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PubMed2026

Exploring precision risk in pediatric vesicoureteral reflux: Innate immune gene variations and reflux outcomes in the RIVUR cohort.

INTRODUCTION: Children with vesicoureteral reflux (VUR) are at increased risk for morbidity from recurrent urinary tract infections (UTIs), yet the factors influencing spontaneous VUR resolution remain poorly defined. This study evaluates whether genetic variations in key urinary innate immune effectors (DEFA1A3, DMBT1, and RNASE7) influences VUR resolution and interacts with prophylaxis to alter clinical response. METHODS: We conducted a secondary analysis of 303 RIVUR participants with available DEFA1A3 and DMBT1 copy number variation (CNV) data and RNASE7 rs1263872 genotype. Primary outcomes were (1) VUR improvement (decrease in grade) and (2) VUR resolution at study exit. Multivariable logistic regression models included genotype, treatment, and their interactions, adjusting for age, sex, baseline grade (high vs low), laterality, bowel/bladder dysfunction, and any UTI. Internal validation used 2000-sample bootstrap with bias-corrected and accelerated confidence intervals and influence diagnostics. RESULTS: Clinical covariates did not significantly predict VUR improvement. Children with DEFA1A3 CNV >5 had higher odds of improvement (OR 2.36, 95% CI 1.12-4.96, p = 0.023), an effect that remained significant in bootstrap analyses. High-grade VUR was associated with lower odds of resolution (OR 0.34, 95% CI 0.12-0.94, p = 0.038). A significant interaction was observed between prophylaxis and high DMBT1 copy number for VUR resolution (interaction OR 2.99, 95% CI 1.11-8.04, p = 0.031); no interaction was seen for improvement. RNASE7 rs1263872 was not associated with either outcome. CONCLUSION: Innate immune gene variation may contribute to heterogeneity in VUR outcomes. High DEFA1A3 copy number was associated with reflux improvement and a DMBT1-prophylaxis interaction was associated with reflux resolution. The results of this study is hypothesis-generating and prompt further evaluation to assess whether a subset of children may experience structural benefit from prophylaxis or have a more favorable natural history based on their innate immune genotype.

Humans↗

PubMed1992

NMR studies of defensin antimicrobial peptides. 1. Resonance assignment and secondary structure determination of rabbit NP-2 and human HNP-1.

Two-dimensional nuclear magnetic resonance spectroscopy has been used to make resonance assignments of the proton spectra of two defensin antimicrobial peptides, human neutrophil peptide HNP-1 and rabbit neutrophil peptide NP-2. The secondary structures of these peptides were determined from analysis of the proton-proton NOEs and from the positions of slowly exchanging amide protons. Both peptides contain a long stretch of a double-stranded antiparallel beta-sheet in a hairpin conformation that contains a beta-bulge, a short region of triple-stranded beta-sheet, and several tight turns. The NMR results clearly show that HNP-1 forms a dimer or higher order aggregate in solution and that Pro8 exists as a cis peptide bond. The NMR data on these peptides are compared with NMR data for a homologous peptide NP-5 [Bach, A. C., Selsted, M. E., & Pardi, A. (1987) Biochemistry 26, 4389-4397]. Analysis of the conformation-dependent proton chemical shifts shows that it is not possible to confidently judge the structural similarity of the three defensins from chemical shift data alone. However, comparison of the 3JHN alpha coupling constants in NP-2 and NP-5 indicates that the backbone conformations for these peptides are very similar. A more detailed comparison of the solution conformations of the defensins peptides is made in the following paper in this issue where the NMR data are used as input for distance geometry and molecular dynamics calculations to determine the three-dimensional structures of HNP-1 and NP-2.

Amino Acid Sequence↗

PubMed1992

NMR studies of defensin antimicrobial peptides. 2. Three-dimensional structures of rabbit NP-2 and human HNP-1.

The solution structure of two homologous naturally occurring antimicrobial peptides, rabbit defensin NP-2 and human defensin HNP-1, have been determined by two-dimensional nuclear magnetic resonance spectroscopy, distance geometry, and restrained molecular dynamics calculations. The structure of these defensins consists of an antiparallel beta-sheet in a hairpin conformation, a short region of triple-stranded beta-sheet, several tight turns, and a loop region that has a well-defined local structure but with a global orientation that is not well-defined with respect to the rest of the molecule. The solution structures of these two peptides are compared with the solution and crystal structures of two other homologous defensins. The structures for the defensins are also compared with known structures of other naturally occurring antimicrobial peptides.

