Scanning ion conductance microscopy reveals differential effect of PM2.5 exposure on A549 lung epithelial and SH-SY5Y neuroblastoma cell membranes.

Numerous studies have linked a wide range of diseases including respiratory illnesses to harmful particulate matter (PM) emissions indoors and outdoors, such as incense PM and industrial PM. Because of their ability to penetrate the lower respiratory tract and the circulatory system, fine particles with diameters of 2.5 µm or less (PM2.5) are believed to be more hazardous than larger PMs. Despite the enormous number of studies focusing on the intracellular processes associated with PM2.5 exposure, there have been limited reports studying the biophysical properties of cell membranes, such as nanoscale morphological changes induced by PM2.5. Our study assesses the membrane topographical and structural effects of PM2.5 from incense PM2.5 exposure in real time on A549 lung carcinoma epithelial cells and SH-SY5Y neuroblastoma cells that had been fixed to preclude adaptive cell responses. The size distribution and mechanical properties of the PM2.5 sample were characterized with atomic force microscopy (AFM). Nanoscale morphological monitoring of the cell membranes utilizing scanning ion conductance microscopy (SICM) indicated statistically significant increasing membrane roughness at A549 cells at half an hour of exposure and visible damage at 4 h of exposure. In contrast, no significant increase in roughness was observed on SH-SY5Y cells after half an hour of PM2.5 exposure, although continued exposure to PM2.5 for up to 4 h affected an expansion of lesions already present before exposure commenced. These findings suggest that A549 cell membranes are more susceptible to structural damage by PM2.5 compared to SH-SY5Y cell membranes, corroborating more enhanced susceptibility of airway epithelial cells to exposure to PM2.5 than neuronal cells.

Observation of α-Synuclein Preformed Fibrils Interacting with SH-SY5Y Neuroblastoma Cell Membranes Using Scanning Ion Conductance Microscopy.

Parkinson's disease (PD) is the second-most prevalent neurodegenerative disorder in the U.S. α-Synuclein (α-Syn) preformed fibrils (PFFs) have been shown to propagate PD pathology in neuronal populations. However, little work has directly characterized the morphological changes on membranes associated with α-Syn PFFs at a cellular level. Scanning ion conductance microscopy (SICM) is a noninvasive in situ cell imaging technique and therefore uniquely advantageous to investigate PFF-induced membrane changes in neuroblastoma cells. The present work used SICM to monitor cytoplasmic membrane changes of SH-SY5Y neuroblastoma cells after incubation with varying concentrations of α-Syn PFFs. Cell membrane roughness significantly increased as the concentration of α-Syn PFFs increased. Noticeable protrusions that assumed a more crystalline appearance at higher α-Syn PFF concentrations were also observed. Cell viability was only slightly reduced, though statistically significantly, to about 80% but independent of the dose. These observations indicate that within the 48 h treatment period, PFFs continue to accumulate on the cell membranes, leading to membrane roughness increase without causing prominent cell death. Since PFFs did not induce major cell death, these data suggest that early interventions targeting fibrils before further aggregation may prevent the progression of neuron loss in Parkinson's disease.

The neurosteroid pregnenolone is synthesized by a mitochondrial P450 enzyme other than CYP11A1 in human glial cells.

