The conserved TFLK motif of mammary-associated serum amyloid A3 is responsible for up-regulation of intestinal MUC3 mucin expression in vitro

In various mammalian species, an isoform of serum amyloid A is secreted at high concentrations into colostrum. A conserved four-amino-acid motif (TFLK) is contained within the first eight N-terminal amino acid residues of this mammary-associated serum amyloid A isoform 3 (M-SAA3). Peptides derived from the bovine N-terminal amino acid sequence of M-SAA3 were produced and added to cell culture medium of HT29 cells to study the effects on intestinal mucin gene expression. HT29 cells were grown to enhance expression of either MUC2 or MUC3 intestinal mucins. After incubation, total RNA was isolated for Northern blot analyses using MUC2 or MUC3 mucin cDNA probes. Signals were detected by autoradiography with mRNA levels expressed relative to 28S rRNA. The 10-mer peptides containing the intact TFLK-motif or a TFLK 4-mer peptide increased MUC3 mRNA expression compared with control cells (p < 0.05). There was no effect of these peptides on MUC2 mRNA expression. Cells that were incubated with 10-mer N-terminal derived peptides containing a scrambled TFLK motif, with all 10 amino acid residues scrambled or derived from a C-terminal region of M-SAA3, did not show increased MUC3 expression. Inhibition of enteropathogenic Escherichia coli strain E2348/69 adhesion to HT29 cells grown to enhance MUC3 expression was reduced by a similar amount when either peptides containing the intact TFLK motif or probiotic microbes were added to cell culture medium compared with control cells. M-SAA3 is a bioactive peptide secreted into colostrums that can up-regulate mucin expression and thereby may enhance innate protective mechanisms that limit access of deleterious microbes to intestinal mucosal epithelial cells in the postparturition period.     

Expression and secretion of cathelicidin antimicrobial peptides in murine mammary glands and human milk

Mammalian milk possesses inherent antimicrobial properties that have been attributed to several diverse molecules. Recently, antimicrobial peptides that belong to the cathelicidin gene family have been found to be important to the mammalian immune response. This antimicrobial is expressed in several tissues and increased in neonatal skin, possibly to compensate for an immature adaptive immune response. We hypothesized that the mammary gland could produce and secrete cathelicidin onto the epithelial surface and into milk. Human cathelicidin hCAP18/LL-37 mRNA was detected in human milk cells by PCR. Quantitative real-time PCR demonstrated an increase in relative expression levels at 30 and 60 d after parturition. Immunohistochemistry of mouse breast tissue identified the murine cathelicidin-related antimicrobial peptide in lobuloacinar and ductules. Western blot analysis of human milk showed that LL-37 was secreted and present in the mature peptide form. The antimicrobial activity of LL-37 against Staphylococcus aureus, group A Streptococcus, and enteroinvasive Escherichia coli O29 in the human milk ionic environment was confirmed by solution colony-forming assay using synthetic peptide. These results indicate that cathelicidin is secreted in mammary gland and human milk, has antimicrobial activity against both Gram-positive and Gram-negative bacteria, and can contribute to the anti-infectious properties of milk.     

Expression,characterization, and biologic activity of recombinant human lactoferrin in rice

BACKGROUND: Lactoferrin has been suggested to have many biologic activities, such as facilitating iron absorption and having antimicrobial and antiinflammatory effects. In humans, several of these activities are likely to only be facilitated by human lactoferrin because they depend on the binding of human lactoferrin to specific receptors. Rice may be a useful vehicle to introduce recombinant human lactoferrin to infant foods because it has low allergenicity and is likely to be safer than using microorganisms or transgenic animals. METHODS: Recombinant human lactoferrin was expressed in the rice cell culture system, and its biologic activity was assessed by iron-binding and -releasing properties, antimicrobial activity, and binding and uptake to Caco-2 cells. The authors also compared the stability of recombinant and native human lactoferrins against heat, low pH, and in vitro digestion. RESULTS: Biologic activity of rice-expressed recombinant human lactoferrin was similar to that of native human lactoferrin. Heat-treated proteins retained their functional activities except with severe treatment at 100 degrees C for 8 seconds, which disturbed the iron-binding capacity of recombinant human lactoferrin. Both types of proteins retained their functional activities between pH 2 and 7.4. After in vitro digestion, 50% of both proteins were detectable by enzyme linked immunosorbent assay. The remaining native and recombinant lactoferrins retained antimicrobial and Caco-2 binding and uptake activities. CONCLUSIONS: The results indicate recombinant human lactoferrin has stability similar to native human lactoferrin when exposed to thermal treatment, pH treatment, and in vitro digestion, suggesting it may be active when added to infant formula.     

