kawasaki Y, Tazume S, Shimizu K, et al. Inhibitory effects of bovine lactoferrin on the adherence of enterotoxigenic Escherichia coli to host cells. Biosci Biotechnol Biochem 2000;64:348–54. 17. Gifford JL, Hunter HN, Vogel HJ. Lactoferricin: a lactoferrinderived peptide with antimicrobial, antiviral, antitumor and immunological properties. Cell Mol Life Sci 2005;62:2588–98. 18. Lupetti A, Brouwer CPJM, Bogaards SJP, et al. Human lactoferrinderived peptide’s antifungal activities against disseminated Candida albicans infection. J Infect Dis 2007;196:1416–24. 19. Di Mario F, Aragona G, Dal Bo` N, et al. Bovine lactoferrin for Helicobacter pylori eradication: an open, randomized, multicentre study. Aliment Pharmacol Ther 2006;23:1235–40. 20. Laffan AM, McKenzie R, Forti J, et al. Lactoferrin for the prevention of post-antbiotic diarrhoea. J Health Popul Nutr 2011; 29:547–51. 21. Gomez HF, Ochoa TJ, Herrera-Insua I, et al. Lactoferrin protects rabbits from Shigella flexneri –induced inflammatory enteritis. Infect Immun 2002;70:7050–3. 22. Tanaka K, Ikeda M, Nozaki A, et al. Lactoferrin inhibits hepatitis C virus viremia in patients with chronic hepatitis C: a pilot study. Jpn J Cancer Res 1999;90:367–71. 23. Puddu P, Borghi P, Gessati S, et al. Antiviral effect of bovine lactoferrin saturated with metal ions on early steps of human immunodeficiency virus type 1 infection. Int J Biochem Cell Biol 1998;30:1055–62. 24. Zuccotti GV, Salvini F, Riva E, Agostoni C. Oral lactoferrin in HIV-1 vertically infected children: an observational follow-up of plasma viral load and immune parameters. J Int Med Res 2006;34: 88–94. 25. de la Rosa G, Yang D, Tewary P, et al. Lactoferrin acts as an alarmin to promote the recruitment and activation of APCs and antigen-specific immune responses. J Immunol 2008;180:6868–76. 26. Sherman MP, Adamkin DH, Radmacher PG, et al. Protective proteins in mammalian milks: lactoferrin steps forward. Neoreviews 2012;13:e293–300.
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27. Zaghouani H, Hoeman CM, Adkins B. Neonatal immunity: faulty T-helpers and the shortcomings of dendritic cells. Trends Immunol 2009;30:585–91. 28. Wolf JS, Li G, Varadhachary A, et al. Oral lactoferrin results in t cell-dependent tumor inhibition of head and neck squamous cell carcinoma in vivo. Clin Cancer Res 2007;13:1601–10. 29. Naot D, Grey A, Reid IR, Cornish J. Lactoferrin – a novel bone growth factor. Clin Med Res 2005;3:93–101. 30. Edde L, Hipolito RB, Hwang FF, et al. Lactoferrin protects neonatal rats from gut related systemic infection. Am J Physiol Gastrointest Liver Physiol 2001;281:1140–50. 31. Sherman MP, Bennett SH, Hwang FF, et al. Neonatal small bowel epithelia: enhancing anti-bacterial defense with lactoferrin and Lactobacillus GG. Biometals 2004;17:285–9. 32. Manzoni P, Rinaldi M, Cattani S, et al. Bovine lactoferrin supplementation for prevention of late onset sepsis in very lowbirth-weight neonates: a randomized trial. JAMA 2009;302: 1421–8. 