Neurosteroids, modulators of neuronal and glial cell functions, are synthesized in the nervous system from cholesterol. In peripheral steroidogenic tissues, cholesterol is converted to the major steroid precursor pregnenolone by the CYP11A1 enzyme. Although pregnenolone is one of the most abundant neurosteroids in the brain, expression of CYP11A1 is difficult to detect. We found that human glial cells produced pregnenolone, detectable by mass spectrometry and ELISA, despite the absence of observable immunoreactive CYP11A1 protein. Unlike testicular and adrenal cortical cells, pregnenolone production in glial cells was not inhibited by CYP11A1 inhibitors DL-aminoglutethimide and ketoconazole. Furthermore, addition of hydroxycholesterols increased pregnenolone synthesis, suggesting desmolase activity that was not blocked by DL-aminoglutethimide or ketoconazole. We explored three different possibilities for an alternative pathway for glial cell pregnenolone synthesis: (1) regulation by reactive oxygen species, (2) metabolism via a different CYP11A1 isoform, and (3) metabolism via another CYP450 enzyme. First, we found oxidants and antioxidants had no significant effects on pregnenolone synthesis, suggesting it is not regulated by reactive oxygen species. Second, overexpression of CYP11A1 isoform b did not alter synthesis, indicating use of another CYP11A1 isoform is unlikely. Finally, we show nitric oxide and iron chelators deferoxamine and deferiprone significantly inhibited pregnenolone production, indicating involvement of another CYP450 enzyme. Ultimately, knockdown of endoplasmic reticulum cofactor NADPH-cytochrome P450 reductase had no effect, while knockdown of mitochondrial CYP450 cofactor ferredoxin reductase inhibited pregnenolone production. These data suggest that pregnenolone is synthesized by a mitochondrial cytochrome P450 enzyme other than CYP11A1 in human glial cells.

Identification, Characterization, and Transcriptional Reprogramming of Epithelial Stem Cells and Intestinal Enteroids in Simian Immunodeficiency Virus Infected Rhesus Macaques.

Epithelial cell injury and impaired epithelial regeneration are considered key features in HIV pathogenesis and contribute to HIV-induced generalized immune activation. Understanding the molecular mechanisms underlying the disrupted epithelial regeneration might provide an alternative approach for the treatment of HIV-mediated enteropathy and immune activation. We have observed a significant increased presence of α defensin5+ (HD5) Paneth cells and proliferating Ki67+ epithelial cells as well as decreased expression of E-cadherin expression in epithelial cells during SIV infection. SIV infection did not significantly influence the frequency of LGR5+ stem cells, but the frequency of HD5+ cells was significantly higher compared to uninfected controls in jejunum. Our global transcriptomics analysis of enteroids provided novel information about highly significant changes in several important pathways like metabolic, TCA cycle, and oxidative phosphorylation, where the majority of the differentially expressed genes were downregulated in enteroids grown from chronically SIV-infected macaques compared to the SIV-uninfected controls. Despite the lack of significant reduction in LGR5+ stem cell population, the dysregulation of several intestinal stem cell niche factors including Notch, mTOR, AMPK and Wnt pathways as well as persistence of inflammatory cytokines and chemokines and loss of epithelial barrier function in enteroids further supports that SIV infection impacts on epithelial cell proliferation and intestinal homeostasis.

Nano- and Macroscale Imaging of Cholesterol Linoleate and Human Beta Defensin 2-Induced Changes in Pseudomonas aeruginosa Biofilms.

The biofilm production of Pseudomonas aeruginosa (PA) is central to establishing chronic infection in the airways in cystic fibrosis. Epithelial cells secrete an array of innate immune factors, including antimicrobial proteins and lipids, such as human beta defensin 2 (HBD2) and cholesteryl lineolate (CL), respectively, to combat colonization by pathogens. We have recently shown that HBD2 inhibits biofilm production by PA, possibly linked to interference with the transport of biofilm precursors. Considering that both HBD2 and CL are increased in airway fluids during infection, we hypothesized that CL synergizes with HBD2 in biofilm inhibition. CL was formulated in phospholipid-based liposomes (CL-PL). As measured by atomic force microscopy of single bacteria, CL-PL alone and in combination with HBD2 significantly increased bacterial surface roughness. Additionally, extracellular structures emanated from untreated bacterial cells, but not from cells treated with CL-PL and HBD2 alone and in combination. Crystal violet staining of the biofilm revealed that CL-PL combined with HBD2 effected a significant decrease of biofilm mass and increased the number of larger biofilm particles consistent with altered cohesion of formed biofilms. These data suggest that CL and HBD2 affect PA biofilm formation at the single cell and community-wide level and that the community-wide effects of CL are enhanced by HBD2. This research may inform future novel treatments for recalcitrant infections in the airways of CF patients.

