Mass Spectrometric and Bio-Computational Binding Strength Analysis of Multiply Charged RNAse S Gas-Phase Complexes Obtained by Electrospray Ionization from Varying In-Solution Equilibrium Conditions.

We investigated the influence of a solvent's composition on the stability of desorbed and multiply charged RNAse S ions by analyzing the non-covalent complex's gas-phase dissociation processes. RNAse S was dissolved in electrospray ionization-compatible buffers with either increasing organic co-solvent content or different pHs. The direct transition of all the ions and the evaporation of the solvent from all the in-solution components of RNAse S under the respective in-solution conditions by electrospray ionization was followed by a collision-induced dissociation of the surviving non-covalent RNAse S complex ions. Both types of changes of solvent conditions yielded in mass spectrometrically observable differences of the in-solution complexation equilibria. Through quantitative analysis of the dissociation products, i.e., from normalized ion abundances of RNAse S, S-protein, and S-peptide, the apparent kinetic and apparent thermodynamic gas-phase complex properties were deduced. From the experimental data, it is concluded that the stability of RNAse S in the gas phase is independent of its in-solution equilibrium but is sensitive to the complexes' gas-phase charge states. Bio-computational in-silico studies showed that after desolvation and ionization by electrospray, the remaining binding forces kept the S-peptide and S-protein together in the gas phase predominantly by polar interactions, which indirectly stabilized the in-bulk solution predominating non-polar intermolecular interactions. As polar interactions are sensitive to in-solution protonation, bio-computational results provide an explanation of quantitative experimental data with single amino acid residue resolution.

DNA-based ribonuclease detection assays.

Ribonucleases are useful as biomarkers and can be the source of contamination in laboratory samples, making ribonuclease detection assays important in life sciences research. With recent developments in DNA-based biosensing, several new techniques are being developed to detect ribonucleases. This review discusses some of these methods, specifically those that utilize G-quadruplex DNA structures, DNA-nanoparticle conjugates and DNA nanostructures, and the advantages and challenges associated with them.

A Highly Specific DNA Aptamer for RNase H2 from Clostridium difficile.

Molecular recognition elements with high specificity are of great importance for the study of molecular interactions, accurate diagnostics, drug design, and personalized medicine. Herein, a highly specific DNA aptamer for RNase H2 from Clostridium difficile (C. difficile) was generated by SELEX and minimized to 40 nucleotides. The aptamer exhibits a dissociation constant (Kd) of 1.8 ± 0.5 nM and an inhibition constant (IC50) of 7.1 ± 0.6 nM for C. difficile RNase H2, both of which are 2 orders of magnitude better for the same enzyme from other control bacteria. The fluorescent version of the aptamer can distinguish C. difficile from several other control bacteria in a cell lysate assay. This work demonstrates that a ubiquitous protein like RNase H2 can still be used as the target for the development of highly specific aptamers and the combination of the protein and the aptamer can achieve the recognition specificity needed for a diagnostic test and drug development.

A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity.

DNA and RNA nucleases play a critical role in a growing number of cellular processes ranging from DNA repair to immune surveillance. Nevertheless, many nucleases have unknown or poorly characterized activities. Elucidating nuclease substrate specificities and co-factors can support a more definitive understanding of cellular mechanisms in physiology and disease. Using fluorescence-based methods, we present a quick, safe, cost-effective, and real-time versatile nuclease assay, which uniquely studies nuclease enzyme kinetics. In conjunction with a substrate library we can now analyse nuclease catalytic rates, directionality, and substrate preferences. The assay is sensitive enough to detect kinetics of repair enzymes when confronted with DNA mismatches or DNA methylation sites. We have also extended our analysis to study the kinetics of human single-strand DNA nuclease TREX2, DNA polymerases, RNA, and RNA:DNA nucleases. These nucleases are involved in DNA repair, immune regulation, and have been associated with various diseases, including cancer and immune disorders.

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.

Quantitative Evaluation of Native Protein Folds and Assemblies by Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS).

