Genetically encoded antibiotic peptides are evolutionarily ancient and widespread effector molecules of immune defence. Mammalian defensins, one subset of such peptides, have been implicated in the antimicrobial defence capacity of phagocytic leukocytes and various epithelial cells, but direct evidence of the magnitude of their in vivo effects have not been clearly demonstrated. Paneth cells, specialized epithelia of the small intestinal crypt, secrete abundant alpha-defensins and other antimicrobial polypeptides including human defensin 5 (HD-5; also known as DEFA5). Although antibiotic activity of HD-5 has been demonstrated in vitro, functional studies of HD-5 biology have been limited by the lack of in vivo models. To study the in vivo role of HD-5, we developed a transgenic mouse model using a 2.9-kilobase HD-5 minigene containing two HD-5 exons and 1.4 kilobases of 5'-flanking sequence. Here we show that HD-5 expression in these mice is specific to Paneth cells and reflects endogenous enteric defensin gene expression. The storage and processing of transgenic HD-5 also matches that observed in humans. HD-5 transgenic mice were markedly resistant to oral challenge with virulent Salmonella typhimurium. These findings provide support for a critical in vivo role of epithelial-derived defensins in mammalian host defence.

Paneth cells represent one of the four major epithellal llneages In the mouse small Intestine. It is the only llneage that migrates downward from the stem-cell zone located in the lower portion of the crypt of Lieberkuhn to the crypt base. Mature Paneth cells release growth factors, digestive enzymes, and antimicrobial peptides from their apical secretor granules. Some of these factors may affect the crypt stem cell, its transit-cell descendants, differentiating villusassociated epithelal lages, and/or the gut microflora. We used single and multllabel immunocytochemical methods to study Paneth cell days (2) before being removed by phagocytosis (3). A variety offunctions have been attributed to Paneth cells. These functions include modulation of the intestinal microflora and maintenance of mucosal defense barriers through production of antimicrobial peptides (cryptdins, lysozyme). The location of Paneth cells at the crypt base, combined with their production of growth factors and other regulatory molecules (4-6), suggests that they may also contribute to the stem-cell niche through short-circuit paracrine loops and/or regulate the proliferation and differentiation programs of other cell lineages.One way of examining Paneth cell function is to study the differentiation program of this lineage during gut development. Morphogenesis of the mouse small intestinal epithelium is not completed until the end of the third postnatal week. A pseudostratified endoderm undergoes conversion to a monolayer overlying nascent villi in a morphologic "wave" of cytodifferentiation. This wave moves from the duodenum to the ileum from embryonic day 15 (E15) through E19. Crypts form from an intervillus epithelium during the first two postnatal weeks (7). Crypt number increases rapidly between the second and third postnatal week through crypt fission (7). Analyses of mouse aggregation chimeras indicate that the perinatal mouse gut contains a polyclonal intervillus epithelium, supplied by stem cells with multiple genotypes (8). A process of cell selection occurs during crypt morphogenesis, yielding monoclonal crypts by postnatal day 14 (P14). An adult mouse crypt appears to be supplied either by a single slowly dividing master stem cell and its more rapidly cycling transit-cell descendants (the stem-cell pedigree concept) or by several equivalent stem cells with similar cycling times and probabilities for self-maintenance (9).We have examined the Paneth cell lineage from E15 to P42 in germ-free, ex-germ-free, and conventional mice and in intestinal isografts. The results of these studies form a basis for interpreting the significance of the pattern of expression of a mouse cryptdin 2/human growth hormone (hGH) transgene in several pedigrees of mice.MATERIALS AND METHODS Animals. FVB/N mice were caged in microisolators and given autoclaved chow (Ralston Purina) ad libitum. Germfree NMRI mice (10) were maintained in gnotobiotic isolator cages. Conventional NMRI animals were housed in a nonsterile but pathogen-free environment. Ex-...

Mammalian epithelial surfaces are remarkable for their ability to provide critical physiologic functions in the face of frequent microbial challenges. The fact that these mucosal surfaces remain infection-free in the normal host suggests that highly effective mechanisms of host defense have evolved to protect these environmentally exposed tissues. Throughout the animal and plant kingdoms, endogenous genetically encoded antimicrobial peptides have been shown to be key elements in the response to epithelial compromise and microbial invasion. In mammals, a variety of such peptides have been identified, including the well-characterized defensins and cathelicidins. A major source of these host defense molecules is circulating phagocytic leukocytes. However, more recently, it has been shown that resident epithelial cells of the skin and respiratory, alimentary, and genitourinary tracts also synthesize and release antimicrobial peptides. Both in vitro and in vivo data support the hypothesis that these molecules are important contributors to intrinsic mucosal immunity. Alterations in their level of expression or biologic activity can predispose the organism to microbial infection. The regulatory and developmental aspects of antimicrobial peptide synthesis are discussed from a perspective that emphasizes the possible relevance to pediatric medicine.