Increased Risk of Thrombocytopenia and Death in Patients with Bacteremia Caused by High Alpha Toxin-Producing Methicillin-Resistant Staphylococcus aureus.

Alpha toxin (Hla) is a major virulence factor of Staphylococcus aureus that targets platelets but clinical data on Hla pathogenesis in bacteremia (SAB) is limited. We examined the link between in vitro Hla activity and outcome. Study isolates obtained from 100 patients with SAB (50 survivors; 50 non-survivors) were assessed for in vitro Hla production by Western immunoblotting in a subset of isolates and Hla activity by hemolysis assay in all isolates. Relevant demographics, laboratory and clinical data were extracted from patients' medical records to correlate Hla activity of the infecting isolates with outcome. Hla production strongly correlated with hemolytic activity (rs = 0.93) in vitro. A trend towards higher hemolytic activity was observed for MRSA compared to MSSA and with high-risk source infection. Significantly higher hemolytic activity was noted for MRSA strains isolated from patients who developed thrombocytopenia (median 52.48 vs. 16.55 HU/mL in normal platelet count, p = 0.012) and from non survivors (median 30.96 vs. 14.87 HU/mL in survivors, p = 0.014) but hemolytic activity of MSSA strains did not differ between patient groups. In vitro Hla activity of MRSA strains obtained from patients with bacteremia is significantly associated with increased risk for thrombocytopenia and death which supports future studies to evaluate feasibility of bedside phenotyping and therapeutic targeting.

American Association of Immunologists Recommendations for an Undergraduate Course in Immunology.

Identifying the "essential" components of an undergraduate immunology lecture course can be daunting because of the varying postgraduate pathways students take. The American Association of Immunologists Education Committee commissioned an Ad Hoc Committee, representing undergraduate, graduate, and medical institutions as well as the biotechnology community, to develop core curricular recommendations for teaching immunology to undergraduates. In a reiterative process involving the American Association of Immunologists teaching community, 14 key topics were identified and expanded to include foundational concepts, subtopics and examples, and advanced subtopics, providing a flexible list for curriculum development and avenues for higher-level learning. Recommendations for inclusive and antiracist teaching that outline opportunities to meet the needs of diverse student populations were also developed. The consensus recommendations can be used to accommodate various course settings and will bridge undergraduate and graduate teaching and prepare diverse students for subsequent careers in the biomedical field.

Human Defensins Inhibit SARS-CoV-2 Infection by Blocking Viral Entry.

Innate immunity during acute infection plays a critical role in the disease severity of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), and is likely to contribute to COVID-19 disease outcomes. Defensins are highly abundant innate immune factors in neutrophils and epithelial cells, including intestinal Paneth cells, and exhibit antimicrobial and immune-modulatory activities. In this study, we investigated the effects of human α- and ß-defensins and RC101, a θ-defensin analog, on SARS-CoV-2 infection. We found that human neutrophil peptides (HNPs) 1-3, human defensin (HD) 5 and RC101 exhibited potent antiviral activity against pseudotyped viruses expressing SARS-CoV-2 spike proteins. HNP4 and HD6 had weak anti-SARS-CoV-2 activity, whereas human ß-defensins (HBD2, HBD5 and HBD6) had no effect. HNP1, HD5 and RC101 also inhibited infection by replication-competent SARS-CoV-2 viruses and SARS-CoV-2 variants. Pretreatment of cells with HNP1, HD5 or RC101 provided some protection against viral infection. These defensins did not have an effect when provided post-infection, indicating their effect was directed towards viral entry. Indeed, HNP1 inhibited viral fusion but not the binding of the spike receptor-binding domain to hACE2. The anti-SARS-CoV-2 effect of defensins was influenced by the structure of the peptides, as linear unstructured forms of HNP1 and HD5 lost their antiviral function. Pro-HD5, the precursor of HD5, did not block infection by SARS-CoV-2. High virus titers overcame the effect of low levels of HNP1, indicating that defensins act on the virion. HNP1, HD5 and RC101 also blocked viral infection of intestinal and lung epithelial cells. The protective effects of defensins reported here suggest that they may be useful additives to the antivirus arsenal and should be thoroughly studied.