Hydrogen deuterium exchange mass spectrometry (HDX-MS) has significant potential for protein structure initiatives but its relationship with protein conformations is unclear. We report on the efficacy of HDX-MS to distinguish between native and non-native proteins using a popular approach to calculate HDX protection factors (PFs) from protein structures. The ability of HDX-MS to identify native protein conformations is quantified by binary structural classification such that merits of the approach for protein modelling can be quantified and better understood. We show that highly accurate PF calculations are not a prerequisite for HDX-MS simulations that are capable of effectively discriminating between native and non-native protein folds. The simulations can also be performed directly on unique structures facilitating high-throughput evaluation of many alternate conformations. The ability of HDX-MS to classify the conformations of homo-protein assemblies is also investigated. In contrast to protein monomers, we show a significant lack of correspondence between the simulated and experimental HDX-MS data for these systems with a subsequent decrease in the ability of HDX-MS to identify native states. However, we demonstrate surprisingly high diagnostic ability of the simulated data for assemblies in which a significant proportion of the individual chains occupy protein-protein interfaces. We relate this to the number of peptides that can sample alternate subunit orientations and discuss these observations within the larger context of applying HDX-MS to evaluate protein structures. Graphical Abstract.

Heart-cut nano-LC-CZE-MS for the characterization of proteins on the intact level.

Multidimensional separation techniques play an increasingly important role in separation science, especially for the analysis of complex samples such as proteins. The combination of reversed-phase liquid chromatography in the nanoscale and CZE is especially beneficial due to their nearly orthogonal separation mechanism and well-suited geometries/dimensions. Here, a heart-cut nano-LC-CZE-MS setup was developed utilizing for the first time a mechanical 4-port valve as LC-CE interface. A model protein mixture containing four different protein species was first separated by nano LC followed by a heart-cut transfer of individual LC peaks and subsequent CZE-MS analysis. In the CZE dimension, various glycoforms of one protein species were separated. Improved separation capabilities were achieved compared to the 1D methods, which was exemplarily shown for ribonuclease B and its different glycosylated forms. LODs in the lower µg/mL range were determined, which are considerably lower compared to traditional CZE-MS. In addition, this study represents the first application of an LC-CE-MS system for intact protein analysis. The nano-LC-CZE-MS system is expected to be applicable to various other analytical challenges.

Ultraviolet (UV) Cross-Linking of Live Cells, Lysate Preparation, and RNase Titration for Cross-Linking Immunoprecipitation (CLIP).

One of the great advantages of RNA CLIP (cross-linking immunoprecipitation) is that RNA-protein complexes can be "frozen" in situ in live cells by ultraviolet (UV) irradiation. This protocol describes UV cross-linking of mammalian tissue culture cells or whole tissues. For the latter, the tissue is typically triturated to allow UV penetration. However, depending on the thickness of the chosen tissue, this may not be necessary. It is preferable to handle the tissue as little as possible, to keep it in ice-cold buffers, and to cross-link as soon after the time of collection as is feasible to preserve native interactions at the time of cross-linking. This protocol also describes cell lysis following cross-linking, as well as treatment with RNase to partially hydrolyze the bound RNA. The first time this protocol is performed, a pilot experiment should be performed to determine the optimal RNase concentration for the particular sample. Once the RNase conditions are optimized this section of CLIP protocol can be repeated on experimental samples before proceeding through the rest of the protocol.

Identification of a perchloric acid-soluble protein (PSP)-like ribonuclease from Trichomonas vaginalis.

A perchloric acid-soluble protein (PSP), named here tv-psp1, was identified in Trichomonas vaginalis. It is expressed under normal culture conditions according to expressed sequence tag (EST) analysis. On the other hand, Tv-PSP1 protein was identified by mass spectrometry with a 40% of identity to human PSP (p14.1). Polyclonal antibodies against recombinant Tv-PSP1 (rTv-PSP1) recognized a single band at 13.5 kDa in total protein parasite extract by SDS-PAGE and a high molecular weight band analyzed by native PAGE. Structural analysis of Tv-PSP1, using dynamic light scattering, size exclusion chromatography, and circular dichroism spectroscopy, showed a trimeric structure stable at 7 M urea with 38% α-helix and 14% ß-sheet in solution and a molecular weight of 40.5 kD. Tv-PSP1 models were used to perform dynamic simulations over 100 ns suggesting a stable homotrimeric structure. Tv-PSP1 was located in the nucleus, cytoplasm, and hydrogenosomes of T. vaginalis, and the in silico analysis by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) showed interactions with RNA binding proteins. The preliminary results of RNA degradation analysis with the recombinant Tv-PSP1 showed RNA partial deterioration suggesting a possible putative ribonuclease function.