Objective: Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer used in medical products made with polyvinyl chloride (PVC) plastic and may be toxic to humans. DEHP is lipophilic and binds non-covalently to PVC, allowing it to leach from these products. Medical devices containing DEHP are used extensively in neonatal intensive care units (NICUs). Among neonates in NICUs, we studied exposure to DEHP-containing medical devices in relation to urinary levels of mono(2-ethylhexyl) phthalate (MEHP), a metabolite of DEHP.Design: We used a cross-sectional design for this study.Participants: We studied 54 neonates admitted to either of two level III hospital NICUs for at least 3 days between 1 March and 30 April 2003.Measurements: A priori, we classified the infants’ exposures to DEHP based on medical products used: The low-DEHP exposure group included infants receiving primarily bottle and/or gavage feedings; the medium exposure group included infants receiving enteral feedings, intravenous hyperalimentation, and/or nasal continuous positive airway pressure; and the high exposure group included infants receiving umbilical vessel catheterization, endotracheal intubation, intravenous hyperalimentation, and indwelling gavage tube. We measured MEHP in the infants’ urine using automated solid-phase extraction/isotope dilution/high-performance liquid chromatography/ tandem mass spectrometry.Results: Urinary MEHP levels increased monotonically with DEHP exposure. For the low-, medium-, and high-DEHP exposure groups, median (interquartile range) MEHP levels were 4 (18), 28 (58), and 86 ng/mL (150), respectively (p = 0.004). After adjustment for institution and sex, urinary MEHP levels among infants in the high exposure group were 5.1 times those among infants in the low exposure group (p = 0.03).Conclusion: Intensive use of DEHP-containing medical devices in NICU infants results in higher exposure to DEHP as reflected by elevated urinary levels of MEHP.

Cryptdins are antimicrobial peptides of the defensin family that are produced by intestinal Paneth cells. mRNAs encoding 17 cryptdin isoforms have been characterized from a cDNA library generated from a single jejunal crypt. Six cryptdin cDNAs correspond to known peptides, and the remainder predict 11 novel Paneth cell defensins. Most cryptdin cDNAs have .93% nucleotide sequence identity overall, except for cryptdin 4 and 5 cDNAs, whose respective mature peptide-encoding regions are only 74 and 78% identical to that of cryptdin 1. Cryptdin cDNAs differ at a small number of nucleotide positions: frequent substitutions were found in codons 38 and 52 of the propiece and in codons 68, 73, 76, 87, and 89 of the deduced peptides; cDNA clones with changes in codons 74, 83, and 88 were found, but there were fewer of these. The antimicrobial activities of cryptdins 1 to 6 were tested against Escherichia coli ML35 in two assays. In an agar diffusion assay, the potencies of cryptdins 1 to 3, 5, and 6 were approximately equivalent to that of rabbit neutrophil defensin NP-1 but cryptdin 4 was 30 times more active than NP-1. In a bactericidal assay system, cryptdins 1 and 3 to 6 were equally active at 10 ,ug/ml but cryptdin 2 and rabbit NP-1 were not active at this concentration. Since cryptdins 2 and 3 differ only at residue 10 (Thr and Lys, respectively), this amino acid appears to function in bactericidal interaction with E. coli. The demonstration that Paneth cells express a diverse population of microbicidal defensins further implicates cryptdins in restricting colonization or invasion of small intestinal epithelium by bacteria.

No abstract

Antimicrobial peptides are part of the host defense systems of plants, insects, fish, amphibia, birds, and mammals. These small proteins were previously thought of as an evolutionarily ancient system of immune protection with little relevance to the normal function of human skin. Recent developments have found that mammalian skin expresses these gene-encoded peptide antibiotics during inflammatory events such as wound repair, contact dermatitis, and psoriasis. The presence of these peptides in the skin forms a barrier for innate host protection against microbial pathogenesis. Furthermore, antimicrobial peptides also act on animal cells by stimulating them to change behaviors such as syndecan expression, chemotaxis, and chloride secretion. The combination of effects on host cells with antimicrobial action in a single molecule represents an efficient defense and response system against injury. Understanding the action of antimicrobial peptides in skin may yield further insight into the mechanism of innate cutaneous disease control and provide new approaches to therapy of wounds and inflammatory dermatitis.

No abstract