Enhanced Intestinal TGF-ß/SMAD-Dependent Signaling in Simian Immunodeficiency Virus Infected Rhesus Macaques.

Transforming growth factor-ß signaling (TGF-ß) maintains a balanced physiological function including cell growth, differentiation, and proliferation and regulation of immune system by modulating either SMAD2/3 and SMAD7 (SMAD-dependent) or SMAD-independent signaling pathways under normal conditions. Increased production of TGF-ß promotes immunosuppression in Human Immunodeficiency Virus (HIV)/Simian Immunodeficiency Virus (SIV) infection. However, the cellular source and downstream events of increased TGF-ß production that attributes to its pathological manifestations remain unknown. Here, we have shown increased production of TGF-ß in a majority of intestinal CD3-CD20-CD68+ cells from acute and chronically SIV infected rhesus macaques, which negatively correlated with the frequency of jejunum CD4+ T cells. No significant changes in intestinal TGF-ß receptor II expression were observed but increased production of the pSMAD2/3 protein and SMAD3 gene expression in jejunum tissues that were accompanied by a downregulation of SMAD7 protein and gene expression. Enhanced TGF-ß production by intestinal CD3-CD20-CD68+ cells and increased TGF-ß/SMAD-dependent signaling might be due to a disruption of a negative feedback loop mediated by SMAD7. This suggests that SIV infection impacts the SMAD-dependent signaling pathway of TGF-ß and provides a potential framework for further study to understand the role of viral factor(s) in modulating TGF-ß production and downregulating SMAD7 expression in SIV. Regulation of mucosal TGF-ß expression by therapeutic TGF-ß blockers may help to create effective antiviral mucosal immune responses.

The Antimicrobial Peptide Human Beta-Defensin 2 Inhibits Biofilm Production of Pseudomonas aeruginosa Without Compromising Metabolic Activity.

Biofilm production is a key virulence factor that facilitates bacterial colonization on host surfaces and is regulated by complex pathways, including quorum sensing, that also control pigment production, among others. To limit colonization, epithelial cells, as part of the first line of defense, utilize a variety of antimicrobial peptides (AMPs) including defensins. Pore formation is the best investigated mechanism for the bactericidal activity of AMPs. Considering the induction of human beta-defensin 2 (HBD2) secretion to the epithelial surface in response to bacteria and the importance of biofilm in microbial infection, we hypothesized that HBD2 has biofilm inhibitory activity. We assessed the viability and biofilm formation of a pyorubin-producing Pseudomonas aeruginosa strain in the presence and absence of HBD2 in comparison to the highly bactericidal HBD3. At nanomolar concentrations, HBD2 - independent of its chiral state - significantly reduced biofilm formation but not metabolic activity, unlike HBD3, which reduced biofilm and metabolic activity to the same degree. A similar discrepancy between biofilm inhibition and maintenance of metabolic activity was also observed in HBD2 treated Acinetobacter baumannii, another Gram-negative bacterium. There was no evidence for HBD2 interference with the regulation of biofilm production. The expression of biofilm-related genes and the extracellular accumulation of pyorubin pigment, another quorum sensing controlled product, did not differ significantly between HBD2 treated and control bacteria, and in silico modeling did not support direct binding of HBD2 to quorum sensing molecules. However, alterations in the outer membrane protein profile accompanied by surface topology changes, documented by atomic force microscopy, was observed after HBD2 treatment. This suggests that HBD2 induces structural changes that interfere with the transport of biofilm precursors into the extracellular space. Taken together, these data support a novel mechanism of biofilm inhibition by nanomolar concentrations of HBD2 that is independent of biofilm regulatory pathways.

Yeast casein kinase 2 governs morphology, biofilm formation, cell wall integrity, and host cell damage of Candida albicans.