33. Ochoa TJ, Cam L, Lianos R, et al. Lactoferrin for prevention of sepsis in Peruvian neonates. Abstract from the Proceedings of Pediatric Academic Societies Meeting in Boston, MA, USA, 2012. Pediatric Academic Societies web site, 2012 Abstracts2View, E-PAS2012:2170.7. 34. Kaufman D, Fairchild KD. Clinical microbiology of bacterial and fungal sepsis in very-low-birth-weight infants. Clin Microbiol Rev 2004;17:638–80. 35. Stoll BJ, Hansen N, Fanaroff AA, et al. Late-onset sepsis in very low birth weight neonates: the experience of the NICHD Neonatal Research Network. Pediatrics 2002;110:285–91. 36. Stoll BJ, Hansen NI, Adams-Chapman I, et al. Neurodevelopmental and growth impairment among extremely low-birth weight infants with neonatal infection. JAMA 2004;292:2357–65. 37. Guntupalli K, Dean N, Morris PE, et al. A phase 2 randomized, double-blind, placebo-controlled study of the safety and efficacy of talactoferrin in patients with severe sepsis. Crit Care Med 2013;41: 706–16. 38. Kaur G, Gathwala G. Efficacy of bovine lactoferrin supplementation in preventing late onset sepsis in low birth weight neonates: a randomised placebo controlled clinical trial. In: Dutta V, Mehendiratatta S, eds. Abstract from proceeding of XXXIII Annnual Convention of National Neonatology Forum meeting. Hyderabad, India: NNF India; 2013:3. 39. Kaufman D, Fairchild KD. Clinical microbiology of bacterial and fungal sepsis in very-low-birth-weight infants. Clin Microbiol Rev 2004;17:638–80. 40. Venkatesh MP, Ron L. Human recombinant lactoferrin acts synergistically with antimicrobials commonly used in neonatal practice against coagulase-negative Staphylococci and Candida albicans causing neonatal sepsis. J Medi Micro 2008;57:1113–21. 41. Paesano R, Pietropaoli M, Berlutti F, Valenti P. Bovine lactoferrin in preventing preterm delivery associated with sterile inflammation. Biochem Cell Biol 2012;90:468–75. 42. Pammi M, Abrams SA. Oral lactoferrin for the prevention of sepsis and necrotizing enterocolitis in preterm infants. Cochrane Database Syst Rev 2011;5:CD007137. 43. Pammi M, Abrams SA. Oral lactoferrin for the treatment of sepsis and necrotizing enterocolitis in neonates. Cochrane Database Syst Rev 2011;5:CD007138.
REVIEW ARTICLE
Use of lactoferrin in the newborn: where do we stand? J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Oregon Health Sciences University on 03/27/15 For personal use only.
Deepak Sharma1, Anuradha Murki2, Srinivas Murki1, and Oleti Tejo Pratap1 1
Department of Neonatology, Fernandez Hospital, Hyderguda, Hyderabad, Andhra Pradesh, India and 2Department of Obstetrics and Gynaecology, Kamineni Hospital, Hyderabad, Andhra Pradesh, India Abstract
Keywords
Sepsis and necrotizing enterocolitis (NEC) cause significant morbidity and mortality in the newborn. Their ill effects persist in spite of appropriate and effective antibiotic therapy. Lactoferrin as an adjunct to antibiotics in the treatment of sepsis or NEC in the newborn may improve the clinical outcomes by enhancing the host defense and modulating the inflammatory response. This review focuses on the various aspects of lactoferrin use in the newborn.