Animals↗

PubMed1989

Solution structures of proteins from NMR data and modeling: alternative folds for neutrophil peptide 5.

The structure of neutrophil peptide 5 in solution has recently been reported (Pardi et al., 1988). The structure determination was accomplished by using a distance geometry algorithm and 107 interproton distance constraints obtained from 2D NMR data. In each of the eight independent solutions to the distance geometry equations, the overall fold of the polypeptide backbone was identical and the root mean square (rms) deviation between backbone atoms of the superimposed structures was small (approximately 2.4 A). In this paper we report additional NP-5 structures obtained by using a new structure generation algorithm: a Monte Carlo search in torsion angle space. These structures have a large rms backbone deviation from the distance geometry structures (approximately 5.0 A). The backbone topologies differ in significant respects from the distance geometry structures and from each other. Structures are found that are pseudo mirror images of part or all of the fold corresponding to that first obtained with the distance geometry procedure. For small proteins, the problem of distinguishing the correct structure among pseudo mirror images is likely to be greater than previously recognized. When a set of test distance constraints constructed from a novel Monte Carlo structure is used as input in the distance geometry algorithm, the fold of the resulting structure does not correspond to that of the target. The results also demonstrate that the previously accepted criteria (the magnitude of the rms deviation between multiple solutions of the distance geometry equations) for defining the accuracy and precision of a peptide structure generated from NMR data are inadequate. An energetic analysis of structures corresponding to the different folding topologies has been carried out. The molecular mechanics energies obtained by minimization and molecular dynamics refinement provide sufficient information to eliminate certain alternative structures. On the basis of a careful comparison of the different trial structures with the experimental data, it is concluded that the NP-5 peptide fold which was originally reported is most consistent with the data. An alternative fold corresponding to structures with low energies and small total distance violations is ruled out because for this fold predicted NOEs are not observed experimentally.

Blood Proteins↗

PubMed1996

Interactions of monomeric rabbit neutrophil defensins with bilayers: comparison with dimeric human defensin HNP-2.

Human antimicrobial neutrophil defensin HNP-2 has been shown to form large multimeric pores in pure 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG) bilayers that lead to all-or-none release of vesicle contents [Wimley et al. (1994) Protein Sci.3, 1362-1373]. Because human neutrophil defensins form natural dimers in solution, the question arises as to the role of dimerization in pore formation. However, the dimers are so stable that this question is not easily answered directly. Rabbit neutrophil defensins, whose three-dimensional structures are very similar to those of human defensins, are monomeric in aqueous solution and thus provide an opportunity to test the hypothesis that dimerization may play a role in multimeric pore formation. We therefore examined the interactions of the six known rabbit neutrophil defensins with large unilamellar vesicles (LUV) under the conditions known to lead to stable pore formation by HNP-2. We find that the rabbit defensins bind strongly to LUVs formed from pure POPG or mixtures of POPG with neutral (zwitterionic) phospholipid but induce leakage of vesicle contents only from pure POPG vesicles. Rabbit defensin NP-4 does not cause leakage under any conditions examined. The remaining defensins, NP-1, NP-2, NP-3A, NP-3B, and NP-5, cause graded release of the contents of pure POPG vesicles as does a mixture of the six defensins. The graded release indicates that the rabbit defensins do not form stable pores in the membrane. This result thus suggests that the structural features of human defensins that permit dimer formation in aqueous solution are likely to be important in the formation of multimeric pores.

Amino Acid Sequence↗

PubMed1995

Inheritance of unequal numbers of the genes encoding the human neutrophil defensins HP-1 and HP-3.