The regulatory networks governing morphogenesis of a pleomorphic fungus, Candida albicans are extremely complex and remain to be completely elucidated. This study investigated the function of C. albicans yeast casein kinase 2 (CaYck2p). The yck2Δ/yck2Δ strain displayed constitutive pseudohyphae in both yeast and hyphal growth conditions, and formed enhanced biofilm under non-biofilm inducing condition. This finding was further supported by gene expression analysis of the yck2Δ/yck2Δ strain which showed significant upregulation of UME6, a key transcriptional regulator of hyphal transition and biofilm formation, and cell wall protein genes ALS3, HWP1, and SUN41, all of which are associated with morphogenesis and biofilm architecture. The yck2Δ/yck2Δ strain was hypersensitive to cell wall damaging agents and had increased compensatory chitin deposition in the cell wall accompanied by an upregulation of the expression of the chitin synthase genes, CHS2, CHS3, and CHS8. Absence of CaYck2p also affected fungal-host interaction; the yck2Δ/yck2Δ strain had significantly reduced ability to damage host cells. However, the yck2Δ/yck2Δ strain had wild-type susceptibility to cyclosporine and FK506, suggesting that CaYck2p functions independently from the Ca+/calcineurin pathway. Thus, in C. albicans, Yck2p is a multifunctional kinase that governs morphogenesis, biofilm formation, cell wall integrity, and host cell interactions.

The Role of Defensins in HIV Pathogenesis.

Profound loss of CD4+ T cells, progressive impairment of the immune system, inflammation, and sustained immune activation are the characteristics of human immunodeficiency virus-1 (HIV-1) infection. Innate immune responses respond immediately from the day of HIV infection, and a thorough understanding of the interaction between several innate immune cells and HIV-1 is essential to determine to what extent those cells play a crucial role in controlling HIV-1 in vivo. Defensins, divided into the three subfamilies α-, ß-, and θ-defensins based on structure and disulfide linkages, comprise a critical component of the innate immune response and exhibit anti-HIV-1 activities and immunomodulatory capabilities. In humans, only α- and ß-defensins are expressed in various tissues and have broad impacts on HIV-1 transmission, replication, and disease progression. θ-defensins have been identified as functional peptides in Old World monkeys, but not in humans. Instead, θ-defensins exist only as pseudogenes in humans, chimpanzees, and gorillas. The use of the synthetic θ-defensin peptide "retrocyclin" as an antiviral therapy was shown to be promising, and further research into the development of defensin-based HIV-1 therapeutics is needed. This review focuses on the role of defensins in HIV-1 pathogenesis and highlights future research efforts that warrant investigation.

Assessment of free fatty acids and cholesteryl esters delivered in liposomes as novel class of antibiotic.

BACKGROUND: Healthcare associated infections (HAI) with multidrug-resistant (MDR) bacteria continue to be a global threat, highlighting an urgent need for novel antibiotics. In this study, we assessed the potential of free fatty acids and cholesteryl esters that form part of the innate host defense as novel antibacterial agents for use against MDR bacteria. METHODS: Liposomes of six different phospholipid mixtures were employed as carrier for six different fatty acids and four different cholesteryl esters. Using a modified MIC assay based on DNA quantification with the fluoroprobe Syto9, formulations were tested against Gram-positive and Gram-negative bacteria implicated in HAI. Formulations with MIC values in the low µg/mL range were further subjected to determination of minimal bactericidal activity, hemolysis assay with sheep erythrocytes, and cytotoxicity testing with the human liver cell line HepG2. The potential for synergistic activity with a standard antibiotic was also probed. RESULTS: Palmitic acid and stearic acid prepared in carrier 4 (PA4 and SA4, respectively) were identified as most active lipids (MIC against MDR Staphylococcus epidermidis was 0.5 and 0.25 µg/mL, respectively; MIC against vancomycin resistant Enterococcus faecalis (VRE) was 2 and 0.5 µg/mL, respectively). Cholesteryl linoleate formulated with carrier 3 (CL3) exhibited activity against the S. epidermidis strain (MIC 1 µg/mL) and a Pseudomonas aeruginosa strain (MIC 8 µg/mL) and lowered the vancomycin MIC for VRE from 32-64 µg/mL to as low as 4 µg/mL. At 90 µg/mL PA4, SA4, and CL3 effected less than 5 % hemolysis over 3 h and PA4 and CL3 did not exhibit significant cytotoxic activity against HepG2 cells when applied at 100 µg/mL over 48 h. CONCLUSIONS: Our results showed that selected fatty acids and cholesteryl esters packaged with phospholipids exhibit antibacterial activity against Gram-positive and Gram-negative bacteria and may augment the activity of antibiotics. Bactericidal activity could be unlinked from hemolytic and cytotoxic activity and the type of phospholipid carrier greatly influenced the activity. Thus, fatty acids and cholesteryl esters packaged in liposomes may have potential as novel lipophilic antimicrobial agents.