Antimicrobial polypeptides of human vernix caseosa and amniotic fluid: implications for newborn innate defense

Antimicrobial peptides/proteins are widespread in nature and play a critical role in host defense. To investigate whether these components contribute to surface protection of newborns at birth, we have characterized antimicrobial polypeptides in vernix caseosa (vernix) and amniotic fluid (AF). Concentrated peptide/protein extracts were obtained from 11 samples of vernix and six samples of AF and analyzed for antimicrobial activity using an inhibition zone assay. Proteins/peptides in all vernix extracts exhibited strong antibacterial activity against Bacillus megaterium (strain Bm11), in addition to antifungal activity against Candida albicans, whereas AF-derived proteins/peptides showed only the former activity. Fractions obtained after separation by reverse-phase HPLC exhibited antibacterial activity, with the most pronounced activity in a fraction containing alpha-defensins (HNP1-3). The presence of HNP1-3 was proved by dot blot analysis and confirmed by mass spectrometry. Lysozyme and ubiquitin were identified by sequence analysis in two fractions with antibacterial activity. Fractions of vernix and AF were also positive for LL-37 with dot blot and Western blot analyses, and one fraction apparently contained an extended form of LL-37. Interestingly, psoriasin, a calcium-binding protein that is up-regulated in psoriatic skin and was found recently to exhibit antimicrobial activity, was characterized in the vernix extract. The presence of all of these antimicrobial polypeptides in vernix suggests that they are important for surface defense and may have an active biologic role against microbial invasion at birth.     

Defined formula diets alter characteristics of the intestinal transport of amino acid and peptide in growing rats

Recently defined formula diets are widely used for patients with digestive diseases. Long-term administration of such diets is presumed to change the absorptive characteristics of the small intestine. We investigated the influence of the defined formula diet on the absorptive capacity of growing rats by measuring the potential difference of sugars, amino acid, half-maximum concentration (kt) of these substrates, the activities of disaccharidase and dipeptidase, and the portal amino acid concentrations. There was no significant difference in the body weight of rats fed amino acid or peptide diets and those given the normal chow, but the administration of the defined formula diets reduced the absorption of amino acid and small peptide per serosal area and kt. On the other hand, absorption of sugars was not significantly influenced by the type of the diets. The differences in the absorptions of amino acid and peptide following the administration of the defined formula diet might be associated with the change in the resistance of the unstirred water layer or the alteration in the active transport system of amino acids or peptides in the small intestine. No significant differences were observed between the influences of the amino acid and the peptide diets.     

The response of bovine beta‐lactoglobulin‐specific T‐cell clones to single amino acid substitution of T‐cell core epitope

Cow’s milk is one of the most common food allergens in the first year of life, with approximately 2.5% of infants experiencing an allergic reaction to it. Beta‐lactoglobulin (BLG) is one of the major allergens in cow’s milk. Previously, we reported that four of six T‐cell clones (TCC) which were established from cow’s milk allergy patients recognized BLGp97‐117 as the core sequence and also recognized BLG in association with the human leucocyte antigen (HLA)‐DRB1*0405 allele. Using two of these four TCCs, we evaluated the T‐cell response to BLG peptides with single amino acid substitution or deletion and identified BLGp102‐112 as the minimum essential region in BLGp97‐117. In the alanine‐scan assay, the proliferative responses of TCCs to pE108A disappeared, and the proliferative responses of TCCs to pC106A decreased. In the analog peptide proliferation assay, pY102S had retained some T‐cell response to the two TCCs. Collecting these results, we propose a motif for the interaction between the HLA‐DRB1*0405 allele and antigen peptide, and suggest that BLGp105‐108 are important residues to retain the TCR/BLG‐peptide/HLA complex. pY102A and pY102S are partial agonists for the T‐cell receptor. These peptides might be considered as candidate peptides for the modification of the T‐cell response to BLG in cow’s milk allergy.     