It is unclear whether the six known human defensin peptides are all encoded by separate genes or whether some of them are allelic. Three of the peptides, HP-1, HP-2, and HP-3, differ by only one amino acid, and it is thought that HP-2 may represent a proteolytic product of HP-1 and/or HP-3. To help determine the relationship of these three proteins, we isolated a nearly full-length cDNA encoding HP-1 with a sequence very similar to, but different from, the previously isolated HP-1 and -3 cDNAs. Gene copy number experiments established that there were at least two but fewer than five defensin genes with a high level of similarity to the HP-1 cDNA (HP-1/3-like). Three genomic clones were isolated that contained two different configurations of the HP-1/3-like sequences. Sequencing established that one encoded the HP-1 peptide, whereas the other encoded HP-3. Analysis of DNAs obtained from 18 unrelated individuals by Southern blot analysis revealed the expected fragments as well as additional fragments that were not present in the genomic clones. This suggested the possibility of alleles; however, when DNAs from families were examined, these fragments did not segregate in an obvious Mendelian fashion. The HP-1/3-like defensin genes are on human chromosome 8. Surprisingly, somatic cell hybrid mapping showed that the number of HP-1/3-like genes on isolated copies of chromosome 8 was variable. We conclude that individuals can inherit versions of chromosome 8 harboring either two or three copies of the genes that encode the HP-1, HP-2, and/or HP-3 peptides.

Animals↗

PubMed1996

Identification of defensin-1, defensin-2, and CAP37/azurocidin as T-cell chemoattractant proteins released from interleukin-8-stimulated neutrophils.

Reports that interleukin-8 (IL-8) induces the infiltration of neutrophils followed by T-cells into injection sites led us to postulate that by stimulation of neutrophil degranulation IL-8 may cause the release of factors with chemoattractant activity for T-lymphocytes. Extracts of human neutrophil granules were chromatographed to isolate and purify T-lymphocyte chemoattractant factors. Two major peaks of T-cell chemotactic activity were purified by C18 reversed phase high pressure liquid chromatography (HPLC). The first peak was resolved further by C4 reversed phase HPLC and yielded an active fraction shown by NH2-terminal amino acid sequence analysis to contain defensins HNP-1, HNP-2, and HNP-3. Purified defensins HNP-1 and HNP-2 (kindly provided by Dr. R. I. Lehrer, UCLA) were also potent chemoattractants for human T-cells, while HNP-3 was inactive. The second peak of T-cell chemoattractant activity was also further purified to homogeneity by C4 reversed phase HPLC and identified by NH2-terminal sequence analysis as CAP37/azurocidin, a protein with sequence homology to serine proteases. 0.1 100 ng of defensins and 1.0 100 ng/ml CAP37 were able to stimulate in vitro T-cell chemotaxis. Neutrophil activating factors, i.e. IL-8, phorbol 12-myristate 13-acetate/ionomycin, and formylmethionylleucylphenylalanine each induced the release of CAP37 and defensins from neutrophil granules. Subcutaneous administration of defensins or CAP37/azurocidin into BALB/c mice resulted in a moderate neutrophil and mononuclear cell infiltrate by 4 h, which was greater by 24 h at the site of injection. Additionally, subcutaneous injection of defensins into chimeric huPBL-SCID mice resulted in significant infiltration by human CD3+ cells within 4 h. These results identify the antimicrobial proteins, CAP37/azurocidin and defensins HNP-1 and HNP-2, as potent neutrophil-derived chemoattractants for T-cells. These proteins represent primordial antimicrobial peptides which may have evolved into acute inflammatory cell-derived signals that mobilize immunocompetent T-cells and other inflammatory cells.

Amino Acid Sequence↗

PubMed1995

A flow cytometric assay reveals a suppression of phagocytosis by rabbit defensin NP-3A in mouse peritoneal macrophages.

Phagocytosis of fluorescein isothiocyanate (FITC)-labeled polystyrene microparticles by peritoneal macrophages from thioglycollate-elicited mice was examined by means of flow cytometry (FCM). This assay revealed that rabbit defensin NP-3A suppressed the phagocytosis in a dose-dependent manner. The present results suggest that NP-3 released from neutrophils is one of the mediators which modulates the activity of macrophages in response to infection.

Animals↗

PubMed1994

The in vitro effect of human polymorphonuclear leukocyte azurophil granule components on natural killer cell cytotoxicity.