Cholesteryl esters are elevated in the lipid fraction of bronchoalveolar lavage fluid collected from pediatric cystic fibrosis patients.

BACKGROUND: Host-derived lipids including cholesteryl esters (CEs) such as cholesteryl linoleate have emerged as important antibacterial effectors of innate immunity in the airways and cholesteryl linoleate has been found elevated in the context of inflammation. Cystic fibrosis (CF) patients suffer from chronic infection and severe inflammation in the airways. Here, we identified and quantified CEs in bronchoalveolar lavage fluid (BALF) from CF patients and non-CF disease controls, and tested whether CE concentrations are linked to the disease. MATERIALS AND METHODS: CEs in BALF from 6 pediatric subjects with CF and 7 pediatric subjects with non-CF chronic lung disease were quantified by mass spectral analysis using liquid chromatography coupled with tandem mass spectrometry and multiple reaction monitoring. BALFs were also examined for total lipid, total protein, albumin, and, as a marker for inflammation, human neutrophil peptide (HNP) 1-3 concentrations. Statistical analysis was conducted after log 10 transformation of the data. RESULTS: Total lipid/protein ratio was reduced in CF BALF (p = 0.018) but the concentrations of CEs, including cholesteryl linoleate, were elevated in the total lipid fraction in CF BALF compared to non-CF disease controls (p < 0.050). In addition, the concentrations of CEs and HNP1-3 correlated with one another (p < 0.050). CONCLUSIONS: The data suggests that the lipid composition of BALF is altered in CF with less total lipid relative to protein but with increased CE concentrations in the lipid fraction, likely contributed by inflammation. Future longitudinal studies may reveal the suitability of CEs as a novel biomarker for CF disease activity which may provide new information on the lipid mediated pathophysiology of the disease.

Detection of antimicrobial (poly)peptides with acid urea polyacrylamide gel electrophoresis followed by Western immunoblot.

Antimicrobial (poly)peptides (AMPs) are ancient key effector molecules of innate host defense and have been identified in mammals, insects, plants, and even fungi (Nakatsuji and Gallo, J Invest Dermatol, 132: 887-895, 2012). They exhibit a cationic net charge at physiological pH and are rich in hydrophobic amino acids (Dufourc et al., Curr Protein Pept Sci, 13: 620-631, 2012). Their mode of action has been best investigated in bacteria. When assuming secondary structure the cationic and hydrophobic amino acids are sequestered creating a bipartitioned molecule in which the cationic amino acids mediate initial electrostatic interaction with the negatively charged bacterial surface and the hydrophobic amino acids mediate embedding into the bacterial membranes followed by a multitude of effects interfering with bacterial viability (Nicolas, FEBS J, 276: 6483-6496, 2009; Padovan et al., Curr Protein Pept Sci, 11: 210-219, 2010). However, immunomodulatory, antitumor, and other effects have been added to the ever increasing list of AMP functions (Pushpanathan et al., Int J Pept, 2013: 675391, 2013). Several classes of AMPs have been distinguished based on structure, namely anti-parallel beta-sheet, alpha-helical, circular, as well as disulfide bridge connectivity (Bond and Khalid, Protein Pept Lett, 17: 1313-1327, 2010). Many of the AMPs undergo posttranslational modification including further proteolysis. Biochemical analysis at the protein level is of great interest for a wide range of scientists and important when studying host-pathogen interaction, for example Salmonella invasion of the small intestine. Acid-urea polyacrylamide gel electrophoresis (AU-PAGE) followed by Western immunoblotting is an important tool for the identification and quantification of cationic AMPs. The protocol for these procedures outlined here describes, in detail, the necessary steps; including pouring the AU-gels, preparing the test samples, performing the electrophoretic separation and protein transfer to the membrane, and conducting the immunodetection using an alkaline phosphatase/NBT/BCIP system. A standard SDS-PAGE in comparison with AU-PAGE and the corresponding Western immunoblot are depicted in Fig. 1.