Enhanced spectral density mapping through combined multiple-field deuterium 13CH2D methyl spin relaxation NMR spectroscopy.

Quadrupolar relaxation of 2H (D) nuclear spins is a powerful probe of conformational dynamics in biological macromolecules. Deuterium relaxation rate constants are determined by the spectral density function for reorientation of the C-D bond vector at zero, single-quantum, and double-quantum 2H frequencies. In the present work, 2H relaxation rate constants were measured for an E. coli ribonuclease H [U-2H, 15N] ILV-[13CH2D] sample using 400, 500, 800, and 900 MHz NMR spectrometers and analyzed by three approaches to determine spectral density values. First, data recorded at each static magnetic field were analyzed independently. Second, data recorded at 400 and 800 MHz were analyzed jointly and data recorded at other fields were analyzed independently. Third, data recorded at 400 and 500 MHz were interpolated to 450 MHz, and the resulting two pairs of data, corresponding to 400 MHz/800 MHz and 450 MHz/900 MHz, were analyzed jointly. The second and third approaches rely on the identity between the double quantum frequency at the lower field and the single quantum frequency at the higher field. Spectral density values for 32 of the 48 resolvable ILV methyl resonances were fit by the Lipari-Szabo model-free formalism and used to validate the three methods. The three spectral density mapping methods performed equally well in cross validation with data recorded at 700 MHz. However, the third method yielded approximately 10-15% more precise estimates of model-free parameters and consequently provides a general strategy for analysis of 2H spin relaxation data in biological macromolecules.

Analysis of ribonuclease activity in sub-nanoliter droplets by label-free fluorescence measurements.

We report the results of a label-free analysis of ribonuclease activity using droplet-based microfluidics. The ribonucleolytic activity of ribonucleases (RNases) plays a critical role in cellular functions such as development, survival, growth and differentiation. Altered ribonucleolytic activity and/or the expression level of the RNase A family are known to be associated with pancreatic, bladder, ovarian and thyroid cancers among others. For this reason, the RNase A family is a meaningful protein biomarker that can be used in the diagnosis of cancer and as a target for new drug screening. There are some successful traditional methods for analysing the RNase activity, such as radioactive label-based assay, methylene blue-based assay, gel zymography, as well as other more recently developed methods such as electrochemical assay and fluorescence resonance energy transfer (FRET). However, these methods require analytical samples with a volume ranging from microliters to milliliters, and are not suitable for high-throughput analysis. Therefore, we integrated ethidium bromide (EtBr), which intercalates the chemical itself to nucleic acid, to droplet-based microfluidics for a cost-effective, high-throughput analysis. Put simply, this method is dependent on the amount of intercalated EtBr molecules on RNA. Our assay also uses visible light that is harmless to humans, unlike previous methods that used harmful UV rays, to excite the EtBr molecules. Specifically, we monitored the ribonucleolytic activity of less than 10 nM RNase A in droplets of about 330 picoliters. Also, half the maximal inhibitory concentration (IC50) of the RNase inhibitor was successfully measured in the same volume of droplets at a frequency of 40 hertz.

MCPIP1 contributes to the inflammatory response of UVB-treated keratinocytes.