We previously demonstrated that human polymorphonuclear leukocyte (PMN) secretions are capable of activating and inhibiting natural killer cell (NK) cytotoxicity depending on the eliciting PMN stimulus. Serum-opsonized zymosan induced PMN to secrete substances that enhanced NK activity in vitro. Serum-opsonized zymosan stimulates the release of PMN azurophil granules, which contain both human neutrophil peptides (HNPs) and neutral serine proteases (NSPs). When HNPs and NSPs were tested for their ability to activate NK cells in peripheral blood lymphocytes, all but cathepsin G consistently enhanced cytotoxicity above control values. HNP-induced cytotoxicity was significantly enhanced within 12 h, peaking at approximately 24 h. Of the HNPs, HNP-1 was the most potent activator, enhancing NK activity at 1.25 micrograms/ml. Interleukin-2 and interferon-gamma were not involved in this activational process, as antibodies to interleukin-2 and interferon-gamma did not block activation by HNPs and NSPs, and interleukin-2 receptor expression was unaltered after 24 h of incubation. Enzymatically inactivated elastase and cathepsin G produced equivalent activational effects to their active counterparts. Antisera to elastase and cathepsin G decreased but did not eliminate NK activation over untreated peripheral blood lymphocytes. These data suggest that certain PMN azurophil granule components, including HNPs and NSPs directly increase the cytotoxic activity of NK cells.

Animals↗

PubMed1994

Neutrophil lysosomal nonoxidative microbicidal proteins in early-onset periodontitis.

The purpose of this study was to evaluate whether the reduced microbicidal activity of polymorphonuclear leukocytes (neutrophils) in patients with early-onset periodontitis is associated with a deficiency of nonoxidative microbicidal proteins. Neutrophils from 10 patients with early-onset periodontitis and 8 healthy control subjects were assessed for elastase isozymes 1 through 4, cathepsin G isozymes 1 through 4 and defensins (HNP-1, HNP-2 and HNP-3) using cationic and acid urea polyacrylamide gel electrophoresis. The results showed that both the total content and the relative distribution of elastase and cathepsin G isozymes was normal in neutrophils of patients with early-onset periodontitis. However, the HNP-3 content was significantly reduced in neutrophils from patients with generalized early-onset periodontitis. These findings indicate that the impaired microbicidal activities of neutrophils in patients with early-onset periodontitis does not appear to be based on an elastase or cathepsin G abnormality in neutrophils. Due to the high variability of HNP-1 + 2 and HNP-3 in neutrophils of control subjects, the reduced HNP-3 content in neutrophils probably plays a minor role in the pathogenesis of generalized early-onset periodontitis.

Adult↗

PubMed1993

Purification and characterization of new anti-adrenocorticotropin rabbit neutrophil peptides (defensins).

Neutrophil peptides (NPs, defensins), which consist of approximately 30 amino acids with a highly conserved backbone of six Cys residues, possess several biological activities, such as antimicrobial, antiviral, cytotoxic, and anti-adrenocorticotropin (corticostatic) activity in vitro. In the rabbit, six NPs, i.e., NP-1, -2, -3A, -3B, -4, and -5, have been isolated, among them, NP-3A has the most potent corticostatic activity, which involves the inhibition of adrenocorticotropin-stimulated corticosterone synthesis in adrenal cells. Using a sensitive radioimmunoassay for NP-3A and reverse-phase HPLC, we found a novel component in rabbit tissue extracts with NP-3A-like immuno-reactivity. We further purified and characterized the component and found two novel peptides. One of these peptides, designated NP-6, has an amino acid sequence identical to that of NP-3A except for three amino acids, i.e. Leu11, Phe13, and Gln15, in the central portion of the sequence. The other has a sequence corresponding to that of NP-6 except that the N-terminal Gly was deleted and is thus named des-G1-NP-6. Using chemically synthesized NP-6 and des-G1-NP-6, we measured their putative corticostatic activities by a dispersed rat adrenal cell bioassay. NP-6 showed corticostatic activity comparable to that of NP-3A while des-G1-NP-6 showed weak activity. These findings are also compatible with the notion that the N-terminal Gly, but not the central portion, is important for the anti-adrenocorticotropin activity of the NPs.

Adrenocorticotropic Hormone↗

PubMed1990

Defensins.