Assessment of epithelial innate antimicrobial factors in sinus tissue from patients with and without chronic rhinosinusitis.

BACKGROUND: Airway secretions contain endogenous antimicrobial factors (AMFs) that contribute to the innate host defense of the respiratory tract. Antibacterial peptides as well as host-derived lipids including cholesteryl esters have been detected in maxillary lavage fluid. Sterol O-acyltransferase 1 (SOAT1) is a key enzyme in cholesteryl ester production. The purpose of this study is to determine if such intrinsic microbicidal molecules are acutely expressed within sinus tissue and to compare levels of expression between patients with and without chronic rhinosinusitis (CRS). METHODS: Sinus tissue was obtained from subjects with (24) and without (9) a history of CRS. Six CRS patients had nasal polyposis (CRSwNP). Immunofluorescence staining for human neutrophil peptide (HNP) was done as a marker for inflammation. Real-time polymerase chain reaction (RT-PCR) following RNA extraction was used to quantify the expression of SOAT-1, the epithelial beta-defensins (HBD2 and HBD3), and the cathelicidin LL37 with ribosomal protein, large, P0 (RPLP0) as the housekeeping gene. RESULTS: Immunofluorescence showed significant increase in HNP staining in CRS patients without nasal polyposis (CRSsNP) vs non-CRS specimens (p = 0.010), in agreement with clinical inflammation status. SOAT1 messenger RNA (mRNA) expression was also upregulated in CRSsNP compared to non-CRS (p = 0.041) and CRSwNP (p = 0.005) patients, whereas increases for HBD2 and HBD3 were less prominent. LL37 was either absent or expressed at very low levels in all samples. CONCLUSION: Increased biosynthesis of SOAT1, a key enzyme for antimicrobial cholesteryl ester production, was observed in the sinus tissue of CRSsNP patients but not in CRSwNP patients. This further supports the novel concept of lipid-mediated innate mucosal defense and delineates CRS with and without nasal polyposis as distinct subtypes.

Effects of aerobic exercise on lipid-effector molecules of the innate immune response.

INTRODUCTION: Consistent, moderate-to-vigorous-intensity exercise has been associated with a lower risk of upper respiratory tract infection (URI). However, the molecular basis for this apparent protection has not yet been fully resolved. Host-derived lipids such as cholesteryl esters (CE) have emerged as important effector molecules of innate defense against infections. Here, we compared antimicrobial CE in nasal fluid before and after moderate-to-vigorous exercise between active and inactive subjects. METHODS: Nasal fluid was collected from 14 healthy, recreationally active subjects (32 ± 11 yr, 7 men and 7 women) and 14 healthy, inactive subjects (25 ± 3 yr, 7 men and 7 women) before and after treadmill exercise at 70% heart rate reserve. Nasal fluid was analyzed for lysozyme, cholesteryl linoleate (CL), cholesteryl arachidonate (CA), and albumin (Alb) concentrations. RESULTS: Baseline concentrations (mean ± SEM, inactive vs active) of lysozyme (117.7 ± 31.1 vs 122.9 ± 15.5 µg·mL), CL + CA (15.3 ± 1.8 vs 26.2 ± 10.05 µg·mL), and Alb (156.6 ± 54.5 vs 126.9 ± 32.8 µg·mL) were similar to previously reported levels and did not differ significantly between study groups. However, postexercise, CL + CA concentration was significantly lower in inactive compared with active subjects (7.8 ± 1.5 vs 20.1 ± 4.8 µg·mL, P = 0.036) dropping below the antimicrobial effective range. Once adjusted to Alb concentrations, the changes were no longer significant, suggesting that plasma transudation accounted for the increased CA + CL concentration postexercise in the active group relative to the inactive group. CONCLUSIONS: Moderate-to-vigorous aerobic exercise acutely decreases the antimicrobial CE response in inactive subjects but does not modify baseline levels of CE between active and inactive subjects. This suggests that compared with active individuals, inactive individuals may be at greater risk for upper respiratory tract infection immediately postexercise.