BACKGROUND: Monocyte chemoattractant protein-1-induced protein-1 (MCPIP1), also known as regnase-1, negatively regulates many cellular processes including the cellular response to inflammatory agents, differentiation, viability, and proliferation. It possesses a PilT N-terminus (PIN) domain that is directly involved in regulating the stability of transcripts and miRNAs by recognizing stem loop structures and degrading them by endonucleolytic cleavage. OBJECTIVE: We investigated the role of MCPIP1 in the response of human primary keratinocytes to UVB stress. METHODS: Keratinocytes were treated with UVB, siRNA against MCPIP1, pharmacological inhibitors of signaling pathways, or subjected to control treatments. The mRNA and protein levels of MCPIP1 and MCPIP1-dependent changes gene expression were analyzed by quantitative (Q)-RT-PCRs and Western blots. Secretion of TNFα and IL-8 was determined by ELISA. RESULTS: UVB treatment of keratinocytes induced upregulation of MCPIP1 at the mRNA level after 4-8h and at the protein level after 8-16h. MCPIP1 abundance depended on NF-κB activity. Using an siRNA strategy, we found that diminished MCPIP1 resulted in an up-regulation of transcripts coding for IL-8, TNFα, COX-2, and BCL-2, as well as an enhanced release of IL-8. Moreover, decreased phosphorylation of NF-κB and p38 signaling pathways were observed in addition to a slight up-regulation of ERK1/2 directly after UVB treatment. Twenty-four hours later, decreased phosphorylation was observed only for NF-κB and p38. Furthermore, in MCPIP1-suppressed cells, the levels of pro-apoptotic Puma, the phosphorylated form of p53 and the abundance of its target p21 as well as the activity of caspase 3 decreased, while the level of cyclin D1 increased. CONCLUSION: MCPIP1 contributes to the UVB response of keratinocytes by altering metabolic and apoptotic processes and the release of inflammatory mediators.

Highly sensitive derivatization reagents possessing positively charged structures for the determination of oligosaccharides in glycoproteins by high-performance liquid chromatography electrospray ionization tandem mass spectrometry.

We have developed three kinds of novel derivatization reagents (4-CEBTPP, 4-CBBTPP, 5-COTPP) with triphenylphosphine (TPP) as a basic structure carrying a permanent positive charge for resolution of the oligosaccharides in glycoprotein using high-performance liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The synthesized reagents reacted with the sialylglycosylamine of the sialylglycopeptide after treatment by PNGase F. The final derivatives were analyzed by ESI-MS and sensitively detected in the selected reaction monitoring (SRM) mode. Furthermore, the limits of detection (S/N=3) on the SRM chromatograms were at the fmol level (30fmol). Therefore, we used the limit of detection of the reagent products detected by the SRM and evaluated the utility of each reagent. Among the reagents, the positively charged 4-CEBTPP derivative's peak area was the highest; 4-CEBTPP with a positively charged structure showed about a 20 times greater sensitivity for the glycosylamine of the SGP product compared to the conventional fluorescence reagent, Fmoc-Cl. In addition, various fragment ions based on the carbohydrate units also appeared in the MS/MS spectra. Among the fragment ions, m/z 627.37 (CE=40eV) corresponding to 4-CEBTPP-GlcNAc and m/z 120.09 (CE=100eV) corresponding to 4-CEBTPP are the most important ones for identifying the oligosaccharide. 4-CEBTPP-SGA was easily identified by the selected-ion chromatogram in the product ion scan (m/z 120.09) and in the precursor ion scan (m/z 627.37) by MS/MS detection. The derivatized analytes have a high ionization efficiency and they are detected with a high sensitivity in the electrospray ionization. The novel derivatization reagent with a multi-function provided a higher sensitivity for the oligosaccharide analysis, as well as a better specificity and feasibility. Furthermore, several oligosaccharides in fetuin and ribonuclease B were successfully identified by the proposed procedure.

Hybrid Approach Combining Boronate Affinity Magnetic Nanoparticles and Capillary Electrophoresis for Efficient Selection of Glycoprotein-Binding Aptamers.