Anti-bacterial, anti-fungal, anti-viral, fungal sepsis, immunomodulation, lactoferrin, necrotizing enterocolitis, neonatal sepsis
Introduction The term ‘‘Lactoferrin’’ (LF) is derived from its past classification as a major iron-binding protein in milk. LF, also referred to as lacto-transferrin, was first identified in 1939 in bovine milk [1] and it was isolated from human milk in 1960 [2]. LF is produced mainly from the neutrophils [3] but other cells also contribute to its levels in milk. The complete amino acid sequences of human LF contain 703amino acid residues [4]. LF is present in human plasma in relatively low concentrations, but high levels are observed in colostrum, human breast milk, and seminal plasma. Markedly higher levels occur in cord blood, tears, and vaginal mucus. Masson et al, when studying ten different mammalian species, reported the highest levels of LF in human breast milk [5]. In human milk, its concentration peaks in the colostrum (7 mg/ mL) and then decrease (1 mg/mL) in mature milk, the rate of reduction being slower in the milk of mothers of premature neonates [6,7]. Bovine lactoferrin (BLF) and human lactoferrin (HLF) share a high degree (77%) of amino-acid homology, and also have the same N-terminal peptide, called lactoferricin. Both BLF and HLF resist proteolysis through the infant’s digestive tract, bind to specific receptors on enterocytes, and may be found intact in stools that are poorly absorbed in the gut [8,9]. LF receptors have been identified in the gastrointestinal tract, on leukocytes and macrophages, platelets, and on bacteria. BLF is transferred from the intestine into
Address for correspondence: Dr Srinivas Murki, Consultant Fernandez Hospital, Hyderguda, Hyderabad 500029, Andhra Pradesh, India. E-mail: [email protected]
History Received 27 January 2014 Revised 19 September 2014 Accepted 19 September 2014 Published online 8 October 2014
peripheral blood in a form with intact molecular weight (80 kDa] and localized within 10 to 20 min after oral administration in the liver, kidneys, gall bladder, spleen, and brain [10]. 54LF has been known to have antimicrobial properties, immunomodulatory and trophic activities on the developing gut. LF has broad spectrum antimicrobial activity with bacteriostatic activity against Escherichia coli, Klebsiella pneumoniae, Streptococcus mutans and Candida albicans [11]. LF antimicrobial activity acts by several mechanisms.
Antimicrobial and anti-fungal action LF has direct antimicrobial action against all types of bacteria, fungi, viruses, and parasites. LF acts by prevention of biofilm formation by Pseudomonas aeruginosa [12] and oral pathogens [13] and proteolysis of virulence factors of Haemophilus influenzae [14]. LF blocks bacterial adhesion to host cells by binding to glycosaminoglycans on bacteria and/or host cell membranes [15,16]. Prophylactic LF fed to neonatal rats before they are enterally infected with E coli initiates a mechanism called anoikis wherein cells containing viable bacteria develop apoptosis [17]. Human lactoferrin-derived peptide, HLF [1–11], exerts potent in-vitro candidacidal activity, in order to display antifungal activity against disseminated Candida albicans infections [18]. BLF has been proved as an effective adjuvant to 7-day triple therapy for eradication of Helicobacter pylori infection [19].
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BLF has also been seen useful in antibiotic associated diarrhea caused by Clostridium difficile [20] and enteritis caused by Shigella Flexnieria [21] in adults. LF has bifidogenic activity, enhancing the growth of the normal commensal bifidogenic microflora in the gut.
Anti-viral properties
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LF is found to decrease serum alanine transaminase and Hepatitis C Virus (HCV) RNA concentrations in patients with chronic hepatitis C [22]. LF has anti-viral activity against Human Immunodeficiency Virus (HIV] virus [23] and is shown to increase the percentage of CD4+ cells count [24].
Immunomodulation properties Talactoferrin (TLF) which is human recombinant LF acts on alarmin receptors and promotes activation and recruitment of antigen presenting cells [25]. TLF increases the ability of dendritic cells to trigger proliferation and release of interferon-gamma. It is proposed that activated dendritic cells promote secretion of interleukin (IL)-12 [26] which in turn inhibits production of IL-4. This protects the T helper cell Th1 cells from apoptosis mediated by IL-4 and helps to overcome the neonatal bias for Th2 cells over Th1 cells [27].
Immature gut growth LF promotes growth and differentiation of immature gut epithelium. The higher the LF levels the better the effects. LF is found in high concentrations in preterm human milk.
Anticancer properties LF can inhibit the growth of head and neck squamous cell carcinoma via direct cellular inhibition as well as systemically via immune modulation [28].
Bone growth properties LF has also been reported as a novel bone growth factor. At physiological concentrations, LF potently stimulates the proliferation and differentiation of primary osteoblasts and also acts as a survival factor inhibiting apoptosis induced by serum withdrawal [29].