Probiotics in neonatology

Probiotics are micro-organisms that confer health benefits on the host. Postulated mechanisms include: increasing resistance of the mucosal barrier to migration of bacteria and their toxins by strengthening intestinal cell junctions, modification of host response to microbial products, augmentation of immunoglobulin A mucosal responses, enhancement of enteral nutrition to inhibit the growth of pathogens; production of antimicrobial proteins; and competitive exclusion of potential pathogens. Published meta-analyses and systematic reviews report the effects of probiotics on important clinical outcomes in neonates. This paper will review the evidence for probiotic supplementation in neonatology, with a focus on preterm infants.     

Surveillance cultures in pediatric allogeneic hematopoietic stem cell transplantation

The value of surveillance cultures in predicting systemic infections and in guiding antimicrobial treatment is controversial. We investigated 57 pediatric allo‐SCTs between 2007 and 2009. ALL (34), AML (5), and severe aplastic anemia (4) were the largest patient groups. Conditioning was TBI‐based in 87% and 54% developed GVHD (21% grade III‐IV). Of the 2594 weekly colonization samples, 24% were positive (fecal bacteria 86%, fecal fungi 16%, Clostridium difficile 16%; throat bacteria 17% and throat fungi 4%). Enterobacteria and enterococci were the most common fecal findings, staphylococci and streptococci in the throat. Of the bacterial stool samples pretransplant, 74% (mostly enterococci) were resistant to our first‐line antibiotics (ceftazidime and cloxacillin). Candida species accounted for the majority of the fungal findings: 62% of the fecal and 78% in the throat. A total of 170 clinical infection episodes were recorded, and in 12 of these, the bacterial blood culture was positive. In 4/12 cases, the pathogen was detected in surveillance culture previously, leading to sensitivity and specificity of 33.3 and 47.4%, respectively. Positive predictive value of bacterial surveillance cultures was 0.9%. The antimicrobial treatment was changed in only five cases based on the surveillance culture results. Weekly surveillance cultures seldom provided clinical benefit and were not cost‐effective.     

Plasma concentrations of defensins and lactoferrin in children with severe sepsis.

BACKGROUND: We hypothesized that systemic release of endogenous leukocyte-derived polypeptide antimicrobial defensins (polymorphonuclear leukocyte-specific) and lactoferrin (polymorphonuclear leukocyte and epithelial cell derived) occurs in nonneutropenic children with severe sepsis. METHODS: We performed a prospective cross-sectional and longitudinal study in a university children's hospital pediatric intensive care unit. Ninety-two consecutive children meeting criteria for sepsis and 14 critically ill children without sepsis (controls) were enrolled, and plasma defensins and lactoferrin concentrations were measured on Days 1 and 3 of sepsis. RESULTS: Nonneutropenic sepsis patients (n = 71) had increased defensins and lactoferrin plasma concentrations compared with critically ill control patients [defensins, 450 ng/ml vs. 150 ng/ml; lactoferrin, 332 ng/ml vs. 176 ng/ml (median values); P < 0.05] and neutropenic sepsis patients [n = 21; defensins, 450 ng/ml vs. 50 ng/ml; lactoferrin, 332 ng/ml vs. 20 ng/ml (median values); P < 0.05]. Neutropenic sepsis patients had similar plasma defensin concentrations and a decrease in plasma lactoferrin concentrations compared with control patients (P < 0.05). Defensins and lactoferrin plasma concentrations correlated to total white blood cell and absolute neutrophil count (P < 0.05). There was no association between plasma defensin concentration and organ failure or outcome; however, increased plasma lactoferrin concentrations were observed with the development of organ failure (P < 0.05). CONCLUSION: These data suggest that increased circulating defensins and lactoferrin release are dependent in part on neutrophil count and might play a role in host defense in children with severe sepsis.     