Capillary electrophoresis (CE) and magnetic beads have been widely used for the selection of aptamers owing to their efficient separation ability. However, these methods alone are associated with some apparent drawbacks. CE suffers from small injection volumes and thereby only a limited amount of aptamer can be collected at each round. While the magnetic beads approach is often associated with tedious procedure and nonspecific binding. Herein we present a hybrid approach that combines the above two classical aptamer selection methods to overcome the drawbacks associated with these methods alone. In this hybrid method, one single round selection by boronate affinity magnetic nanoparticles (BA-MNPs) was first performed and then followed by a CE selection of a few rounds. The BA-MNPs-based selection eliminated nonbinding sequences, enriching effective sequences in the nucleic acid library. While the CE selection, which was carried out in free solutions, eliminated steric hindrance effects in subsequent selection. Two typical glycoproteins, Ribonuclease B (RNase B) and alkaline phosphatase (ALP), were used as targets. This hybrid method allowed for efficient selection of glycoprotein-binding aptamers within 4 rounds (1 round of BA-MNPs-based selection and 3 rounds of CE selection) and the dissociation constants reached 10-8 M level. The hybrid selection approach exhibited several significant advantages, including speed, affinity, specificity, and avoiding negative selection. Using one of the selected ALP-binding aptamers as an affinity ligand, feasibility for real application of the selected aptamers was demonstrated through constructing an improved enzyme activity assay.

RNase 7 in Cutaneous Defense.

RNase 7 belongs to the RNase A superfamily and exhibits a broad spectrum of antimicrobial activity against various microorganisms. RNase 7 is expressed in human skin, and expression in keratinocytes can be induced by cytokines and microbes. These properties suggest that RNase 7 participates in innate cutaneous defense. In this review, we provide an overview about the role of RNase 7 in cutaneous defense with focus on the molecular mechanism of the antimicrobial activity of RNase 7, the regulation of RNase 7 expression, and the role of RNase 7 in skin diseases.

Electrokinetic effect for molecular recognition: A label-free approach for real-time biosensing.

We present a simple and inexpensive method for label-free detection of biomolecules. The method monitors the changes in streaming current in a fused silica capillary as target biomolecules bind to immobilized receptors on the inner surface of the capillary. To validate the concept, we show detection and time response of different protein-ligand and protein-protein systems: biotin-avidin and biotin-streptavidin, barstar-dibarnase and Z domain-immunoglobulin G (IgG). We show that specific binding of these biomolecules can be reliably monitored using a very simple setup. Using sequential injections of various proteins at a diverse concentration range and as well as diluted human serum we further investigate the capacity of the proposed technique to perform specific target detection from a complex sample. We also investigate the time for the signal to reach equilibrium and its dependence on analyte concentration and demonstrate that the current setup can be used to detect biomolecules at a concentration as low as 100pM without requiring any advanced device fabrication procedures. Finally, an analytical model based on diffusion theory has been presented to explain the dependence of the saturation time on the analyte concentration and capillary dimensions and how reducing length and inner diameter of the capillary is predicted to give faster detection and in practice also lower limit of detection.

Metal cation control of electroosmotic flow magnitude in phospholipid-coated capillaries.

CZE has become widespread for the separation and analysis of biomolecules such as proteins and peptides, due to factors such as, the speed of the separations, low sample volume, and high resolution associated with the technique. However, the separation of biomolecules by CZE does present a significant challenge due to the electrostatic attraction and adsorption of cationic, or cation containing, biomolecules to the capillary surface. To that end numerous methods have been developed to passivate, or protect the surface, in order to prevent the adsorption of analytes. Yet, in the process of protecting the capillary surface, the potential for further modification of the EOF, a factor crucial to effective analyte resolution, is greatly diminished. In seeking to overcome this limitation we have explored the potential of incorporating a range of metal cations into a phospholipid bilayer capillary coating. It has previously been established that the inclusion of calcium into the separation buffer with a phospholipid coating will reverse the EOF in the capillary. Here, we present our investigation of a broader range of metal cations included in the separation buffer (Ca(2+) , Mg(2+) , Co(2+) , Ni(2+) , Sr(2+) , Ba(2+) , and Ce(3+) ) revealing that the choice of metal cation can drastically influence the EOF, with observed values between -3.80 × 10(-4) and -5.74 × 10(-5) cm(2) /V·s.

[Cytochemical localization and properties of selected nucleolytic enzymes]. / Cytochemiczna lokalizacja i wlasciwosci wybranych enzymów nukleolitycznych.