Clinical uses of Lactoferrin Prevention of necrotizing enterocolitis (NEC). In the preclinical trial, feeding human recombinant LF to neonatal rats before an intestinal infection with Escherichia coli (E. coli) significantly reduced translocation, bacteremia, and death [30]. In another preclinical trial feeding recombinant human LF and Lactobacillus rhamnosus GG (LGG) had more effect than feeding LGG alone in reducing gut-related translocation after an enteral infection with E. coli; recombinant HLF enhanced intestinal colonization with LGG [31]. This research was the basis for the first clinical trial of LF in preterm infants. Manzoni et al. in their prospective, multicenter, double-blind, placebo controlled randomized trial found out that oral LF alone did not reduce the incidence of NEC (1.9% versus 6%, p ¼ 0.09, but a significant reduction in
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NEC was noted with the combination of LF with LGG (n ¼ 0/151 versus n ¼ 10/168, p ¼ 0.002). This study was not powered for detection of a difference in NEC in the intervention and the placebo groups [32]. In a Peruvian study that reported its results at the 2012 Pediatric Academic Society meeting, 190 infants weighing less than 2500 g at birth were randomized to BLF or maltodextrin, 200 mg/d in 3 divided doses over the first 4 weeks of age. Although the incidence of sepsis and NEC was lower in the BLF group the study was powered to detect the intended results of decrease in sepsis and NEC [33]. One clinical trial is underway in Ankara University, Turkey ‘‘Oral Lactoferrin Prophylaxis to Prevent Sepsis and Necrotizing Enterocolitis of Very Low Birth Weight Neonates in Neonatal Intensive Care Unit and Effect on T-regulatory Cells’’ (grant NCT01287507). The primary outcome is late onset sepsis (LOS), i.e. sepsis occurring ‘‘more than 72 hours after birth’’ with isolation of any pathogen from blood or from peritoneal or cerebrospinal fluid and NEC. This study is in the phase of recruiting the patients. Multi-center studies of LF to prevent NEC are needed and those investigations should be sufficiently powered to demonstrate effectiveness in preterm infants weighing less than 1000 g and 1000–1500 g at birth. Lactoferrin and sepsis LOS is an increasing concern in neonatal intensive care units (NICUs) worldwide, given the associated high morbidity and related mortality. Infections are frequently related to severe late neuro-developmental impairment and are the primary cause of death in pre-term infants and a major risk of poor outcomes [34–36]. Manzoni et al. conducted the study ‘‘Bovine Lactoferrin Supplementation for Prevention of Late-Onset Sepsis in Very Low-Birth-Weight Neonates’’. The primary outcome measured was first episode of LOS. In their study, infants were randomly assigned to receive orally administered BLF (100 mg/d) alone (n ¼ 153), BLF plus Lactobacillus rhamnosus GG (LGG) (6 109 colony-forming units/d) (n ¼ 151), or placebo (n ¼ 168) from birth until day 30 of life (day 45 for neonates 51000 g at birth). Overall LOS occurred less frequently in the BLF and BLF plus LGG group than in the control group (RR 0.34, 95% CI: 0.17–0.70, p ¼ 0.002 for BLF versus control; RR 0.27, 95% CI: 0.12– 0.60, p50.001 for BLF plus LGG versus control). When stratification for birth weight was done, the decrease in LOS was significant in extremely low birth-weight (ELBW) neonates (RR 0.31, 95% CI: 0.14–0.70; p ¼ 0.002 for BLF versus control and RR 0.30, 95% CI: 0.13–0.69; p ¼ 0.002 for BLF plus LGG versus control), whereas no significant difference was seen in neonates weighing 1001 to 1500 g [32]. Death from sepsis was significantly lower in very low birth-weight (VLBW) infants when supplemented with BLF. In a recent study by Kaur et al. 121 low birth weight infants with birth weight 52000 grams were randomized in the first 12 hours of life to either BLF or placebo. BLF was supplemented until day 28 of life. The primary outcome was LOS. In their study there was a significant reduction in the incidence of LOS in the BLF group than in the placebo group [n ¼ 2/59 versus n ¼ 9/62, p ¼ 0.033). There was a trend towards lower sepsis attributable mortality in the BLF
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DOI: 10.3109/14767058.2014.968548