Defensins are a family of small, variably cationic proteins which are highly abundant in the granules of mammalian phagocytes. Three defensins, HNP-1, 2, and 3, comprise 30-50% of total protein in azurophil granules of human neutrophils. Some defensins are broadly antimicrobial, antiviral and cytotoxic, while others are chemotactic, opsonic, or may modulate hormonal responses. The defensin molecule typically consists of 29-34 amino acids with a conserved pattern of disulfide linkage among its 6 cysteines. The three-dimensional fold of defensins forms a highly amphiphilic molecule. Microbicidal and cytotoxic properties of defensins are most likely a consequence of their ability to insert into biological membranes and to generate pores. Defensins are synthesized by phagocytes or their precursors as a 94-95 amino acid charge-neutralized preprodefensin, an arrangement which may avoid cytotoxic injury to the phagocyte. Although defensins were recognized only recently, the existence of homologs in certain invertebrates suggests that they are ancestral components of the host defense system.

Amino Acid Sequence↗

PubMed1991

Crystal structure of defensin HNP-3, an amphiphilic dimer: mechanisms of membrane permeabilization.

Defensins (molecular weight 3500 to 4000) act in the mammalian immune response by permeabilizing the plasma membranes of a broad spectrum of target organisms, including bacteria, fungi, and enveloped viruses. The high-resolution crystal structure of defensin HNP-3 (1.9 angstrom resolution, R factor 0.19) reveals a dimeric beta sheet that has an architecture very different from other lytic peptides. The dimeric assembly suggests mechanisms by which defensins might bind to and permeabilize the lipid bilayer.

Amino Acid Sequence↗

PubMed1996

Evaluation of susceptibility of gram-positive and -negative bacteria to human defensins by using radial diffusion assay.

Defensins are small cationic bactericidal peptides present abundantly in the granules of polymorphonuclear neutrophils (PMNs). Human PMNs contain four defensins termed HNP-1 to HNP-4. We used a new assay system in agar plates, the radial diffusion assay, to evaluate the effects of human defensins against gram-positive and -negative bacteria. A crude mixture of HNP-1, -2, and -3 (crude HNPs) was purified from human PMN extracts by reversed-phase high-pressure liquid chromatography (RP-HPLC). The different components were later separated by RP-HPLC and gel permeation chromatography. We compared the antibacterial activities of purified HNP-1, -2, and -3 against Escherichia coli, Pseudomonas aeruginosa, methicillin-susceptible Staphylococcus aureus, and methicillin-resistant S. aureus strains using the radial diffusion assay. The antibacterial activities of HNP-1 and HNP-2 against all strains tested were similar to those of the crude HNPs, but the activity of HNP-3 was less than those of the other defensins. To quantitate the activities of HNPs against different bacteria, we defined the minimal dose of crude HNPs forming a detectable clear zone around the bacteria as the minimal inhibitory dose (MID) and determined the MIDs for 10 strains of E. coli, 12 strains of P. aeruginosa, 10 strains of methicillin-susceptible S. aureus, and 12 strains of methicillin-resistant S. aureus isolates, including clinical isolates. In general, the MIDs of the HNPs were similar against similar bacterial species. However, the MIDs for P. aeruginosa were higher than those for the other organisms tested. The radial diffusion assay is suitable as a screening test for measuring the susceptibilities of isolates to defensins, because it is sensitive and simple and has good reproducibility.

Anti-Bacterial Agents↗

PubMed1987

Defensins mediate the microbicidal activity of human neutrophil granule extract against Acinetobacter calcoaceticus.

An acid extract of human neutrophil granules was fractionated on a Sephadex G-100 column and tested for microbicidal activity against Acinetobacter calcoaceticus HO-1 as described previously (M.C. Modrzakowski and C. M. Paranavitana, Infect. Immun. 32:668-674, 1981). The low-molecular-weight protein fraction, peak D, accounted for about 30% of the protein and nearly all of the activity of the crude extract against strain HO-1. Peak D protein proved to be a mixture of the three human defensin peptides HNP1, HNP2, and HNP3. Purified defensins reproduced the microbicidal activity of peak D. The data suggest that defensins could play a major role in the killing of A. calcoaceticus by human neutrophils.