In the article there are shortly outlined studies on cytochemical localization of selected nucleolytic enzymes carried out between 1957-1986 by David Shugar and his coworkers. The histochemical localization of several nucleolytic enzymes in animal and plant tissues was determined by synthesis of specific substrates, alpha-naphthyl esters of 5'- and 3'-nucleotides and their derivatives. In rat tissues phosphodiesterase I was localized in the plasma membrane whereas phosphodiesterase II in the lizosomes, reflecting their physiological roles. The localization of pancreatic type ribonuclease in animal tissues was determined, indicating its role in extracellular digestion. Plant nucleotide pyrophosphatase was localized in several tissues, purified to near homogeneity from potato tubers and its properties and substrate specificity were determined. Application of this enzyme for removal of m7GMP from the "cap" of eukaryotic mRNA allowed to elucidate the role of "cap" in mRNA binding to ribosomes in the process of translation. Furthermore, cyclic nucleotide phosphodiesterase was isolated from potato tubers and its physicochemical properties, oligomeric structure and substrate specificity were elucidated.

Novel Method for the Analysis of Ribonuclease Based on Fluorescence Recovery of a Cationic Aluminum Phthalocyanine-RNA Association Complex as a Red-emitting Fluorogenic Substrate.

The conventional spectrophotometric method that is often applied to determine ribonuclease (RNase) has disadvantages that include cumbersome manipulation, time-consuming processing and a lack of linear range. We had found that a low concentration of RNA could induce cationic aluminum phthalocyanine (tetra(trimethylammonio)aluminum phthalocyanine (TTMAAlPc)), which emitted strong red fluorescence to aggregate in neutral media, resulting in an almost complete quenching of fluorescence from the cationic aluminum phthalocyanine. The RNA is degraded through hydrolysis by RNase, which destroys the induced aggregation of TTMAAlPc on RNA and releases free TTMAAlPc, leading to a significant fluorescence recovery of the reaction system. Based on this new finding, a method to detect RNase by enhanced fluorescence was established using the TTMAAlPc-RNA association complex as a new fluorogenic substrate of RNase. The optimal conditions were determined, and the interfering foreign substances were investigated. Under optimal conditions, the linear range was 0.05 - 50 µg/L, and the detection limit was 0.02 µg/L. This method was applied for the analysis of ribonuclease in urine specimens from normal adults, and the results were consistent with those determined by conventional spectrophotometric methods. The developed method is easy to operate and highly sensitive, and has a wide linear range, thus solving issues with conventional methods. This study applied, for the first time, cationic phthalocyanine as a fluorescent probe in the detection of nuclease, which provides new applications of phthalocyanine as a fluorescent probe emitting at the red wavelength region.

LEAP-2, LL-37 and RNase7 in tonsillar tissue: downregulated expression in seasonal allergic rhinitis.

In the upper airway, the production of antimicrobial peptides (AMPs) protects against bacteria, viruses and fungi. Previous investigations have revealed downregulated expression of AMPs in different manifestations of allergic disease. In this study, we examined the expression of LL-37, Ribonuclease7 (RNase7) and Liver-expressed antimicrobial peptide 2 (LEAP-2) in tonsillar tissue and studied a possible relation to seasonal allergic rhinitis (SAR). Tonsils, obtained from patients with SAR and nonallergic controls, were examined for the occurrence of LL-37, RNase7 and LEAP-2 with real-time RT-PCR and immunohistochemistry. Tonsillar mononuclear cells were cultured in presence or absence of LEAP-2 or LL-37 and analyzed for cytokine levels using ELISA. mRNA and protein for LL-37, RNase 7 and LEAP-2 were found in all tonsils. Immunohistochemistry revealed prominent staining for LL-37 and RNase7 in the tonsillar epithelium, whereas a moderate staining was seen with LEAP-2. Real-time RT-PCR showed a downregulation of RNase7 and LEAP-2 in the allergic as compared to the nonallergic group. Mononuclear cells cultured in presence of LEAP-2 or LL-37 demonstrated reduced levels of IL-10. The present study demonstrates the presence and function of LEAP-2, LL-37 and RNase7 in tonsils. Moreover, a downregulation of LEAP-2 and RNase7 is seen in SAR patients, indicating that allergic individuals may be more susceptible to respiratory tract infections due to an impaired antimicrobial defense.