supplemented group (n ¼ 0/59 versus 5/62, p ¼ 0.058) [38]. The Peruvian study also reported reduction in the cumulative incidence of sepsis in the BLF group as compared with placebo group [33]. Gunutupalli et al. studied the role of Talactoferrin alpha, a recombinant form of HLF, in 194 adult patients within the first 24 hours of onset of severe sepsis. They reported a significant reduction in the all cause mortality at day 28 of hospital admission; 26.9% in the placebo group and 14.4% in the talactoferrin group (p ¼ 0.052) representing 12.5% absolute and 46.5% relative reduction in mortality. Reduction in all-cause mortality was sustained at 6 months (p ¼ 0.039). No adverse effect was noted of drug intake [37]. Ongoing clinical trials of LF in children include (1) LF for prevention of neonatal sepsis (grant NCT01264536), (2) supplementation with LF in preterm newborns (grant NCT01172236), (3) study of TLF oral solution for nosocomial infection in preterm infants (grant NCT00854633), (4) study of biotene oral Balance gel for oral care in critically-ill mechanically ventilated neonates (grant NCT01314742). Oral LF prophylaxis reduces the incidence of LOS in infants weighing less than 1500 g and most effective in infants weighing less than 1000 g. Further studies confirming these findings are warranted in order to establish the role of BLF in the armamentarium of preventative strategies for sepsis. Lactoferrin and fungal sepsis Preterm infants in NICUs are at great risk for fungal sepsis due to a number of specific risk factors, such as immature immunity, prolonged need for intensive care, and disorders in gut micro ecology with proliferation of saprophytes and pathogens including Candida spp. Invasive fungal infections (IFIs) in preterm infants are caused mainly by the various Candida spp, and are responsible for considerable short and long-term morbidity and high attributable mortality [39]. In the study by Manzoni et al. fungal colonization rates were similar in the treatment and control groups. However, IFI occurred less frequently in the treatment groups (p ¼ 0.004 in BLF and p ¼ 0.07 in BLF plus LGG). The rate of progression from colonization to IFI was significantly reduced in BLF group (p ¼ 0.005) in comparison with controls [32]. Kaur et al. in their study reported decreasing trend in incidence of fungal sepsis in intervention group in comparison to control group [38]. In an in-vitro experiment, Venketesh et al. evaluated the synergic effect of talactoferrin with vancomycin (VAN) and naficillin (NAF) on coagulase negative Staphylococcus aureus (CoNS) and with amphotericin (AMB) or fluconazole (FLU) on Candida albicans. It was found that TLF acted synergistically with NAF and VAN against CoNS, and with AMB and FLC against C. albicans, at multiple dose effects and drug-dose ratios. They concluded that it is a promising agent and should be further evaluated [40]. The role of lactoferrin in prevention of fungal sepsis has to be evaluated further by more randomized controlled trials (RCTs). Lactoferrin and threshold Retinopathy of prematurity Manzoni et al. found decreasing trends in incidence of threshold Retinopathy of prematurity (ROP) requiring surgery
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in BLF group and in BLF plus LGG group as compared to control group. Further trials are needed to prove the beneficial effect of LF on this issue [32]. Lactoferrin and Bronchopulmonary dysplasia Manzoni et al. found no statistical difference in Bronchopulmonary dysplasia (BPD) in both BLF and BLF with LGG group after oral lactoferrin supplementation though there was decrease in incidence in intervention group [32]. Further research is needed to support this hypothesis. Lactoferrin and prevention of preterm delivery LF decreases the level of mediators which are involved in preterm delivery like IL-6 in both serum and cervico-vaginal fluids, prostaglandin F2a in cervico-vaginal and suppresses uterine contractility preventing preterm delivery. Paesano et al. in a cohort of 161 pregnant women with iron deficiency and iron deficient anemia evaluated the role of oral BLF in improving the hematological parameters and the role of oral with intravaginal BLF in a sub-cohort at threat of preterm delivery (PTD). In their study, BLF significantly improved the hematological parameters of anemia, iron deficiency and in the sub-cohort BLF resulted in better cervical length, lower values of IL-6 and PGF2a; both markers of preterm delivery. None of the women in the sub-cohort delivered before 37 weeks of gestation. No side effects were noted in the study. All the newborns exposed to BLF in the antenatal period had normal birth weights and good Apgar scores. These results provide strong evidence for a role of BLF in PTD treatment, thus extending the therapeutic potential of this multifunctional natural protein [41]. There are currently no studies showing effect of LF on neurological outcome at two years of age or more, periventricular leucomalacia, and duration of assisted ventilation through an endotracheal tube or length of hospital stay.