Acinetobacter↗

PubMed1988

Killing of Proteus mirabilis by polymorphonuclear leukocyte granule proteins: evidence for species specificity by antimicrobial proteins.

Low-molecular-weight (Mr, ca. 3,800) polypeptides containing human defensins HNP-1 and HNP-2 (T. Ganz, M. S. Selsted, D. Szlarek, S. L. Harwig, K. Daher, D. F. Bainton, and R. I. Lehrer, J. Clin. Invest. 76:1427-1434, 1985) prepared in our laboratory from acid extracts of human polymorphonuclear granulocyte granules and purified human defensins were found to exert potent bactericidal action against Proteus mirabilis. The antimicrobial action of the extracts of human polymorphonuclear leukocytes granules against P. mirabilis appears to be due to the presence of the defensins. Because P. mirabilis resists the antimicrobial action of other granule proteins, we interpret the present results to mean that the various antimicrobial proteins display species specificity in their microbicidal action.

Blood Bactericidal Activity↗

PubMed1990

Characterization of defensin resistance phenotypes associated with mutations in the phoP virulence regulon of Salmonella typhimurium.

The defensin sensitivities of Salmonella typhimurium strains with mutations in the phoP/phoQ two-component virulence regulon were tested by using purified defensins NP-1 and NP-2. Strains with mutations in either gene of the regulatory pair (phoP [transcriptional activator] or phoQ [membrane sensor kinase]) had increased sensitivities to defensin. The predicted periplasmic domain of the PhoQ protein contained a markedly anionic domain that could interact with cationic proteins and that could be responsible for resistance to defensin. Because insertion mutations in phoP are polar on phoQ, we constructed strains that expressed the PhoQ protein in the absence of PhoP to test whether resistance to defensin requires only the phoQ gene product. We found that resistance to defensin requires the function of both components of this regulatory system, because strains expressing PhoQ without PhoP were still markedly sensitive to defensins. This implied that a pag (phoP-activated gene) product is responsible for defensin resistance. We also tested for the ability of defensins NP-1, NP-5, and HNP-1 to activate pag expression and found that these peptides have no effect. Defensin resistance is not the only virulence characteristic controlled by the PhoP-PhoQ regulon because mutations in pagC, as well as ones in the phoP locus that resulted in constitutive pag activation (phenotype PhoPc), had no effect on defensin resistance, even though they rendered the organism avirulent and deficient in survival within macrophages. The virulence defect conferred by mutations in the phoP-phoQ two-component regulatory system is not completely explained by alterations in resistance to cationic proteins and involves the control of other proteins necessary for S. typhimurium survival within macrophages.

Animals↗

PubMed1990

In vitro sensitivity of oral, gram-negative, facultative bacteria to the bactericidal activity of human neutrophil defensins.

Neutrophils play a major role in defending the periodontium against infection by oral, gram-negative, facultative bacteria, such as Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Capnocytophaga spp. We examined the sensitivity of these bacteria to a mixture of low-molecular-weight peptides and highly purified individual defensin peptides (HNP-1, HNP-2, and HNP-3) isolated from human neutrophils. Whereas the Capnocytophaga spp. strains were killed significantly by the mixed human neutrophil peptides, the A. actinomycetemcomitans and E. corrodens strains were resistant. Killing was attributable to the defensins. The bactericidal activities of purified defensins HNP-1 and HNP-2 were equal, and both of these activities were greater than HNP-3 activity against strains of Capnocytophaga sputigena and Capnocytophaga gingivalis. The strain of Capnocytophaga ochracea was more sensitive to defensin-mediated bactericidal activity than either C. sputigena or C. gingivalis was. The three human defensins were equipotent in killing C. ochracea. C. ochracea was killed under aerobic and anaerobic conditions and over a broad pH range. Killing was most effective under hypotonic conditions but also occurred at physiologic salt concentrations. We concluded that Capnocytophaga spp. are sensitive to oxygen-independent killing by human defensins. Additional studies will be required to identify other components that may equip human neutrophils to kill A. actinomycetemcomitans, E. corrodens, and other oral gram-negative bacteria.

Blood Bactericidal Activity↗