Analysis of the Ribonuclease a superfamily of antimicrobial peptides in patients undergoing chronic peritoneal dialysis

Infectious peritonitis is a common complication in patients undergoing chronic peritoneal dialysis (PD), limiting the duration of PD as a modality for renal replacement therapy and increasing patient morbidity and mortality. Antimicrobial peptides (AMPs) serve critical roles in mucosal defense, but their expression and activity during peritonitis are poorly understood. We hypothesized that AMPs belonging to the Ribonuclease (RNase) A Superfamily are present in peritoneal fluid and increase during peritonitis in patients undergoing chronic PD. In the absence of peritonitis, we detected RNase 3, RNase 6, and RNase 7 in cell-free supernatants and viable cells obtained from peritoneal fluid of chronic PD patients. The cellular sources of these RNases were eosinophils (RNase 3), macrophages (RNase 6), and mesothelial cells (RNase 7). During peritonitis, RNase 3 increased 55-fold and RNase 7 levels increased 3-fold on average, whereas RNase 6 levels were unchanged. The areas under the receiver-operating characteristic curves for RNase 3 and RNase 7 were 0.99 (95% confidence interval (CI): 0.96–1.0) and 0.79 (95% CI: 0.64–0.93), respectively, indicating their potential as biomarkers of peritonitis. Discrete omental reservoirs of these RNases were evident in patients with end stage kidney disease prior to PD initiation, and omental RNase 3 reactive cells increased in patients undergoing PD with a history of peritonitis. We propose that constitutive and inducible pools of antimicrobial RNases form a network to shield the peritoneal cavity from microbial invasion in patients undergoing chronic PD

Pinless frameless electromagnetic image-guided neuroendoscopy in children

Frameless imaged-guided neuronavigation is a useful adjunct to neuroendoscopy in paediatric patients, especially those with abnormal or complex ventricular or cyst anatomy. The development of electromagnetic neuronavigation has allowed the use of image-guided navigation in the very young patient in whom rigid fixation in cranial pins is contraindicated. The technique and the authors' experience of its use in a series of paediatric patients are described

Expression and Significance of the HIP/PAP and RegIIIγ Antimicrobial Peptides during Mammalian Urinary Tract Infection

<div><p>Recent evidence indicates that antimicrobial peptides (AMPs) serve key roles in defending the urinary tract against invading uropathogens. To date, the individual contribution of AMPs to urinary tract host defense is not well defined. In this study, we identified <i>Regenerating islet-derived 3 gamma</i> (RegIIIγ) as the most transcriptionally up-regulated AMP in murine bladder transcriptomes following uropathogenic <i>Escherichia coli</i> (UPEC) infection. We confirmed induction of RegIIIγ mRNA during cystitis and pyelonephritis by quantitative RT-PCR. Immunoblotting demonstrates increased bladder and urinary RegIIIγ protein levels following UPEC infection. Immunostaining localizes RegIIIγ protein to urothelial cells of infected bladders and kidneys. Human patients with UTI have increased urine concentrations of the orthologous Hepatocarcinoma-Intestine-Pancreas / Pancreatitis Associated Protein (HIP/PAP) compared to healthy controls. Recombinant RegIIIγ protein does not demonstrate bactericidal activity toward UPEC <i>in vitro</i>, but does kill <i>Staphylococcus saprophyticus</i> in a dose-dependent manner. Kidney and bladder tissue from RegIIIγ knockout mice and wild-type mice contain comparable bacterial burden following UPEC and Gram-positive UTI. Our results demonstrate that RegIIIγ and HIP/PAP expression is induced during human and murine UTI. However, their specific function in the urinary tract remains uncertain.</p></div

The human RegIIIγ orthologue, HIP/PAP, is secreted in infected human urine.

<p>(<b>A</b>) HIP/PAP levels were quantified by ELISA and adjusted for urine creatinine to control for differences in urine concentration. The resulting PAP/Cr ratio is significantly elevated in urine from patients with positive urine cultures, compared to sterile urine (n = 26 urines/group; * <i>P</i> < 0.0001, Mann-Whitney U test). The horizontal line indicates the mean. (<b>B</b>) Western blotting demonstrates absent of HIP/PAP in sterile (S) urine, whereas infected (I) urine from two children with UTI exhibits immunoreactivity.</p

Regulation of AMP mRNA expression during experimental cystitis.

<p>Genes encoding known or putative AMP with ≥ 4-fold change (FC) are depicted. False discovery rate (FDR) < 10 is considered significant; n.s. = not significant.</p

RegIIIγ protein localizes to urothelium following UPEC infection.

<p>RegIIIγ immunoreactivity is absent in uninfected bladders. Non-specific staining of UPEC is seen 2 and 6 hpi. Beginning 16 hpi, patchy urothelial immunoreactivity is observed. Urothelial RegIIIγ expression is most prominent 24 hpi and persists to a lesser extent 48 hpi. All figures are 20x magnification.</p

<i>RegIIIγ</i> mRNA and RegIIIγ protein expression are induced during UPEC cystitis, and RegIIIγ is secreted into the urinary stream following UPEC infection.

<p>(<b>A</b>) Fold-change in bladder <i>RegIIIγ</i> mRNA levels in response to intravesical inoculation of 10⁸ CFU of UPEC strain UTI89. * <i>P</i> = 0.0286, Mann-Whitney U test versus uninfected bladders (n = 4 bladders/group). (<b>B</b>) Relative <i>RegIIIγ</i> mRNA levels increase 24 hpi following inoculation with 10⁸ CFU of UPEC, but not in response to Gram-positive uropathogens. * <i>P</i> = 0.0286, Mann-Whitney U test, UPEC versus uninfected bladders (n = 4 bladders/group). (<b>C</b>) RegIIIγ protein (arrow) is induced in wild-type (WT) bladder 16 hpi with UPEC, but expression is absent at earlier times, and as well as in <i>RegIIIγ</i>^-/- bladders. Bladder RegIIIγ co-migrates with recombinant (r) RegIIIγ protein. A non-specific reactive band (n.s., arrowhead) migrates more rapidly and is present in WT and <i>RegIIIγ</i>^<i>-/-</i> bladder lysates. (<b>D</b>) RegIIIγ protein is undetectable in sterile WT urine, but secreted 24 hpi with UPEC. Gram-positive inocula, <i>E</i>. <i>faecalis</i> (EF) and <i>S</i>. <i>saprophyticus</i> (SS), fail to induce RegIIIγ secretion, and RegIIIγ immunoreactivity is absent in <i>RegIIIγ</i>^<i>-/-</i> urine 24 hpi with UPEC.</p

Induction of <i>RegIIIγ</i> mRNA and RegIIIγ protein by UPEC in C3H/HeOuJ kidneys.

<p>(<b>A</b>) Significant <i>RegIIIγ</i> mRNA induction by UPEC in C3H/HeOuJ kidneys, with progressive accumulation over time. (* P = 0.0286, Mann-Whitney U test versus uninfected C3H/HeOuJ kidneys, n = 4 kidneys/time). (<b>B</b>) Absent RegIIIγ immunostaining in uninfected C3H/HeOuJ urothelium, which exhibits uniform apical expression of Uroplakin 3a (Upk3a; red). U: Urinary space. (<b>C</b>) RegIIIγ protein (green) localizes to the hypertrophied urothelium of C3H/HeOuJ kidneys 28 dpi, with reduced expression of Upk3a. Urothelial border is indicated by dashed line. Both images are 20x magnification.</p