Future and conclusion A review published by Cochrane advised for further studies for evaluating the effect of LF on NEC and sepsis [42,43]. Future studies should answer the questions which are unanswered until now. As the primary outcome of most neonatal clinical trials is long-term neurodevelopmental outcomes, the impact of BLF on subsequent neurodevelopment and growth need to be studied. Infants in resource poor areas of the developing world have a higher frequency of infections and higher case fatality rate, role of LF need to be studied, as well as, the impact of LF use in the larger late preterm infant population. While the overall neonatal mortality rate is low for late preterm infants, infections among these infants increase the risk of complications, prolonged hospital stay, and increased mortality. Lastly, there is a need to understand the optimal dose and duration of LF supplementation and include infants in resource poor areas of the developing world which have higher frequencies of infections and higher case fatality rates. Ongoing clinical trials will hopefully be able to give answers to many of these unanswered questions Table 1.
4
D. Sharma et al.
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Table 1. Showing various lactoferrin studies done. Study Population
Intervention group
Control group
[32]
472 very low birth weight (VLBW) neonates
[33]
190 low birth weight (LBW) neonates
[37]
194 adults within 24 h of the onset of severe sepsis
[38]
121 low birth weight (52000 grams) neonates
First episode of late onset Control group received Infants were randomly sepsis was taken as placebo (2 mL of a assigned to receive outcome. There was 5% glucose solution) orally administered significant reduction of Bovine Lactoferrin incidence of Late onset (BLF) (100 mg/d) sepsis (LOS) in both alone (n ¼ 153), BLF the intervention group plus Lactobacillus in compare to control rhamnosus GG (LGG) group. Both fungal and (6 109 colony-forming bacterial sepsis was units/d) (n ¼ 151), or decreased. placebo (n ¼ 168) from birth until day 30 of life (day 45 for neonates 51000 g at birth). There was reduction in BLF was given enterally at Control group received incidence of neonatal placebo (maltodextrin) 200 mg/d in 3 divided sepsis and NEC in interenterally at 200 mg/d in doses over the first 4 vention group though the 3 divided doses over the weeks of life. difference was not first 4 weeks of life significant. Enterally administered pla- Intention-to-treat analysis Enterally administered was used to assess 28-day cebo every 8 h for up to talactoferrin 1.5 g every all-cause mortality. 28 days or until discharge 8 h for up to 28 days or Enteral administration from the ICU until discharge from the of talactoferrin reduced Intensive care unit (ICU). 28-day all-cause mortality in patients with severe sepsis. Primary outcome measure Bovine Lactoferrin [BLF] Control group (n ¼ 62) were incidence of culture received placebo daily (n ¼ 59) was suppleproven gram positive, from first to 28th day mented daily from first to gram negative and of life. 28th day of life. Candida sepsis at any day, after 72 hours of life. Secondary outcome measure were sepsis attributable mortality after 72 hours of life. They found that incidence of first episode of culture proven LOS was significantly lower in the BLF group than in the placebo group [2/59 (3.4%) versus 9/62 (14.5%); p ¼ 0.033]. The sepsis attributable mortality after 72 hours of life was comparable among the BLF supplemented and placebo group [0/59 (0) versus 5/62 (8.1%); p ¼ 0.058]
Declaration of interest There is no conflict of interest and there was no funding involved.
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Limitations Generalizability was a potential limitation. Trial was underpowered to detect adverse events or to compare the 2 treatment groups.
The study number was small
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Small sample size. Study can only be extrapolated to similar set up.
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