Human alpha defensin 5 expression in the human kidney and urinary tract.

BACKGROUND: The mechanisms that maintain sterility in the urinary tract are incompletely understood. Recent studies have implicated the importance of antimicrobial peptides (AMP) in protecting the urinary tract from infection. Here, we characterize the expression and relevance of the AMP human alpha-defensin 5 (HD5) in the human kidney and urinary tract in normal and infected subjects. METHODOLOGY/PRINCIPAL FINDINGS: Using RNA isolated from human kidney, ureter, and bladder tissue, we performed quantitative real-time PCR to show that DEFA5, the gene encoding HD5, is constitutively expressed throughout the urinary tract. With pyelonephritis, DEFA5 expression significantly increased in the kidney. Using immunoblot analysis, HD5 production also increased with pyelonephritis. Immunostaining localized HD5 to the urothelium of the bladder and ureter. In the kidney, HD5 was primarily produced in the distal nephron and collecting tubules. Using immunoblot and ELISA assays, HD5 was not routinely detected in non-infected human urine samples while mean urinary HD5 production increased with E.coli urinary tract infection. CONCLUSIONS/SIGNIFICANCE: DEFA5 is expressed throughout the urinary tract in non-infected subjects. Specifically, HD5 is expressed throughout the urothelium of the lower urinary tract and in the collecting tubules of the kidney. With infection, HD5 expression increases in the kidney and levels become detectable in the urine. To our knowledge, our findings represent the first to quantitate HD5 expression and production in the human kidney. Moreover, this is the first report to detect the presence of HD5 in infected urine samples. Our results suggest that HD5 may have an important role in maintaining urinary tract sterility.

Expansion of Paneth cell population in response to enteric Salmonella enterica serovar Typhimurium infection.

Paneth cells residing at the base of the small intestinal crypts contribute to the mucosal intestinal first line defense by secreting granules filled with antimicrobial polypeptides including lysozyme. These cells derive from the columnar intestinal stem cell located at position 0 and the transit amplifying cell located at position +4 in the crypts. We have previously shown that Salmonella enterica serovar Typhimurium (ST), a leading cause of gastrointestinal infections in humans, effects an overall reduction of lysozyme in the small intestine. To extend this work, we examined small-intestinal tissue sections at various time points after ST infection to quantify and localize expression of lysozyme and assess Paneth cell abundance, apoptosis, and the expression of Paneth cell differentiation markers. In response to infection with ST, the intestinal Paneth cell-specific lysozyme content, the number of lysozyme-positive Paneth cells, and the number of granules per Paneth cell decreased. However, this was accompanied by increases in the total number of Paneth cells and the frequency of mitotic events in crypts, by increased staining for the proliferation marker PCNA, primarily at the crypt side walls where the transit amplifying cell resides and not at the crypt base, and by apoptotic events in villi. Furthermore, we found a time-dependent upregulation of first ß-catenin, followed by EphB3, and lastly Sox9 in response to ST, which was not observed after infection with a Salmonella pathogenicity island 1 mutant deficient in type III secretion. Our data strongly suggest that, in response to ST infection, a Paneth cell differentiation program is initiated that leads to an expansion of the Paneth cell population and that the transit amplifying cell is likely the main progenitor responder. Infection-induced expansion of the Paneth cell population may represent an acute intestinal inflammatory response similar to neutrophilia in systemic infection.