Human neutrophil peptide-2

Antimicrobial peptides (AMPs) are critical to the protection of the urinary tract of humans and other animals from pathogenic microbial invasion. AMPs rapidly destroy pathogens by disrupting microbial membranes and/or augmenting or inhibiting the host immune system through a variety of signaling pathways. We have previously demonstrated that alpha-defensins 1-3 (DEFA1A3) are AMPs expressed in the epithelial cells of the human kidney collecting duct in response to uropathogens. We also demonstrated that DNA copy number variations in the DEFA1A3 locus are associated with UTI and pyelonephritis risk. Because DEFA1A3 is not expressed in mice, we utilized human DEFA1A3 gene transgenic mice (DEFA4/4) to further elucidate the biological relevance of this locus in the murine urinary tract. We demonstrate that the kidney transcriptional and translational expression pattern is similar in humans and the human gene transgenic mouse upon uropathogenic Escherichia coli (UPEC) stimulus in vitro and in vivo. We also demonstrate transgenic human DEFA4/4 gene mice are protected from UTI and pyelonephritis under various UPEC challenges. This study serves as the foundation to start the exploration of manipulating the DEFA1A3 locus and alpha-defensins 1-3 expression as a potential therapeutic target for UTIs and other infectious diseases.

A quantitative assay for simultaneous measurement of individual human neutrophil peptide-1, -2 and -3 concentrations will aid in exploring the potential of these antimicrobial peptides as biomarkers for various diseases. Therefore, a liquid chromatography-tandem mass spectrometry method has been developed and validated to allow separate quantification of the three human neutrophil peptides in human plasma and serum. Plasma and serum samples (100 microl) were deproteinized by precipitation, followed by chromatographic separation on a Symmetry 300 C18 column (50 mm x 2.1mm I.D., particle size 3.5 microm), using a water-methanol gradient containing 0.25% (v/v) formic acid and human alpha-defensin 5 as internal standard. Tandem mass spectrometric detection was performed on a triple quadrupole mass spectrometer equipped with electrospray ionization. Despite low fragmentation efficiency of the antimicrobial peptides, multiple reaction monitoring was used for detection, though selecting the quaternary charged ions as both precursor and product. The method was linear for concentrations between 5 and 1000 ng/ml with a limit of detection around 3 ng/ml for all peptides. Intra- and inter-assay precisions were 14.8 and 19.1%, respectively, at the lowest measured endogenous concentration (6.4 ng/ml of HNP-1 in plasma), representing the lower limit of quantification of the assay. Recoveries of HNP-1, -2 and -3 from plasma and serum ranged between 85 and 128%. Analysis of serum samples from intensive care patients showed average concentrations of 362, 570 and 143 ng/ml for HNP-1, -2 and -3, respectively.

Increased total serum concentrations of human neutrophil peptide-1, -2 and -3 (HNP-1, -2 and -3) have been associated with colorectal cancer (CRC). Owing to a recently developed and fully validated liquid-chromatography coupled to tandem-mass spectrometry (LC-MS/MS) assay, individual serum concentrations of these antimicrobial peptides were quantified to evaluate their role as serum markers in CRC. Serum was obtained from patients with indications for colonoscopy, subsequently diagnosed as normal colon or hyperplastic polyp (CON; n= 368), adenomatous polyp (AP; n = 179) or colorectal cancer (CRC; n = 69). Comparison of HNP-1, -2 and -3 concentrations between CRC and CON (130 ± 90 vs. 105 ± 80; 264 ± 140 vs. 206 ± 99 and 62 ± 56 vs. 54 ± 59 for HNP-1, -2 and -3, respectively) revealed that reported up-regulated total HNP-concentrations can be largely attributed to increased HNP-2 (P=0.0006) and HNP-1 (P=0.024) levels. Although receiver operating characteristics (ROC) analyses showed low specificity of the peptides for CRC and no significant changes in serum levels were observed after surgical removal of the tumor (n=23), the established differentiation between the HNP-subtypes may be particularly useful to completely elucidate the role of these antimicrobial peptides in CRC.