Isolation and characterization of rhesus monkey milk lactoferrin

Rhesus monkey milk lactoferrin was isolated and its characteristics compared with those of human milk lactoferrin in order to assess the feasibility of using the rhesus monkey as an animal model for the study of iron absorption from milk. Monkey lactoferrin was isolated from pooled monkey milk by two chromatographic steps. Concentration of lactoferrin in milk, determined by rocket immunoelectrophoresis, demonstrated similar concentrations in both human and monkey milk, 1-2 mg/ml. Immunodiffusion of lactoferrins from several species using an antibody raised to money lactoferrin resulted in a cross-reaction only with monkey and human lactoferrin. Lactoferrins from cow, sheep, goat, dog, and rat milk were not recognized by the antibody. Amino acid analysis of monkey lactoferrin showed a composition very similar to human lactoferrin, as well as a similarity in the unusual amino acid sequence at the N-terminal of the protein. The carbohydrate moiety of monkey lactoferrin was investigated and shown to contain monosaccharides in similar proportions to those reported for human lactoferrin. In our opinion, the rhesus monkey is a promising model for the study of the role of lactoferrin in iron absorption in the infant, as well as of the other proposed actions of lactoferrin.     

Epithelial antimicrobial peptides and proteins: their role in host defence and inflammation

Various antimicrobial mechanisms act in concert to protect the lung from infection by forming an efficient host defence system. Most microbial challenges are counteracted by elements of the innate immune system and antimicrobial peptides and proteins have been identified as key components of innate immunity. Although phagocytes are an important cellular source of these so-called endogenous antibiotics, it is now recognized that the airway epithelium is also a major site of synthesis. Antimicrobial peptides and proteins kill a wide variety of micro-organisms. Their importance is illustrated by the observation that in cystic fibrosis changes in the airway surface fluid may result in a dysfunction of these components. Recent studies have revealed other functions of these molecules showing they may link innate and adaptive immunity and appear to be involved in the regulation of inflammation and tissue repair.     

Origin of intact lactoferrin and its DNA-binding fragments found in the urine of human milk-fed preterm infants. Evaluation by stable isotopic enrichment

The origin of intact (78-kD) lactoferrin found in the urine of human milk-fed preterm infants was investigated using human milk containing proteins enriched with [13C]leucine and [15N2]lysine or [2H4]lysine. Mothers of infants selected for the study were infused i.v. with [13C] leucine and [15N2]lysine or [2H4]lysine to label milk proteins. The labeled milk was collected from each mother, pooled, fortified with a lyophilized human milk fraction, and fed to her preterm infant by continuous orogastric infusion for a period of 48 h. Urine was collected from each infant for 96 h. Intact lactoferrin (78 kD) and DNA-binding lactoferrin fragments (51 and 39 kD) were purified from the urine by affinity chromatography on columns of immobilized single-stranded DNA-agarose. The concentration and isotopic enrichment of the intact lactoferrin and DNA-binding fragments were determined separately after their isolation by high-performance reverse-phase (phenyl) chromatography. Mass spectral analyses indicated that the isotopic enrichment of the purified urinary lactoferrin was 87 to 100% of that in the labeled human milk lactoferrin. Similar results were obtained for the isolated DNA-binding lactoferrin fragments. The ratios of isotopically labeled leucine to lysine in the purified milk lactoferrins and urinary lactoferrins were similar for each mother/infant pair. Isotopically labeled lysine, added to the milk as free amino acid, was not incorporated into the purified urinary lactoferrin. These results demonstrate that undegraded (78-kD) lactoferrin of maternal origin is absorbed by the gut and excreted intact in the urine of preterm infants; nearly all of the urinary lactoferrin was of maternal origin. The possible immunoregulatory functions of the absorbed intact, maternal lactoferrin are discussed.     

Enhanced peptide-binding capacities of small intestinal brush border membranes in celiac disease

In pathogenesis of celiac disease, the significance of prolamin peptide interactions with enterocytes is controversial. Changes in cellular metabolism induced by gliadin peptides, as well as uptake and presentation by enterocytes, are discussed. We analyzed peptide binding to enterocytic membranes as a potential key event. Binding capacities of brush border membranes isolated from small intestinal biopsies of untreated (n = 49) and treated celiac patients on a gluten-free diet (n = 30), as well as control subjects (n = 43), were measured with a dot blot chemiluminescence assay. Synthetic gliadin peptides comprising amino acid position 8-19 (G XIV) and 30-41 (G XI) of alpha-gliadins, a peptic-tryptic digest of gliadin (PT-GLI), and a synthetic zein peptide were used. Comparing treated celiac patients with controls, we observed significantly enhanced membrane-binding of PT-GLI [mean 122.4 densitometric units/microg (95% confidence interval 116.0-128.9) vs 108.9 (102.1-115.7)] and of zein peptide [50.2 (38.4-61.9) vs 28.8 (13.4-44.2)], but only slightly increased binding of the synthetic gliadin peptides G XIV [65.5 (60.6-70.5) vs 62.4 (56.3-68.5) and G XI [75.2 (69.8-80.6) vs 65.9 (55.2-76.5)]. Independent of patient group, membrane-binding capacities for celiac-active gliadin peptides exceeded those of the zein peptide. Thus, interaction of gliadin peptides with the apical enterocytic membrane was not found exclusively in celiac disease. Furthermore, increased binding capacities in treated celiac disease were not confined to celiac-active peptides. Quantitative differences in gliadin peptide binding as a primary characteristic in celiac disease might contribute to pathogenetic effects exerted on small intestinal epithelial cells.     

Current concepts on pulmonary host defense mechanisms in children

The respiratory tract is exposed continuously to noxious agents, microbial organisms, particles, and allergens. It has therefore evolved both innate and specific defense mechanisms. The innate host defense mechanisms include components such as collectins, beta-defensins, lactoferrin, and complement, all of which have an important role in modulating the immune response. Immune protection of the lungs by specific antibody is reviewed. The airways are protected by alveolar macrophages, neutrophils, and lymphocytes, and their origins, regulation, functions, and antimicrobial activity are summarized. Antimicrobial peptides and immune-modulating peptides are likely to have a significant therapeutic role for infection and inflammation in the respiratory tract.     

Inhibition of adhesion of S-fimbriated E. coli to buccal epithelial cells by human skim milk is predominantly mediated by mucins and depends on the period of lactation

Expression of S-fimbriae is frequent in Escherichia coli strains causing sepsis and meningitis in the newborn period. We analysed the ability of human skim milk to inhibit adhesion of S-fimbriated E. coli to human buccal epithelia. Adhesion was inhibited by up to 90% using colostrum (5%) and up to 50% with mature milk (5%), indicating that this anti-infective mechanism depends on the period of lactation. Elimination of up to 99% of immunoglobulins and 91% of lactoferrin by affinity chromatography had no effect on the inhibition of adhesion. After separation of high- (> 10 kD) and low-molecular-weight fractions of skim milk, only the fraction > 10 kD was found to be able to inhibit bacterial adhesion. In order to further characterize receptor molecules for bacteria, we investigated binding of isolated S-fimbriae to glycoprotein bands on Western blot strips. Fimbriae mainly bound to a high-molecular-weight band (> 200 kD). According to molecular weight and staining behaviour, this band most likely represents mucins. We conclude that carbohydrate residues on secreted mucins of human skim milk are able to inhibit bacterial adhesion to mucosal surfaces. This could provide protection against neonatal sepsis and meningitis caused by E. coli .     

Bacteremia in childhood cancer

Infection-related mortality affects the overall survival rates of children who are receiving treatment for cancer. The leading cause of mortality is bacteremia and sepsis related to it in febrile neutropenic patients. All positive blood cultures of febrile neutropenic patients treated in the Department of Pediatric Hematology-Oncology, Cerrahpasa Medical School, between January 1995 and January 2001 were reviewed. Cultures grew 159 micro-organisms, 95 (60 per cent) of which were Gram-positive bacteria, 56 (35 per cent) were Gram-negative bacteria and eight (5 per cent) were fungi. Coagulase-negative staphylococci (63, 40 per cent) and S. aureus (8, 5 per cent) were the most frequent Gram-positive pathogens. Klebsiella, E. coli, Enterobacter and Pseudomonas infections were the primary Gram-negative pathogens. Twenty cases were lost because of sepsis: in 11 cases (55 per cent) Gram-negative bacteria, in eight cases (40 per cent) Gram-positive bacteria, and in only one case a fungus were the causative organisms. Although vancomycin was not included in the first-line treatment, the mortality rate of Gram-positive bacteremia was 8 per cent. In Gram-negative bacteremia it was 20 per cent. Gram-negative pathogens, which were resistant to multiple antibiotics, caused the mortality. Drug resistance and mortality due to micro-organisms must be taken into consideration while febrile neutropenia protocols are prepared.     

Diet and faecal flora in the newborn: lactoferrin.

The faecal flora of breast fed babies differs from that of bottle fed babies. We have shown that the use of a whey predominant formula rather than a casein predominant one induced a faecal flora generally closer to that of breast fed babies but substantial differences remained. The whey proteins of breast milk include much more lactoferrin than is found in cows'' milk. Observations both in animals and in vitro suggest that lactoferrin could be responsible for some of these differences between bottle and breast fed babies. This study was designed to determine the effects on faecal flora of the addition of bovine lactoferrin to the diet of bottle fed babies while holding other qualities of their diet constant. As lactoferrin is an iron binding protein three test formulas were used: (a) no added iron and no added lactoferrin (basic), (b) no iron but added lactoferrin (L), and (c) added iron and lactoferrin (LF). The addition of lactoferrin had little effect upon the faecal microflora and did not move the pattern of the faecal flora in the direction of the breast fed baby. The addition of iron to the formula had more effect on the faecal flora than did lactoferrin. At day 4 it encouraged Escherichia coli and discouraged staphylococcal faecal colonisation. At day 14 the addition of iron to the formula discouraged bifidobacteria. The reasons why bovine lactoferrin was ineffective in vivo in this study are discussed.     

T cell epitope mapping study with insulin overlapping peptides using ELISPOT assay in Japanese children and adolescents with type 1 diabetes

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease. Insulin seems to be a critical antigen recognized by autoreactive T cells. In this study, we performed T cell epitope mapping of insulin using serial overlapping peptides in Japanese patients with T1D. Serial overlapping insulin peptides comprising 23 peptides, which were each 15-amino acid long, were prepared based on insulin sequence. Cytokine secretion from peripheral T cells against these peptides was studied by enzyme-linked immunospot (ELISPOT) assay in 18 patients with recent-onset T1D and 12 patients with established T1D, and compared with 17 healthy control subjects. In ELISPOT assay, IFN-gamma-secreting T cells, but not IL-4, against several insulin peptides were observed in 77.8% of patients with recent-onset T1D, 50.0% of patients with established T1D, and 0% of healthy control subjects. All epitopes recognized by T cells were identified in the B-chain of insulin. The most frequent epitope existed at the B10-24 region (9/18), followed by B1-15 and B11-25 regions (6/18, each), with B4-18, B9-23, and B12-26 identified in some patients. These data did not correlate with insulin autoantibodies or HLA-DRB1 of the patients. This is the first report of T cell epitope mapping using one amino acid serial overlapping peptides of insulin in T1D. ELISPOT assay revealed the frequent existence of insulin peptide-specific T cells in patients with recent-onset and established T1D. The T cell epitopes of insulin were similar but not identical in our cohort, which probably explains the difficulty encountered in prevention of human T1D by using insulin.